Encyclopedia of Cancer [4 ed.]
 9783662468746, 9783662468753, 9783662474242, 2017933328

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  • eBook

Table of contents :
Preface to the Fourth Edition
Preface to the Third Edition
Preface to the Second Edition
Preface to the First Edition
Editor-in-Chief
Contributors
A
284461-73-0
85622-93-1
17-1A
A Disintegrin and Metalloprotease
AAMP
Definition
Characteristics
References
See Also
AAPC
AAV
Definition
Characteristics
AAV Genome Structure
AAV Life Cycles
Clinical Relevance
Are Wildtype AAVs Pathogenic in Humans?
Is There a Natural Connection Between AAV Infection and Cancer?
What Are Recombinant AAV Vectors?
Is AAV Unique as a Human Gene Therapy Vector?
What Are Advances in AAV Vector Technology?
What Are Clinically Relevant rAAV Applications in Cancer Treatment?
Anti-Angiogenesis
Immunotherapy
Tumor Suppressors
Suicide Gene Therapy
Drug Resistance
Repair Strategies
Purging of Tumor Cells from Autologous Transplants
RNAi
Future Applications
Cross-References
References
See Also
Ab (Latin: Away) -Scopus (Greek: Target) Effects
ABC (ATP-Binding Cassette) Superfamily
ABC Drug-Transporters
Synonyms
Definition
Cross-References
See Also
ABC Transporter
ABCC Transporters
Synonyms
Definition
Characteristics
Members and Functional Properties
Multidrug Resistance-Associated Proteins (MRPs)
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7)
Sulfonylurea Receptors SUR1 (ABCC8) and SUR2A/B (ABCC9)
Glutathione Transport, Redox Signaling, and Apoptosis
References
ABC-Transporters
Synonyms
Definition
Characteristics
Human ABC-Transporters
ABC-Transporters and Multidrug Resistance of Cancer
ABC-Transporters as Anticancer Drug Targets
Cross-References
References
See Also
Abraxas
Definition
Cross-References
See Also
Abscopal Effect
Abscopal Effects
Synonyms
Definition
Characteristics
Conclusion
Cross-References
References
ABVD
Definition
Characteristics
References
AC1L50CF
ACDC
2-Acetoxybenzenecarboxylic Acid
2-Acetoxybenzoic Acid
Acetylsalicylic Acid
Achneiform Rash
Definition
Cross-References
Acral Metastasis
ACRP30
Actinic Keratosis
Definition
Cross-References
Activated Natural Killer Cells
Synonyms
Definition
Characteristics
Biology of NK Cells
Role of NK Cells in Human Cancer
Markers of NK Cells
NK Cell Receptors
NK Cell and Cytokines
Cytotoxicity of NK Cells Against Tumor or Infected Cells
NK Cells and Cancer Immunotherapy
Cross-References
References
Activation-Induced Cytidine Deaminase
Synonyms
Definition
Characteristics
Identification of AID
AID Deamination Mechanism
AID Targeting
AID Expression in Cancer
B Cell Lineage Malignancies
Non-B Cell Lineage Malignancies
AID Is Required for Recurrent Chromosomal Translocations
AID Expression Associated with Cancer Progression and Poor Prognosis
AID as a DNA Demethylase
References
Active Cell Death
Active Specific Immunization
Synonyms
Definition
Cross-References
See Also
Activin
Synonyms
Definition
Characteristics
Signaling Cascade
Functions
Activin Antagonists
The Role of Activin A in Cancer Development and Progression
Conclusion
References
Activin A
Activin B
Activin C
Activin E
Activin Receptor Type 2
Activin Receptor Type 1
Activin Receptor-Like Kinase
Activin Receptors
Synonyms
Definition
Characteristics
Structure and Signaling
Function and Expression
Activin Receptors in Cancer
Conclusion
References
ACTR
ActRI
ActRII
Acute Granulocytic Leukemia
Acute Lymphoblastic Leukemia
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Acute Megakaryoblastic Leukemia
Synonyms
Definition
Characteristics
Epidemiology
Clinical and pathologic features
Cytogenetic and Biological Features
Prognosis and Treatment
References
Acute Megakaryoblastic Leukemia M7
Acute Myelogenous Leukemia
Acute Myeloid Leukemia
Synonyms
Definition
Characteristics
Classification
Epidemiology
Etiology
Signs and Symptoms of AML
Prognostic Factors
Therapy
Cross-References
References
See Also
Acute Myeloid Leukemia 1
Acute Nonlymphocytic Leukemia
Acute Promyelocytic Leukemia
Definition
Characteristics
Clinical and Laboratorial Presentation
Molecular Characterization
Modeling APL in Mice
Therapeutics
References
Acute-Phase Response Factor
ACVR1
ACVR2
1-acyl-sn-glycerol-3-phosphate
2-acyl-sn-glycerol-3-phosphate
ADAbp
ADA-CP
ADAM Molecules
Synonyms
Definition
Characteristics
Metalloprotease Function
Adhesion Function
Signaling Function
Other Functions
Cross-References
References
ADAM17
Synonyms
Definition
Characteristics
Structure
Expression and Regulation
Biological Function
Clinical Relevance
Summary
Cross-References
References
See Also
Adaptive Immunity
Definition
Cross-References
Adaptor Proteins
Definition
Characteristics
References
Adducts to DNA
Synonyms
Definition
Characteristics
Rationale for Using DNA Adducts as Biomarkers for Exposure and Adverse Effects
Advantages and Disadvantages of DNA Adducts Compared to Other Biomarkers
Cellular Defense: Repair of DNA Adducts
Adduct Measurements in Disease Epidemiology
Association of DNA Adducts with Cancer Risk
Background DNA Adduct Levels: Sources, Variations, and Cancer Risk Prediction
Contributions of DNA Adduct Measurements to Disease Etiology and Pathogenesis
Cross-References
References
Adenine Nucleoside
Adenine-9-beta-d-Ribofuranoside
Adenocarcinoma
Definition
Cross-References
See Also
Adenoma
Definition
Cross-References
See Also
Adenomas
Adenomatous Polyposis Coli
Adenomatous Polyps
Adenomucinosis
Adenopathy
Definition
Adenosine
Adenosine and Tumor Microenvironment
Synonyms
Definition
Characteristics
Adenovirus
Definition
Characteristics
Infection and Viral Transcription
Adenoviral Functions and Oncogenesis
Gene Therapy: First- and Second-Generation Adenoviral Vectors
``Gutless´´ Adenoviral Vectors
Replication-Competent Adenoviral Vectors for Cancer Gene Therapy
Adenoviral Vectors for Genetic Vaccination
References
ADEPT
ADF
Adherens Junctions
Synonyms
Definition
Characteristics
Implications in Cancer
Cross-References
References
See Also
Adhesion
Definition
Characteristics
Cell Adhesion Receptors
Adhesion and Cancer
Adhesion in Metastasis
Adhesion Within The Tumor Mass
Malignant Tumor Cells in the Blood Stream: Adhesion to Blood Cells and Platelets
Adhesion in the Target Organ
Adhesion to Endothelial Cells (EC)
Adhesion to Extracellular Matrix Components
Cross-References
References
See Also
Adhesion Molecules
ADI
Adipocyte C1q and Collagen Domain Containing
Adipocyte Complement-Related Protein of 30 kDa
Adipocytic Tumors
Adiponectin
Synonyms
Definition
Characteristics
Adiponectin and Carcinogenesis
Prostate Cancer
Breast Cancer
Endometrial Cancer
Lung Cancer
Kidney Cancer
Pancreatic Cancer
Liver Cancer
Esophageal Cancer
Gastric and Colorectal Cancer
Leukemia and Myeloma
Mechanisms
AMPK
Glycogen Synthase Kinase (GSK) 3beta/beta-Catenin Signaling Pathway
Other Pathways
Adiponectin-based Therapeutics
Cross-References
References
See Also
AdipoQ
Adipose Most Abundant Gene Transcript 1
Adipose Tissue-Specific Secretory Factor (ADSF)
Adipose Tumors
Synonyms
Definition
Characteristics
Benign Adipose Tumors
Malignant Adipose Tumors
Cross-References
References
See Also
Adjuvant Chemoendocrine Therapy
Synonyms
Definition
Characteristics
Breast Cancer
Adjuvant Hormone Therapy
Adjuvant Chemotherapy
Planning the Adjuvant Treatment: Adjuvant Chemoendocrine Therapy
Prostate Cancer
References
Adjuvant Chemohormonal Therapy
Adjuvant Cytotoxic Therapy
Adjuvant Hormonal Therapy
Adjuvant Therapy
Definition
Characteristics
Cross-References
See Also
ADMET Screen
Definition
Characteristics
References
Adopted Orphan Nuclear Receptors
Adoptive Cellular Transfer
Adoptive Immunotherapy
Synonyms
Keywords
Definition
Characteristics
Unmanipulated T Cells
NK Cells
Cytokine-Induced Killer Cells
Lymphokine-Activated Killer Cells
Tumor-Infiltrating Lymphocytes
Antigen-Specific Cytotoxic T Lymphocytes
Enhancing the Function of Adoptively Transferred Cells
Lymphodepletion
TCR and Chimeric Antigen Receptors
Cross-References
References
See Also
Adoptive T-Cell Transfer
Synonyms
Definition
Characteristics
TILs
Cancer Antigen-Induced T Cells
Antigen-Specific TCR-Transduced T Cells
CAR-Transduced T Cells
Conclusions
Cross-References
References
Adrenocortical Cancer
Synonyms
Definition
Characteristics
Epidemiology of Adrenocortical Cancer
Pathophysiology of Adrenocortical Cancer
IGF-II (Insulin-Like Growth Factor II)
beta-Catenin Activation in Adrenocortical Cancer
TP53
Diagnosis and Treatment of Adrenocortical Cancer
Clinical and Hormonal Investigations
Imaging of Adrenocortical Cancer
Pathology and Molecular Analysis
Prognosis of Adrenocortical Cancer
Treatment of Adrenocortical Cancer
References
Adrenomedullin
Definition
Characteristics
Adrenomedullin: Peptide and Gene Structure
Signal Transduction
AM Serves as a Common Language Between the Different Cellular Components of the Tumor Microenvironment
Conclusion
References
Adriamycin
Synonyms
Definition
Characteristics
Chemical Properties
Clinical Aspects
Therapeutic Applications
Pharmacokinetics
Clinical Toxicities
Pharmacological Mechanisms
Mechanisms of Action
Mechanisms of Resistance
Mechanisms for Development of Cardiotoxicity
References
ADT
Adult Stem Cells
Synonyms
Definition
Characteristics
Adult Stem Cell Differentiation
Therapeutic Potential
Cancer Stem Cells
Cross-References
References
See Also
Adult T-Cell Leukemia
Synonyms
Definition
Cross-References
See Also
Adult T-Cell Leukemia-Derived Factor
Adult Type
Aerobic Glycolysis
Afinitor (marketed by NOVARTIS)
Aflatoxins
Definition
Characteristics
Biotransformation
Carcinogenesis
Repair
Species/Tissue Susceptibility
Cross-References
References
Aflibercept
Synonyms
Definition
Cross-References
AFP
Aggressive Fibromatosis
Aggressive Fibromatosis in Children
Synonyms
Definition
Characteristics
Clinical Presentation
Diagnostic Approach
Pathogenesis
Treatment
Side Effects in Survivors
Conclusion
Cross-References
References
See Also
Aggrus
Aging
Definition
Characteristics
Aging
Epithelial Cancers
Cellular Senescence
Alterations in the Microenvironment
Tumor Progression in Aging
Implications for Treatment
Cross-References
References
See Also
Aging-Associated Gene 4 Protein (AAG4)
Aging-Associated Inflammation
Synonyms
Definition
Characteristics
References
Agnogenic Myeloid Metaplasia
Agranulocytosis
AHNP
AHR
AIB1
AICDA
AID
AIDS-129717
AIDS-Associated Cancers
AIDS-Associated Malignancies
Synonyms
Definition
Characteristics
HIV Infection and AIDS Increase the Risk for Certain Cancers
The Human Oncogenic Viruses EBV and KSHV and HIV/AIDS
Kaposi Sarcoma Herpesvirus and Oncogenesis of AIDS-KS
KSHV Oncogenesis and Immunosuppression
AIDS-Associated Lymphomas
Current Therapies and Clinical Challenges
Treatments for AIDS-Associated Kaposi Sarcoma
Treatments for AIDS Lymphomas
Conclusion
Cross-References
References
AIDS-Related Cancers
AIE2
AIF-Mediated Cell Death
AIK
AIK2
AIK3
AIM1
AIM-1
Akt Signal Transduction Pathway
Definition
Characteristics
Akt in Human Malignancy and Different Functions of Akt Family Members
Signal Transduction
Akt Pathway as a Target for Cancer Intervention
Cross-References
References
Alcohol Consumption
Definition
Characteristics
Epidemiology
Cancer of the Upper Aerodigestive Tract
Hepatocellular Cancer (HCC)
Breast Cancer
Colorectal Cancer
Mechanisms of Alcohol-Mediated Carcinogenesis
Acetaldehyde
Oxidative Stress
Altered Methyl Transfer
Reduced Retinoic Acid
Specific Mechanisms (Cirrhosis, Gastroesophageal Reflux Disease, Estrogens)
Cross-References
References
See Also
Alcoholic Beverages Cancer Epidemiology
Definition
Characteristics
Epidemiology of Alcohol-Related Cancer
Mechanisms of Alcohol Carcinogenicity
Conclusions
Cross-References
References
Alcoholic Pancreatitis
Definition
Characteristics
Incidence
Symptoms
Treatment
Etiology
Summary
Cross-References
Aldehyde Dehydrogenases
Synonyms
Definition
Chracteristics
Nomenclature System
ALDH Activity and Other Assays
ALDH Activity as a Marker for Stem Cells
ALDH in Retinoic Acid Synthesis
Alcohol Metabolism
ALDH and Drug Metabolism
ALDH Related Diseases
Cross-References
References
See Also
ALDH
Aldo-Keto Reductases
ALK
ALK Protein
Synonyms
Definition
Characteristics
ALK Protein and Cancer
Full-Length ALK
ALK Fusion Proteins
Structure
Distribution
Function
Tumor Types
Therapeutic Options
Cross-References
References
See Also
Alkylating Agents
Definition
Characteristics
Classification
Mechanism of Action
DNA Cross-Links
Molecular Pharmacology, Drug Resistance, and Clinical Efficacy
Metabolism
Toxicology
Cross-References
References
See Also
ALL
Allele Imbalance
Definition
See Also
Allelic Association
Allergic Asthma
Allergic Conjunctivitis
Allergic Rhinitis
Allergy
Synonyms
Definition
Characteristics
Epidemiological Studies
Biological Details
References
Allogeneic Bone Marrow Transplantation
Allogeneic Cell Therapy
Synonyms
Definition
Characteristics
Rationale
Procedure
Mechanisms
Clinical Aspects
Cross-References
References
Allogeneic Cellular Immunotherapy
Allogeneic Hematopoietic Stem Cell Transplantation
Alpha1-Fetoglobulin
Alpha-Fetoprotein
Synonyms
Definition
Characteristics
Immunochemical Techniques
AFP in Pathology
Fetuin Versus AFP
AFP in Laboratory Animals
Cross-References
References
See Also
Alpha-Fetoprotein Diagnostics
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Alternative Reading Frame
Alu Elements
Definition
Characteristics
Structure
Function
Role in Human Cancer
Cross-References
References
See Also
AME Transcription Factor
Synonyms
Definition
Characteristics
Cross-References
References
Amine Oxidases
Definition
Characteristics
Functions
Cross-References
References
See Also
Amino-Bisphosphonate
4-Amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one
AML1
AML1/ETO
AML1/EVI-1
AML1/MTG8
AMN107
Amph II
Amphibian Gastrin-Releasing Peptide
Amphiphysin II
Amphiphysin-Like
Amphiregulin
Synonyms
Definition
Characteristics
Amphiregulin Expression and Function
Cross-References
References
See Also
AMPL
Amplaxin
Amplification
Definition
Characteristics
Cellular Regulation
Clinical Relevance
Cross-References
References
See Also
Amplified in Breast Cancer 1
Synonyms
Definition
Characteristics
Molecular Structure and Functional Domains
Functional Mechanisms
Physiological Function
Role in Cancer
References
Amrubicin
Synonyms
Definition
Characteristics
Preclinical Studies
Clinical Studies
Amrubicin Monotherapy
Amrubicin Combination Chemotherapy
Conclusion
Cross-References
References
Anaerobic
Anaplastic Astrocytoma
Anaplastic Large Cell Lymphoma
Synonyms
Definition
Characteristics
Diagnosis
Genetics
Therapy
Cross-References
References
See Also
Anaplastic Lymphoma Kinase
Definition
Cross-References
Anaplastic Thyroid Carcinomas
Anchorage-Independent
Definition
Cross-References
See Also
Androgen Ablation Therapy
Synonyms
Definition
Cross-References
See Also
Androgen Deprivation Therapy
Androgen Insensitivity Syndrome (AIS)
Androgen Receptor
Synonyms
Definition
Characteristics
Structure-Function Relationships Between Different Domains of Androgen Receptor that Are Required for its Transcriptional Activity
Regulation of Unliganded AR
Regulation of Ligand-Bound AR
Nongenomic Function of the AR
Androgen Receptor in Human Physiology and Pathology
References
Androgen Suppression Therapy
Androgen-Independent Prostate Cancer (AIPC)
Synonyms
Definition
Cross-References
Aneuploidy
Definition
Characteristics
Aneuploidy in Cancer
Aneuploidy as a ``Driving Force´´ and Not a ``Consequence´´ in Cancer
Genetic Mechanisms of Aneuploidy in Cancer
Conclusions
Cross-References
References
Angiogenesis
Synonyms
Definition
Characteristics
Clinical Aspects
Cross-References
References
Angiogenesis-Inhibiting Agents
Angiogenin
Synonyms
Definition
Characteristics
Angiogenin is the Fifth Member of the Human Ribonuclease A Superfamily
Angiogenin Is an Angiogenic Factor
Angiogenin Is a Tumorigenic Factor
Angiogenin Is a Neuroprotective Factor
Angiogenin Acts in Other Diseases
Summary
Acknowledgments
References
Angiopoietins
Definition
Characteristics
Structure of Angiopoietins
Angiopoietin 1: A Protective Ligand
Angiopoietin 2 Promotes Vascular Destabilization
Therapeutic Target for Tumor Angiogenesis
Cross-References
References
Angiotensin
Angiotensin II Signaling
Synonyms
Definition
Characteristics
Angiotensin II Signaling in Carcinogenesis
Clinical Aspects
Epidemiological Study of the Effect of ACE Inhibitors on Cancer Risk
References
ANLL
Anoikis
Synonyms
Definition
Characteristics
Mechanisms
Anoxia
Synonyms
Definition
Literal Definition
Conceptual Definition
Characteristics
In Vitro Creation of Hypoxia and Anoxia
Causes and Consequences of Tumor Anoxia
Information Flow Within the Cell
Cell Fate in Anoxia
Anoxia-Induced Dormancy
Interplay Between HIF1α and p53 Determining Cell Fate in Anoxia
p53 and Cell Death
p53 and Cell Survival
Oxygen Sensing/Signaling Pathways Specific to Anoxia
Therapeutic Implications of Anoxia
Cross-References
References
See Also
Anoxic
Ansamycin Class of Natural Product Hsp90 Inhibitors
Synonyms
Definition
Characteristics
Antitumor Activity
References
Anthracyclines
Definition
Cross-References
Antiangiogenesis
Definition
Characteristics
Rationale for Antiangiogenesis for Therapy
Potential Targets for Antiangiogenesis
Antiangiogenesis Strategy in Cancer Patients
Antiangiogenesis for Ocular Diseases
Mechanisms of Action of Pharmacologic Antiangiogenic Agents
Biomarkers of Antiangiogenesis
Toxicity of Antiangiogenesis
Future Directions in Antiangiogenesis
Cross-References
References
See Also
Anti-angiogenic Chemotherapy
Antibodies to Self-antigens
Antibody Toxin Fusions or Conjugates
Antibody-Dependent Cellular Cytotoxicity
Definition
Cross-References
Antibody-Directed Enzyme Prodrug Therapy
Synonyms
Definition
Characteristics
References
Anticancer Drugs
Anti-Cancer Peptides
Definition
Characteristics
Structure
Peptides as Tools to Manipulate Protein Functions
Extracellular Acting Peptides
Intracellular Acting Peptides
References
Anti-c-erB-2
Anti-c-erbB2 Monoclonal Antibody
Anti-ERB-2
Anti-erbB-2
Anti-erbB2 Monoclonal Antibody
Antigen of the Cromer Blood Group
Antiglycolytics and Cancer
Synonyms
Definition
Characteristics
2-Deoxy-d-glucose
3-Bromopyruvate
Lonidamine
Imatinib
Oxythiamine
Inhibitors of Lactate Dehydrogenase
Other Antiglycolytics
Cross-References
References
See Also
Anti-HER2/c-erbB2 Monoclonal Antibody
Anti-Her2/Neu Peptide Mimetic
Synonyms
Definition
Characteristics
Her2 Regulation
AHNP Characteristics
Rational for Design of AHNP
AHNP
AHNP Analogs
AHNP as a Drug Carrier
AHNP in Breast Cancer Diagnosis
Relevance to Cancer Therapy
Conclusions
Cross-References
References
Antihormonal Therapy
Definition
Cross-References
Antihormone Therapy
Antihormones
Definition
Cross-References
Antihuman p185neu Receptor Immunoglobulin G1
Anti-idiotype Vaccination
Anti-inflammatory Drugs
Synonyms
Definition
Characteristics
Steroid Anti-inflammatory Drugs
Nonsteroidal Anti-inflammatory Drugs
Cross-References
References
See Also
Antimalarial
Antimitotic Drugs
Definition
Characteristics
What are the Current Antimitotic Drugs in Clinical Use to Treat Cancer?
How do Antimitotic Drugs Exert Their Anticancer Activity?
What Happens to Cells Treated with Antimitotic Drugs?
What are the Drawbacks of Antimitotic Drugs?
What is the Future of Antimitotic Drugs?
Cross-References
Antioxidant Enzymes
Synonyms
Definition
Characteristics
Antioxidant Enzymes
Superoxide Dismutases
Copper Zinc Superoxide Dismutase
Manganese Superoxide Dismutase
Extracellular Superoxide Dismutase
Catalase
Glutathione Peroxidases
The Thiol-Selenocysteine-Peroxidase Connection
Peroxiredoxins
Thioredoxins
Thioredoxin Reductase
Conclusions
Cross-References
References
See Also
Antioxidants
Anti-p185-HER2
Antisense DNA Therapy
Definition
Characteristics
Target Choice and Oligonucleotide Design
Delivery, Subcellular Trafficking, and Pharmacodynamics of ODNs
Clinical Applications of Antisense ODNs in Hematological Malignancies
Prospects for Antisense DNA Therapy
Cross-References
References
Antiviral Defenses
Definition
Characteristics
Evolution
Prerequisites
Virus Recognition
Toll-like Receptors (TLRs)
RIG-like Receptors (RLRs)
NOD-like Receptors (NLRs)
Response to Virus Infection
Type I Interferon (IFN)
The Antiviral State and Effector Mechanisms
Interferon Stimulated Genes (ISGs)
Cell Death
RNA Interference
Virus-Virus Interference
Activation of Adaptive Immune Responses
Side-Effects of Antiviral Defenses
Virus Antagonism of Antiviral Defenses
Antiviral Defenses in Cancer
Cross-References
References
Antizyme Inhibitor
Definition
Characteristics
Cross-References
References
See Also
AP-1
Definition
Characteristics
General Structure of the AP-1 Subunits
DNA-Binding Domain
Transactivation Domain
Transcriptional and Posttranslational Control of AP-1 Activity
Transcriptional Activation
Regulation of AP-1 Activity
AP-1 in Physiology and Pathology
AP-1 Subunits in Cancer
Cross-References
References
See Also
Apactin (Mouse)
APAF-1 Signaling
Synonyms
Definition
Characteristics
APAF-1 Gene Structure and Regulation
APAF-1 Protein Structure
Assembly and Structure of the Apoptosome
Positive and Negative Regulation of Apoptosome Function
APAF-1 Expression and Apoptosome Regulation in Cancer
Apoptosome-Dependent and -Independent Pathways of Apoptosis in Normal and Neoplastic Cells
Cross-References
References
See Also
APC
APC Gene in Familial Adenomatous Polyposis
Synonyms
Definition
Characteristics
Genotype-Phenotype Correlations
Cross-References
References
See Also
APC/beta-Catenin Pathway
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
References
API3
API4
Apico-Basal Polarity
Definition
aPM1
APO2 Ligand
APO2L
Apolipoprotein J (APO-J)
Apoptosis
Synonyms
Definition
Characteristics
Cellular Regulation
Clinical Relevance
Cross-References
References
See Also
Apoptosis Induction for Cancer Therapy
Definition
Characteristics
Reactivation of Intracellular Pro-apoptotic Systems
Inhibition of Intracellular Anti-apoptotic Systems
Selective Delivery of Additional External Pro-apoptotic Stimuli
Perspectives for Selectively Tipping the Apoptotic Balance in Cancer
Cross-References
References
See Also
Apoptosis Inhibitor 4
Apoptosis Regulator Bcl2
Apoptotic Peptidase Activating Factor-1
Appendiceal Epithelial Neoplasms
Synonyms
Definition
Characteristics
Epithelial Appendiceal Neoplasms
Pseudomyxoma Peritonei Syndrome
Treatment
Management in Absence of Peritoneal Dissemination
Management of Mucinous Neoplasms with Peritoneal Dissemination
Outcomes
Approaches to Management
A New Standard of Care
Summary of Changes in Management of Appendiceal Epithelial Neoplasms
Cross-References
References
See Also
Appendiceal Mucinous Tumor of Uncertain Malignant Potential
APRF
Aptamer Bioconjugates for Cancer Therapy
Characteristics
Cross-References
References
Apudomas
2ar
AR
9-beta-D-Arabinosyl-2-fluoroadenine (F-ara-A) monophosphate
Arachidonate 5-Lipoxygenase
Arachidonic Acid Pathway
Synonyms
Definition
Characteristics
Fatty Acids as Dietary Precursors
Production of Eicosanoids: From AA and EPA by COX and LOX Pathways
Signaling by Eicosanoids
NSAIDS and Eicosanoid Signaling
Interactions with Cancer Signaling Pathways
References
AREG
ARF
ARF Tumor Suppressor Protein
Synonyms
Definition
Characteristics
p53-Dependent Functions of ARF
The ARF-MDM2-p53 Pathway
ARF and Replicative Senescence
p53-Independent Functions of ARF
ARF Negatively Regulates Ribosome Biogenesis
ARF and the Control of Transcription
Conclusion
Cross-References
References
See Also
Argentaffin Carcinoma
Arginine
Definition
Characteristics
Arginine Metabolism
Nitric Oxide, Polyamines, Carcinogenesis, and Chemoprevention
Arginine and Cancer: Experimental Studies
Arginine and Cancer: Epidemiologic Studies
Arginine and Cancer: Clinical Aspects
Cross-References
References
See Also
Arginine-Depleting Enzyme Arginine Deiminase
Synonyms
Definition
Characteristics
Sensitivity of Cancer Cells to ADI Treatment
Mechanisms of Antitumor Activity of ADI
Clinical Trials
References
ARHGAP7
ARK1
ARK2
Aromatase
Aromatase and Its Inhibitors
Synonyms
Definition
Characteristics
Composition
Distribution and Regulation
Expression
Aromatization Reaction
Type I Inhibitors of Aromatase
Formestane
Exemestane
Type II Inhibitors of Aromatase
Applications and Indications
Cross-References
References
Aromatic Amine
Definition
Cross-References
ARP2
Array CGH
Synonyms
Definition
Characteristics
Clinical Applications
Cross-References
References
Array-Based Comparative Genomic Hybridization
Arrhenoblastoma
Synonyms
Definition
Cross-References
Arsenic
Definition
Characteristics
History of Use
Paradox of Arsenic
Mechanisms of Action: Cancer-Causing Effects of Arsenic
Genotoxicity
Possible Involvement of Reactive Oxygen Species (ROS)
Aberrations in Signal Transduction
Arsenic as a Therapeutic Agent
Cross-References
References
See Also
Artemisinin
Synonyms
Definition
Characteristics
Biological Activity
Artemisinin Monomers
Mechanism of Action
Other Artemisinin-Like Compounds
Synergism with Other Chemotherapeutic Agents
Effective on Drug-Resistant Cancer Cells
Case Reports and Human Clinical Trials
Toxicity
Production of Artemisinin
Cross-References
References
Aryl Hydrocarbon Receptor
Synonyms
Definition
Characteristics
Historical Perspective
Xenobiotic Stress
Physiological Effects
A Link Between Environment and Cancer?
The Signaling Pathway
Gene Expression and Biological Pathways
Mechanisms of Carcinogenicity of AhR Ligands
AhR as a Pharmacological Target
Cross-References
References
See Also
Arylamine N-Acetyltransferases
Synonyms
Definition
Characteristics
Acetylation Polymorphism
Clinical Relevance
Cross-References
References
See Also
Arylsulfatase C
Asbestos
Definition
Characteristics
Carcinogenic Action of Iron
Asbestos: A Vehicle for Iron
Asbestos: Usage and Banishment
Cross-References
References
See Also
Ascites
Definition
Characteristics
Pathophysiology
Diagnosis
Treatment
References
ASI
Askin Tumor
Definition
Cross-References
Aspiration Cytology
Aspirin
Synonyms
Definition
Characteristics
Antipyretic Activity
Analgesic Activity
Antiplatelet Activity
Anticancer Activity
Clinical Evidences
Experimental Studies
Mechanism of Action
COX-Dependent Pathways
COX-Independent Pathways
Side Effect of Aspirin
Modified Aspirin
Conclusion
Cross-References
References
See Also
Assessment of Anaplasia of a Tumor
Assessment of the Degree of Tumor Differentiation
Asthma
Astrocytic Tumor
Astrocytoma
Synonyms
Definition
Characteristics
Epidemiology
Clinical Features
Diagnosis
Prognosis
Treatment
Pilocytic Astrocytoma
Subependymal Giant Cell Astrocytoma (SEGA)
Pleomorphic Xanthoastrocytoma (PXA)
Diffuse Astrocytoma
Anaplastic Astrocytoma
Glioblastoma
Diffuse Intrinsic Pontine Glioma (DIPG)
References
Asymmetric Cell Division
Synonyms
Definition
Cross-References
Asymmetric Cytokinesis
Ataxia-Telangiectasia Variant 1 and Variant 2
Ataxia-Telangiectasia, Mutated
ATL
ATM
ATM Protein
Synonyms
Definition
Characteristics
Cellular Functions and Molecular Regulation
Clinical Relevance
Cross-References
References
Atopic Dermatitis
Atopy
ATP-Binding Cassette-Transporters
ATP-Binding-Cassette Transporters Sub-family C
Atrial Myxoma
Atrophy
Attenuated Adenomatous Polyposis Coli
ATX
Atypical Congenital Mesoblastic Nephroma
Atypical Neurocytoma
AURA
AurB
AurC
AURKB
AURKC
Aurora Kinases
Synonyms
Definition
Characteristics
Mechanisms
Aurora Kinase as Target for Cancer Intervention
References
AURORA2
Aurora-A
Aurora-B
Aurora-C
Autoantibodies
Definition
Characteristics
Autoantibodies Can Contribute to Produce Tissue Injury and May Influence Tumor Behavior
Autoantibodies as Diagnostic and Prognostic Serologic Biomarkers of Cancer
Cross-References
References
See Also
Autocrine Signaling
Definition
Cross-References
See Also
Autoimmunity and Cancer
Synonyms
Definition
Characteristics
Evidence for Autoreactivity in Patients with Cancer
Regulation of Autoreactivity
Immunotherapy May Induce Autoimmunity
Autoantigens as a Link Between Cancer and Autoimmunity
References
Autologous (Hematopoietic) Stem Cell Transplantation
Autologous Bone Marrow Transplantation
Autophagocytosis
Autophagy
Synonyms
Definition
Characteristics
Cell Biology of Autophagy
Autophagosome formation
Signal Transduction
Autophagy and Cancer
Modulation of Autophagy for Cancer Treatment
Cross-References
References
See Also
Autotaxin
Synonyms
Definition
Characteristics
Physiological Functions
Protective Functions
Pathophysiology
Crystal Structure
Substrate Binding Specificity
References
Auxilin-2
Avian Erythroblastic Leukemia Viral Oncogene Homolog 2
Away from the Target Effects
5-aza-2 Deoxycytidine
Synonyms
Definition
Characteristics
History
Contemporary Research
Clinical Research
Cross-References
References
See Also
5-Azadeoxycytidine
Azaepothilone B
B
Bacillus Calmette„-Gu„érin
Synonyms
Definition
Characteristics
Microbiological Background on’BCG and’Tuberculosis Vaccination
BCG in’Cancer Immunotherapy
Mode of’Action in’Antitumor Activity
References
Bacteriophage Display
Baculoviral IAP Repeat Containing Protein 5
BAF47
BARD1
Synonyms
Definition
Characteristics
BARD1 Structure, Conservation, and’Expression„
Functions „of’the’BARD1„-BRCA1„ Heterodimer and’BARD1 Isoforms
BRCA1„-Independent„ Tumor Suppressor Function of’BARD1
Mutations and’Differential Splicing of’BARD1 in’Cancer
References„
Barrett Epithelium
Barrett Esophagus
Synonyms
Definition
Cross-References
See Also
Barrett Adenocarcinoma
Basal Cell Carcinoma
Definition
Characteristics
Risk Factors and’Therapy
Squamous Cell Carcinoma
Risk Factors and’Therapy
Cross-References
References
See Also
Basalioma
Definition
BAY 43-9006
BAY 73-4506
BAY-43-9006
Bbc3
BCDF
B-Cell CLL/Lymphoma 2
B-Cell Differentiation Factor
B-Cell Interferon
B-Cell Leukemia/Lymphoma-2 Gene (Bcl-2)
B-Cell Leukemias
B-Cell Lymphoid Neoplasm
B„-Cell„ Lymphoma
Synonyms
Definition
Characteristics
B„-Cell„ Lymphoma Pathology, Diagnosis, and Classification
Molecular Biology of „B-„Cell„ Lymphoma
B„-Cell„ Receptor Signaling in „B-„Cell„ Lymphoma
Clinical Treatment of „B-„Cell„ Lymphoma
References
B-Cell Lymphoma Protein 2
B-Cell Lymphomas
B-Cell Lymphoproliferative Disorder
B-Cell Lymphoproliferative Disorders/Diseases
B-Cell Malignancy
B-Cell Stimulating Factor-2
B„-Cell„ Tumors
Synonyms
Definition
Characteristics
What Is a B-„Lymphocyte?
Immunoglobulin Gene Rearrangement
Somatic Mutation and Class Switching
What Is a „B-„cell„ Tumor?
Characteristics of „B-„Cell„ Tumors
Immunogenetics of „B-„Cell„ Lymphomas
Cross-References
References
See Also
BCG
Bcl2
Synonyms
Definition
Characteristics
Molecular Anatomy
Biological Functions: Apoptosis, Cell Survival, Differentiation, and Autophagy
Regulation of Gene Expression
Regulation of Protein Function
Bioactivity
Cross-„References
References
See Also
Bcl-2
Bcl-2 Binding Component 3
BCL3
Definition
Characteristics
Structure and’Molecular Function
Regulation
Expression
Physiological Function
Oncological Relevance
Cross-„References
References
BCL6 Translocations „in’B-„Cell„ Tumors
Definition
Characteristics
The BCL6 Gene and’Gene Product
BCL6 Translocation Affecting the’IG „and’Non„-IG„ Loci
The 5′ Noncoding Region of’BCL6 Undergoes Somatic Hypermutation
Mouse Model of’BCL6 Translocation to’Develop Lymphoma
Clinical Relevance
Cross-„References
References
BCR„-ABL1„
Definition
Characteristics
A Somatic Mutation of Bone Marrow Progenitor Cells
Molecular Features of „BCR-„ABL1„ Recombination
Complex „BCR-„ABL1„ Rearrangements
Translocation„-Associated„ Genomic Deletions
Functional Impact of BCR-„ABL1
Clinical Relevance
Anti„-BCR„-ABL1„ Therapies
New„-Generation„ „BCR-„ABL1„ Tyrosine Kinases Inhibitors
BCR„-ABL1„ Kinase Domain Mutations
Compound Mutations
Disease Monitoring
BCR„-ABL1„ in Healthy Individuals
What Causes BCR-„ABL1?
Cross-References
References
See Also
BD
Beckwith„-Wiedemann„ Syndrome
Definition
Cross-References
Beckwith„-Wiedemann„ Syndrome Associated Childhood Tumors
Definition
Characteristics
Diagnostic Criteria
(Epi)Genetics
BWSCR1: ICR1 and’ICR2
BWSCR2
Diagnostics
BWS„-Associated„ Tumors
Wilms Tumor
Adrenocortical Carcinoma
Rhabdomyosarcoma
Hepatoblastoma
Common Genetic Pathways
Cross-References
References
See Also
Behcet Disease
Synonyms
Definition
Benign Prostate Hyperplasia
Definition
Symptoms
Cross-References
See Also
Benzene and’Leukemia
Definition
Characteristics
Benzene Toxicity and’Carcinogenicity
Benzene Metabolism and’Toxicity
Benzene, Chromosome Changes, and’Leukemia
Cross-References
References
See Also
Benzoquinone Ansamycin
Benzpyrene
Definition
Cross-References
Berlin Breakage Syndrome
Beta„-Glucosidase„
Definition
Cross-References
Betulinic Acid
Definition
Characteristics
References
Bevacizumab
Definition
Cross-References
References
See Also
BGP
BH3-Interacting Domain Death Agonist
BHD syndrome
Bid
Synonyms„
Definition
Characteristics
The Bid Molecule
Bid Is „a’Pro„-death„ Sensor for’Specific Protease Activation
Activation of’Mitochondria by Bid
Bid „as’a’Pro„-Life„ Sensor for’Cell Cycle Progression and’DNA Damage
Role of’Bid in’Oncogenesis
Summary
Cross-References
References
2′,3-Biindolinylidene-2,3′-dione: 3-(3-Indolinone-2-ylidene)-indolin-2-one
BIK (BCL-2 Interacting Killer)
BIK Proapoptotic Protein
Synonyms
Definition
Characteristics
Discovery
Protein Structure and’Molecular Functions
Gene Structure, Expression, and’Phenotypes
Relevance to’Cancer Genetics and’Therapy
Cross-References
References
Bilateral Acoustic Neurofibromatosis
Bile Acids
Definition
Characteristics
Toxicity of’Bile Acids
Effects of’Bile Acids on’Organs
Upper Gastrointestinal Tract
Lower Gastrointestinal Tract
Liver, Gallbladder, and’Bile Ducts
Pancreas
Cross-References
References
Bile Duct Carcinoma
Bile Duct Neoplasms
Synonyms
Definition
Characteristics
Benign Bile Duct Neoplasms
Malignant Bile Duct Neoplasms
Cross-References
References
See Also
Biliary Glycoprotein
B-immunoblastic
Bin1
Synonyms
Definition
Characteristics
Cross-References
References
See Also
BING2
Bioactive Lipid Therapy
Bioactive Lipids
Bioavailability
Definition
Bioavailability of Nutrients
Bioavailability of Drugs
Cross-References
See Also
Biochip
Bioconjugate
Definition
Cross-References
Biodribin
Biological Markers
Biological Monitoring
Biological Therapy
Bioluminescence Imaging
Synonyms
Definition
Characteristics
About Luciferases
How Is In Vivo Bioluminescence Detected?
Considerations to Maximize In Vivo Imaging Sensitivity
Oncology Model Applications
Cross-References
References
See Also
Bioluminescent Reporter Gene Assays
Biomarkers
Biomarkers in’Detection of’Cancer’Risk Factors and in Chemoprevention
Definition
Characteristics
Background
Types of’Biomarkers
Biomarker Techniques and’Fields of’Application
Cross-References
References
Biomonitoring
Synonyms
Definition
Characteristics
Biomarkers of Exposure
Biomarkers of Effect
Biomarkers of Susceptibility
Ethical and Social Implications
Cross-References
References
See Also
Bioradiotherapy
Biotechnology-Derived Therapeutic Proteins
BIRC5
BIR-Containing Protein 4 (BIRC4)
Birt„-Hogg„-Dub„é Syndrome
Synonyms
Definition
Characteristics
Diagnostic Criteria
BHD Gene Mutations
Screening and Possible Treatment for BHD
Cross-References
References
See Also
Bispecific Antibodies
Definition
Characteristics
Generation of’Bispecific Antibodies
Effector Cell Retargeting
Clinical Experience with’Bispecific Antibodies
Cross-References
References
Bisphosphonates
Definition
Characteristics
Mechanism of’Action
Pharmacokinetic
Clinical Use
Hypercalcemia of’Malignancy „(HCM)
Treatment of’Bone Metastases
Multiple Myeloma
Bone Metastases from Breast Cancer
Bone Metastases from Prostate Cancer
Bone Metastases from Lung Cancer and’Other Solid Tumors
Prevention of’Bone Metastases
Osteoporosis
Side Effects
Cross-References
References
See Also
Bladder Cancer
Definition
Characteristics
Clinical Epidemiology and Risk Factors
Tumor Biology and Genetics
Characteristics of Nonurothelial Cell Carcinomas
Clinical Presentation
Diagnosis and Staging
Management of Noninvasive Disease
Management of Invasive and Metastatic Disease
Cross-References
References
Bladder Cancer Molecular Therapy
Synonyms„
Definition
Characteristics
Molecular Therapy
Genetic Alterations in’the’Course of’UC Development
Aberrantly„-Regulated„ Signaling Pathways Targetable for’Molecular Therapy
Potential Anticancer Agents for’Molecular Therapy of’UCs
Cross-References
References
Bladder Cancer Pathology
Synonyms
Definition
Characteristics
Morphologic Aspects
Noninvasive Lesions
Flat Lesions
Papillary or Exophytic Lesions
Invasive Lesions
Histologic Variants
Other Histological Types
Neuroendocrine Tumors
Molecular Alterations
Diagnostic Markers
Tumor Staging
Primary Tumor „(T)
Regional Lymph Nodes „(N)
Distant Metastases „(M)
Morphological Parameters of Prognostic Value
Lymphovascular Invasion
Surgical Margins
References
B-anaplastic
Blast Crisis
Definition
Characteristics
Clinical Features
Biological Basis
Leukemia Stem Cells „(LSCs)
Genomic Instability Marks Progression to’Blast Crisis
Cytogenetic Evolution
High„-Resolution„ DNA Copy Number Profiling/Deep Sequencing
Mechanisms of’Genomic Instability in’Blast Crisis CML
Failure of’DNA Damage/Repair Responses
Centrosome Aberrations
Telomere Instability
Functional Profiling
Self„-Renewal„
Differentiation Arrest
Survival: Apoptosis
Epigenetic Pathways
Treatment of’Blast Crisis CML
Future Promise
Cross-References
References
See Also
Bleomycin
Definition
Cross-References
BLI
Blood„-Brain„ Barrier
Definition
Characteristics
The BBB Junctional Complexes
Permeability Properties of the BBB
In Vitro BBB Models
BBB in Disease
Cross-References
References
See Also
Bloom Syndrome
Synonyms
Definition
Characteristics
Clinical Description
BLM„-Deficient„ Cells
BLM Gene
Frequency
BLM Protein
Mouse Models
Genetic Counseling
Therapy
Cross-References
References
See Also
Bloom-Torre-Mackacek Syndrome
Blue Nevi
BM-40
BM90, FBLN-1
BMS-247550
Bombesin Amphibian Gastrin-Releasing Peptide
Bone Loss Cancer Mediated
Synonyms
Definition
Characteristics
Types of’Bone Metastasis
Bone Physiology: Control of’Normal Bone Remodeling
The Bone Is a’Unique Environment for’Metastasis
The Vicious Cycle of’Osteolytic Metastasis
The Vicious Cycle of’Osteoblastic Metastases
Osteolytic Multiple Myeloma
Osteolytic Metastasis from Breast Cancer
Osteoblastic Metastasis in’Prostate Cancer
References
Bone Metastasis
Synonyms
Definition
Characteristics
Biology of’Bone Metastasis
How Cancer Spreads to’the’Bone
How Cancer Grows in’the’Bone
Clinical Characteristics, Presentation, and’Diagnosis
Treatment
Conclusion
Cross-References
References
Bone Metastatic Disease
Bone Mets
Bone Neoplasms
Bone Sarcomas
Bone Sialoprotein
Bone Tropism
Synonyms
Definition
Characteristics
Blood Circulation Patterns
Cellular Adhesive Interactions
Chemoattraction of Cancer Cells
Tissue Conditions Supporting the Growth of Cancer Cells
Cross-References
References
See Also
Bone Tumors
Synonyms
Definition
Characteristics
Etiology
Diagnosis
Staging and Prognosis
Cross-References
References
See Also
Bone-Seeking Malignant Phenotypes
Borderline Appendiceal Mucinous Tumor
BORIS
Synonyms
Definition
Characteristics
BORIS Functions
BORIS in Cancers
Clinical Aspects
BORIS as a Cancer Biomarker
BORIS as a Target for Cancer Immunotherapy
Cross-References„
References
See Also
Bortezomib
Synonyms
Definition
Characteristics
Mechanism of’Action
Pharmacology
Clinical Aspects
In Cancer
In Graft Versus Host Disease and’Immune Disorders
Other Uses
Future Directions
Cross-References
References
Bovine Papillomavirus
Definition
Characteristics
BPV Gene Products
E5
E6
E7
L1 and L2
BPV and Papillomas
BPV and Cancer
BPV and Equine Sarcoids
Cross-References
References
See Also
Bowen Disease
Definition
Cross-References
Bp4
Brachytherapy
Synonyms
Definition
Characteristics
Dose Rate
Dose Calculations
Brachytherapy Techniques
Clinical Applications of’Brachytherapy
Gynecological Malignancies
Prostate Cancer
Other
Cross-References
References
See Also
BRAF
BRaf-Signaling
Synonyms
Definition
Characteristics
Physiological Aspects of „BRaf-Signaling
BRaf-Signaling and Tumor Development
B„-Raf„ Structure and Regulation
B„-Raf„ as a Therapeutic Target
Cross-References
References
See also
BRAF„ Somatic Alterations
Definition
Characteristics
History of the BRAF Proto„-oncogene„
Chromosomal Aberrations
Somatic and Germline Point Mutations and In-frame Deletions/Insertions
Amplification and Overexpression
Cross-References
References
See Also
BRAF1
B-raf-1
Brain Cancer Pathology
Definition
Characteristics
Astrocytic Tumors
Oligodendroglial and’Mixed Oligoastroglial Tumors
Ependymal Tumors
Choroid Plexus Tumors
Neuronal and’Mixed „Neuronal„-Glial„ Tumors
Pineal Tumors
Embryonal Tumors
Cross-References
References
Brain Tumors
Definition
Characteristics
Classification and Pathology
Clinical Presentation of Brain Tumor Patients
Clinical Management of Brain Tumor Patients and Prognosis
Complications of Therapy
Cross-References„
References
See Also
BRCA1/BRCA2 Germline Mutations and’Breast Cancer Risk
Definition
Characteristics
Clinical Characteristics
Molecular and’Cellular Characteristics
Tumor Suppressor Genes
Expression of’BRCA1 and’BRCA2
BRCA1„- „and’BRCA2„-Related„ Breast Cancer
BRCA1 and’BRCA2 as’Caretakers of the’Genome
Clinical Relevance
When to’Take the’DNA„-Test„?
Why Take the’DNA„-Test„?
Interpreting a’Negative Test Result
Cross-References
References
BRCA1-Associated Ring Domain 1
Breast Adenocarcinoma
Breast Cancer
„Definition
Characteristics
Risk Factors
Age
Genetics
Reproductive Factors
Obesity and’Physical Activity
Radiation
Alcohol
Hormone Therapy
Breast Cancer Symptoms and’Diagnosis
Mammary Paget Disease
Targeted Breast Cancer Therapies
Cross-References
References
Breast Cancer Antiestrogen Resistance
Definition
Characteristics
Breast Cancer Subtypes
Luminal Type A’Subtype
Luminal Type B’Subtype
Gene Expression Signatures Predicting Response to Antiestrogens
Challenges in Developing New In Vitro Models to Study Antiestrogen Resistance
Future Directions
Cross-References
References
See Also
Breast Cancer Antiestrogen Therapy
Breast Cancer Carcinogenesis
Definition
Characteristics
Breast Cancer Subtypes
Pathological Grade
Genetic Alterations
Endocrine and Reproductive Risk Factors
Exogenous Hormones
Environmental Factors
Radiation Exposure
Viruses
Clinical Studies
Cross-References
References
See Also
Breast Cancer Drug Resistance
Definition
Intrinsic Drug Resistance
Acquired Drug Resistance
Additional Factors Contributing to Resistance
Characteristics
Mediators of Drug Resistance
Drug Efflux Transporters
Cyclin E
Transcription Factors
p53
BRCA1
Epidermal Growth Factor „(EGF) Receptor Family
Estrogen Receptors
Tumor„-Stroma„ „Interaction„-Induced„ Resistance
Extracellular Matrix Composition and Integrin Signaling
Vessel Integrity, Angiogenesis, and Hypoxia
Tumor„-Stromal„ Cell Interactions
Cancer Stem Cells and Resistance
Resistance of Cancer Stem Cells to Conventional Cancer Therapies
Aldehyde Dehydrogenase
Enhanced DNA Repair Mechanisms
Altered Transcription Factor Activity
The Future of Breast Cancer Therapy: Pharmacogenetics and the Promise of Personalized Medicine
Cross-References
References
See Also
Breast Cancer Epidemiology
Definition
Characteristics
Global Impact of Breast Cancer
Breast Cancer Detection, Staging, and Survival
Pathology
Mechanisms of Breast Carcinogenesis
Risk Factors
Hormones
Family History
Estrogen Replacement Therapy
Body Mass Index
Diet
Prevention
References
Breast Cancer Familial Risk
Definition
Characteristics
High„-Penetrance„ Breast Cancer Susceptibility Genes
BRCA1 and BRCA2
TP53
Intermediate„-Penetrance„ Breast Cancer Susceptibility Genes
PTEN
STK11
CDH1
CHEK2
ATM
BRIP1
PALB2
Low„-Penetrance„ Breast Cancer Susceptibility Alleles
Interaction between Breast Cancer Susceptibility Genes
Conclusion and Outlook
Cross-References
See Also
Breast Cancer Hormonal Therapy
Breast Cancer Hormone Therapy
Breast Cancer Immunotherapy
Definition
Characteristics
Monoclonal Antibodies for the Immunotherapy of Breast Cancer
HER2 and EGFR
Trastuzumab
Cetuximab
VEGF
MUC1
Vaccines for the Immunotherapy of Breast Cancer
Cellular Vaccines
Dendritic Cell Vaccines
Vaccines Based on Defined Antigenic Substrates
HER2
CEA
MUC1
Perspectives
Cross-References
References
See also
Breast Cancer Multifocality
Definition
Characteristics
Natural History of Multifocal Breast Carcinomas
Relationship Between Multifocality and Other Tumor Characteristics
Clinical Importance of Multifocality
Diffuse Cases
Conclusions
Cross-References
References
Breast Cancer Multistep Development
Definition
Characteristics
Multistep of Breast Cancer Progression
Hyperplasia
Atypical Hyperplasia
Carcinoma In Situ
Invasive Carcinoma
Metastasis
Mechanisms that Drive Multistep Development of Breast Cancer
Alternative Model of Breast Cancer Development
Cross-References
References
See Also
Breast Cancer New Therapies: HER2, VEGF, and PARP as Targets
Definition
Characteristics
Anti„-HER2„ Therapy
Anti„-VEGF„ Therapy
PARP Inhibitors
Future Directions
Cross-References
References
See Also
Breast Cancer Prognostic and’Predictive Biomarkers
Definition„
Characteristics
Established Prognostic Biomarkers in’Breast Cancer
Prognostic Markers Following Breast Conservation Surgery and’Neoadjuvant Chemotherapy
Established Predictive Biomarkers in’Breast Cancer
Proposed Biomarkers in’Breast Cancer
Multigene Predictors in’Breast Cancer
Conclusion
References
Breast Cancer Prognostic Biomarkers
Definition
Characteristics
Clinical Prognostic Indicators
Traditional Prognostic Markers
Hormone Receptors
Human Epidermal Growth Factor „Receptor„-2„ „(HER-„2)
Ki„-67„ Proliferation Marker
Emerging Prognostic Markers
Genomic Markers
Proliferation Markers
Anti„-apoptosis„ Markers
Structural Proteins
Angiogenesis„-Associated„ Markers
Plasminogen Activators and Inhibitors
Glycosaminoglycans and Proteoglycans
Multigene Arrays
MicroRNA
Future Directions
Cross-References
References
See Also
Breast Cancer Stem Cells
Synonyms
Definition
Characteristics
Origins of’the’Breast Cancer Stem Cells
Characterization of’Breast Cancer Stem Cells
Signal Transduction Pathways
Breast Cancer Metastasis to’Bone Marrow
Cross-References
References
Breast Cancer Susceptibility Genes
Definition
Characteristics
High Penetrance
BRCA1 and BRCA2
TP53
Intermediate Penetrance
ATM
CHEK2
BRIP1
PALB2
RAD50
NBN
Uncertain Penetrance
PTEN
RAD51C
STK11
CDH1
Low Penetrance
Role in DNA Repair and Damage Signaling as the Common Denominator
Clinical Impact
Cross-References
References
See Also
Breast Cancer-Initiating Cell (Breast’CIC)
Breast Carcinoma
Breast Tumor-Initiating Cell (Breast’TIC)
Brenner Tumor
BRG- and BRM-Associated Factor, 47’kDa
Brilliant Yellow S
BRIT1 Gene
Synonyms
Definition
Characteristics
Significance of’BRIT1 in’the’Development of’Cancer
BRIT1 Function in’DNA Damage Response
BRIT1 Function in’Cell Cycle Control
References
Brivanib
BRMS1
Definition
Characteristics
Cellular and Functional Characteristics
Clinical Relevance
Cross-References
References
See Also
BRN-5547136
Bronchogenic Carcinoma
Brother of the Regulator of Imprinted Sites
Bryostatin„-1„
Definition
Characteristics
Rationale for’Targeting the’PKC
Preclinical Activity of’Bryostatin„-1„
Single Agent Activity of’Bryostatin„-1„
Bryostatin„-Based„ Combinations
Future Directions
Cross-References
References
See Also
BSF-2
BTAK
Burkitt Lymphoma
Definition
Cross-References
Bystander Effect
Synonyms
Definition
Characteristics
Antitumor Strategies
Epigenetic Changes
Cell„-Cell„ Communication
Cross-References
References
See Also
C
C.elegans Cell Death 4 Homolog
C.I. 75300
C/EBP-Epsilon-Regulated Myeloid-Specific Secreted Cysteine-Rich Protein (XCP1)
c-erb-B2
C21H2006
C33
Ca Homeostasis
Definition
Characteristics
Cellular Ca Homeostasis
Cellular Ca Homeostasis During Cell Stimulation
Cellular Ca Homeostasis in Cancer
Cross-References
References
See Also
Ca-Activated Phospholipid-Dependent Protein Kinase
CaBP3
Cachectin
Cachexia
Definition
Characteristics
Weight Loss
Poor Appetite
Increased Metabolism and Energy Expenditure
Loss of Adipose Tissue
Loss of Muscle Protein
Treatment
Cross-References
References
See Also
Cachexia-Inducing Agent
Cadherin-1
Cafe-Au-Lait Macule
Cafe-Au-Lait Spots
Synonyms
Definition
Cajal Bodies
Synonyms
Definition
Characteristics
References
CAK1 Antigen
Calcitonin
Definition
Characteristics
Biosynthesis
Biological Actions
Actions on Bone
Renal Actions
Central Actions
Other Actions
CT in Cancer
Mechanism of CT Action
Receptors
Receptor Isoforms
Modulation of CTR Specificity
CTR Signaling
G Protein-Mediated Signaling
G Protein-Independent Signaling
Significance of CT-CTR Axis in Cancer: Clinical Aspects
CT Is ``Oncogene´´ for Prostate Cancer but ``Tumor Suppressor´´ for Breast Cancer
CT Is an Angiogenic Factor
Cross-References
References
Calcium-Binding Proteins
Definition
Characteristics
EF-Hand Motif Calcium-Binding Proteins
Annexins and Other Non-EF-Hand Motif Proteins
Calreticulin-Like Proteins
Cross-References
References
See Also
Calcium-Binding Reticuloplasmin of Molecular Weight 55 kDa
CALI
CALM
Calpain
Definition
Characteristics
Calpain and Cell Proliferation
Calpain and Cell Migration
Calpain and Apoptosis
Cross-References
References
See Also
Calreticulin
Synonyms
Definition
Characteristics
Structure of CRT
Functions of CRT in the Cell
CRT, a Lectin-Like Molecular Chaperone in the ER
CRT, a Regulator of Ca Homeostasis in the ER
Other Miscellaneous Functions of CRT In and Out of the ER
CRT and Development
CRT and Cancer
Expression of CRT in Cancer
Pathophysiological Relevance of CRT in Malignant Disease
CRT as a Tool for Cancer Therapy
References
Calsequestrin-Like Protein
CAM
Campto
Camptosar
CAMs
CAMTA1
Definition
Characteristics
Clinical Relevance
Cross-References
References
See Also
Cancer
Definition
History
Characteristics
Types of Genetic Damage
Cellular Aspects
Sporadic Versus Familial Cancer
Polygenic Determinants of Risk
Cross-References
Cancer (or Tumor) Stroma
Cancer and Cadmium
Definition
Characteristics
References
Cancer Antigen 3
Cancer Causes and Control
Synonyms
Definition
Characteristics
Tobacco
Trends
Physical Activity
Trends
Weight Control and Obesity Prevention
Trends
Dietary Improvements
Dietary Fat
Red Meat
Calcium
Excess Caloric Intake
Whole Grains
Vitamin A and Carotenoids
Selenium
Vitamin D
Trends
Limitation of Alcohol Use
Safer Sex and Decreased Viral Transmission
Trends and Disparities
Sun Protection
Screening
Trends and Disparities
Conclusion
Cross-References
References
See Also
Cancer Cell Cytotoxicity
Cancer Cell-Platelet Microemboli
Cancer Epidemiology
Synonyms
Definition
Characteristics
Cross-References
References
Cancer Epigenetics
Definition
Characteristics
DNA Hypermethylation
Histone Modification
DNA Hypomethylation
Epigenetic Alterations as Targets for Diagnosis and Therapeutic Intervention
Cross-References
See Also
Cancer Etiology
Cancer Germline Antigens
Synonyms
Definition
Characteristics
Identification
Nomenclature
Regulation of Expression
Clinical Studies
Cross-References
References
See Also
Cancer of B-Lymphocytes
Cancer of the Large Intestine
Cancer of the Lung
Cancer Prevention
Cancer Prevention with Green Tea
Cancer Process of the Large Intestine
Cancer Stem Cell Therapies
Cancer Stem Cells
Cancer Stem Cells Targeted Drug Development
Definition
Characteristics
Cancer Stem Cells
Novel Concept of Carcinogenesis Suggests Alternative Paradigm of Cancer Treatment
Potential Molecular Targets
Targeting CSC Signaling Pathways
Targeting CSC Resistance to Therapies
Targeting Angiogenesis
Targeting CSCs with Phytochemicals (Nutraceuticals)
CSC-Targeted Preclinical Evaluation of the Anticancer Agents
Conclusion
References
See Also
Cancer Stem-Like Cells
Synonyms
Definition
Characteristics
Cross-References
References
Cancer Vaccines
Definition
Characteristics
T Lymphocytes
Tumor Cells
Peptides and Carbohydrates
Recombinant Vaccines Expressing Tumor Antigens
Anti-idiotype Vaccines
Dendritic Cell-based Vaccines
Conclusion
Cross-References
References
See Also
Cancer Without Disease
Cancer/Testis Antigen1b
Cancer-Mediated Bone Loss
Cancers of Hormone-Responsive Organs or Tissues
Cancer-Testis Antigen 1.11
Cancer-Testis Antigens
Candidate of Metastasis 1
Canine Transmissible Tumor (CTVT)
Cannabinoids
Synonyms
Definition
Characteristics
Signaling Pathways Modulated by Cannabinoid Receptors
Palliative Effects of Cannabinoids in Cancer
Mechanism Involved in the Antiemetic Effect of Cannabinoids
Mechanism Involved in Appetite Stimulation by Cannabinoids
Mechanism Involved in the Analgesic Effect of Cannabinoids
Antitumoral Effects of Cannabinoids
Induction of Apoptosis
Inhibition of Tumor Angiogenesis
Therapeutic Potential of Cannabinoids as Antitumoral Agents
Cross-References
References
See Also
CAP20
CAR
8-Carbamoyl-3-methylimidazo(5,1-d)-1,2,3,5-tetrazin-4(3H)-one
Carbohydrate Part of Glycoconjugates
Carbon Metabolism
Definition
Characteristics
Homeostatic Regulation of Carbon Metabolism
References
Carbonyl Metabolism
Definition
Characteristics
Alcohol Dehydrogenases (ADHs)
Aldehyde Dehydrogenases (ALDHs)
Aldo-Keto Reductases (AKRs)
References
Carbonyl Reductases
Carcinoembryonic Antigen
Synonyms
Definition
Characteristics
Protein Structure
The CEA Gene Family
Function of CEA in Normal and Cancerous Tissue
Clinical Aspects
Cross-References
References
See Also
Carcinofetal Proteins
Carcinogen Metabolism
Definition
Characteristics
History
Metabolism
Mechanisms
Cancers
Cross-References
References
See Also
Carcinogenesis
Definition
Characteristics
Causes
Molecular Genetics
Clinical Relevance
Prevention
Cross-References
References
See Also
Carcinogenesis in Colon
Carcinogenic Compounds in Food
Carcinoid
Synonyms
Definition
Incidence and Prevalence of Carcinoid Tumors
Cross-References
See Also
Carcinoid (Well-Differentiated Neuroendocrine Tumor (NET) of the Respiratory and Gastrointestinal Tract)
Carcinoid Tumors
Synonyms
Definition
Characteristics
Cross-References
References
Carcinoma In Situ
Synonyms
Definition
Cross-References
Carcinoma of the Adrenal Cortex
Carcinoma Pathogenesis
Carcinoma with Amine Precursor Uptake Decarboxylation Cell Differentiation
Carcinomatosis
Synonyms
Definition
Characteristics
Etiology of Peritoneal Carcinomatosis
Diagnosis
Treatment
Cross-References
References
Carcinosarcoma
Definition
Cardiac Tumors
Definition
Characteristics
Incidence
Clinical Features
Primary Tumors
Benign
Malignant
Secondary or Metastatic Tumors
Therapy and Prognosis
Cross-References
References
Carney Complex
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Carotenoids
Definition
Characteristics
References
Case Control Association Study
Synonyms
Definition
Characteristics
Potential Problems in Association Studies
Cross-References
References
See Also
Case-Control Association Analysis
Casein Kinase 2
Casein Kinase II
CASH
CASP-8
Caspase
Definition
Cross-References
Caspase Homologue
Caspase-8
Synonyms
Definition
Characteristics
Structure and Physiological Functions of Caspase-8
Caspase-8 and Cancer
Cross-References
References
See Also
Caspase-Eight-Related Protein
Caspase-Independent Apoptosis
Synonyms
Definition
Characteristics
Cellular and Molecular Characteristics of Apoptotic Cells
Molecules Involved in the Caspase-Independent Apoptosis Process
Inter-regulations Between Caspase-Dependent and Caspase-Independent Apoptosis in Cancer Cells
Cross-References
References
Caspase-Independent Cell Death
Caspase-Like Apoptosis-Regulatory Protein
CASPER
CASTing
Castrate-Resistant Prostate Cancer
Keywords
Definition
Characteristics
Genetic Causes of the Disease
Diagnosis of the Disease
Survival
Current Treatment Options
Brief Description of Each Therapy
Cross-References
References
Castration-Resistant Prostate Cancer
Definition
Cross-References
Catechin
Cathepsin-D
Definition
Characteristics
Apoptosis
Regulation
Cancer
Clinical Aspects
Cross-References
References
See Also
Cathepsins
Definition
Cross-References
Caudal Type Homeobox 2
Caveolins
Definition
Characteristics
Clinical Aspects
Cross-References
References
See Also
C-BAS/HAS
CBFA2
CBP/p300 Coactivators
Definition
Characteristics
Cross-References
References
See Also
C-CAM
CCCTC-Binding Factor
Synonyms
Definition
Characteristics
CTCF Functions
Clinical Aspects
Cross-References
References
CCI779
CCI-779
CCRG-81045
CD Antigens
Synonyms
Definition
Characteristics
CD Antigens Provide Immunophenotypes of Leukocytes
Classification of Leukemias Using CD Antigens
CD Antigens as Targets for Therapeutic Antibodies
Methods for Identification of CD Antigens
Cross-References
References
See Also
CD156b Antigen
CD184
CD246
CD26
CD26/DPPIV in Cancer Progression and Spread
Synonyms
Definition
Characteristics
Cross-References
CD314
CD318 (Cluster of Differentiation 318)
CD44
Synonyms
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
Cross-References
References
CD55
CD62 Antigen-Like Family Member E (CD62E)
CD66a
CD66b
CD66c
CD66e
CD82
2-CdA
CdA
CDA2
CDCP1
Synonyms
Definition
Characteristics
Discovery
Protein Structure
Cleavage
CDCP1 Expression
CDCP1 Signaling
Phosphorylation
Downstream Signaling
Known Binding Partners
Cross-References
References
See Also
CDCP1 (CUB Domain-Containing Protein 1)
CDDP
CDK
Cdk1 Kinase
CDK2/Cyclin A-Associated Protein p45
CDK4I
CDKN1A
CDKN2
CDKN2A
Synonyms
Definition
Characteristics
Identification of CDKN2A
Gene Structure of CDKN2A
Tumor Suppressor
P16 Is a CDK Inhibitor
Role of the Alternative Reading Frame (ARF) Product
Senescence
Mouse Models
Clinical Aspects
CDKN2A Mutations and Melanoma
Mutation of ARF
Penetrance
Multiple Primary Melanoma
CDKN2A Mutations and Nonmelanoma Cancers
Modifiers of Penetrance of CDKN2A Mutations
CDKN2a Polymorphisms as Low-Risk Factors
CDKN2A and the Atypical Mole Syndrome
References
CDKN4
cDNA Chips
CDX2
Synonyms
Definition
Characteristics
Structure
Expression, Activity, and Mechanisms of Regulation
Structure Physiological Functions
Mode of Action at the Cellular Level
Mode of Action at the Molecular Level
Clinical Relevance for Colon Cancer
Clinical Relevance for Other Types of Cancer
References
CDX3
CDX-3
CEA
CEA Gene Family
Synonyms
Definition
Characteristics
Expression and Functions of CEA Family Members in Normal and Tumor Tissues
Expression and Functions of CEACAM1
Transcriptional Regulation
The Next Frontier
References
CEACAM1
CEACAM1 Adhesion Molecule
Synonyms
Definition
Characteristics
Properties of CEACAM1
CEACAM1 in Cancer
Loss of CEACAM1 Expression in Tumorigenesis and Tumor Progression
Upregulation of CEACAM1 Expression in Malignant Diseases
CEACAM1 and Tumor Angiogenesis
Studying CEACAM1 in Cancer: Animal Models
References
CEACAM1 = BGP
CEACAM5
CEACAM5 = CEA
CEACAM6 = NCA
CEACAM7 = CGM2
CEACAM8 = CGM6
CEA-Related Cell Adhesion Molecule 1
CED
Celastrol
Synonyms
Definition
Characteristics
Biological Properties
Potential Molecular Targets
Clinical Relevance
Cross-References
References
Celebra
Celebrex
Celecoxib
Synonyms
Characteristics
References
Cell Adhesion Molecules
Synonyms
Definition
Characteristics
Cell Adhesion Molecules and the Cytoskeleton
Classification of Cell Adhesion Molecules
Cadherins
Integrins
The Ig Superfamily
Selectins
Proteoglycans
Role of Adhesion Molecules in Cancer
The Metastatic Cascade
Adhesive Events in Metastasis
Other Cancer-Related Functions of Cell Adhesion Molecules
Cross-References
References
See Also
Cell Biology
Definition
Characteristics
The Cell
Cell Division and Reproduction
Cell Proliferation
Cell Cycle
Regulation of Cell Cycle Progression
Programmed Cell Death
Apoptosis
Necrosis
Autophagy
Signal Transduction
Signaling Targets
Systems Biology
Cell Motility and Migration
Cytoskeleton
Signaling Regulation
Tumor Biology
Initiation and Promotion
Progression
Metastasis
Stem Cell Biology
Self-Renewal and Clonality
Potency
Environmental Regulation
Social Implications
Cross-References
References
See Also
Cell Cycle Checkpoint
Definition
Characteristics
G1 checkpoint
Intra S-phase checkpoint
G2/M checkpoint
Mitotic checkpoint
Checkpoints and cancer
References
Cell Locomotion
Cell Motility
Cell Movement
beta-Cell Tumor of the Islets
Cell-Free Circulating Nucleic Acids (cfDNA)
Cell-Free Nucleic Acids in Plasma and Serum (CNAPS), Circulating Tumor DNA (ctDNA)
Cellular Antigens
Cellular Immunotherapy
Cellular Self-cannibalism
Cellular Self-digestion
Central Neurocytoma
Central Neurofibromatosis
Centroblastic
Centrocytic (Mantle Cell) Lymphoma
Centrosome
Synonyms
Definition
Characteristics
Structure and Function
Centrosome Duplication
Abnormal Amplification of Centrosomes and Chromosome Instability in Cancer
Loss of Tumor Suppressor Proteins and Centrosome Amplification
Centrosome Amplification and Cancer Chemotherapy
Cross-References
References
Ceramide
Definition
Characteristics
Formation of Ceramide
Ceramide-Induced Changes of Biological Membranes
Ceramide in Receptor-Mediated Signaling
Signaling Molecules Regulated by Ceramide
Ceramide in Mitochondria and Cell Death
Genetic Evidence for a Function of Ceramide in Apoptosis
Ceramide in gamma-Irradiation- and UV-A Light-Induced Apoptosis
Ceramide and Chemotherapy
Short Chain Ceramide
Cross-References
References
Cervical Cancers
Definition
Characteristics
Symptoms
Pathology
Staging
Prognosis
Therapy
References
Cetuximab
Definition
Cross-References
See Also
CG Antigens
CGP57148
C-HA-RAS1
Charged Particle Therapy
Checkpoint with Forkhead and RING Finger Domain Protein
Chelation Therapy
Chelators as Anti-Cancer Drugs
Synonyms
Definition
Characteristics
Iron Metabolism in Cancer Cells
Desferrioxamine, an Iron Chelator with Some Anti-Cancer Activity
Other Chelators with Anti-Cancer Potential
PIH Chelators
The DpT Chelators: Dp44mT
Iron Chelation and Cell Cycle Control Molecules
Conclusions
Cross-References
References
See Also
Chemical Biology Screen
Chemical Carcinogenesis
Definition
Characteristics
Normal Cell Types in Animals and the Tumors They Give Rise To
Carcinogens
Mechanisms of Chemical Carcinogenesis
Insights into Mechanisms of Chemical Carcinogenesis from Studies of Chemically Induced Neoplastic Transformation
Significance of Chemical Carcinogenesis
Cross-References
References
See Also
Chemical Genetic Screen
Chemical Mutagenesis
Chemically Induced Cell Transformation
Definition
Characteristics
Normal Growth of Normal Cells
Carcinogens
Chemically Induced Cell Transformation: Description and Mechanisms
Significance of Chemically Induced Neoplastic Transformation
Cross-References
References
See Also
Chemoattractant Cytokine
Chemoattraction
Synonyms
Definition
Characteristics
Chemokines
Chemokines and Cancer
Chemoattraction: A Key Process to Attract Cancer Cells to New Biological Niches
Other Biological Effects of Chemokines on Cancer Cells Apart from Chemoattraction
Chemokines Can Contribute to Regulate the Growth of Cancer Cells
Chemokines Can Contribute to Regulate the Survival of Cancer Cells
Chemokines Can Contribute to Regulate the Adhesion to New Sites in Cancer Cells
Chemokines Can Contribute to Control Protease Secretion in Cancer Cells
Chemokines Can Contribute to Control Angiogenesis in Cancer Cells
Therapeutical Aspects
Summary and Final Conclusions
Cross-References
References
See Also
Chemokine Receptor CXCR4
Synonyms
Definition
Characteristics
Cross-References
See Also
Chemokines
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Chemokinesis
Chemoprevention
Definition
Cross-References
See Also
Chemoprotectants
Synonyms
Definition
Characteristics
Synthetic Chemoprotectants
Natural Chemoprotectants
References
Chemoprotection
Chemoradiation
Chemoradiotherapy
Synonyms
Definition
Characteristics
Chemoradiotherapy for Non-small Cell Lung Cancer
Chemoradiotherapy for Small Cell Lung Cancer
Chemoradiotherapy for Cervical Cancer
Chemo-Bioradiotherapy for Head and Neck Cancer
Chemoradiotherapy for Esophageal Cancer
Chemoradiotherapy for Gastric Cancer
Chemoradiotherapy for Rectal Cancer
Chemoradiotherapy for Anal Cancer
Chemoradiotherapy for Glioblastoma
Concluding Remarks
Cross-References
References
See Also
Chemoresistance
Chemosensibilization
Synonyms
Definition
Characteristics
Chemosensibilization of Tumors by Targeting P-gp
Inhibitors of P-gp Activity
Other Strategies to Inhibit P-gp
Chemosensibilization of Tumors by Triggering Apoptosis
Cross-References
References
See Also
Chemosensitization
Chemotactic Cytokine
Chemotaxis
Chemotherapy
Definition
Characteristics
The Challenge
The Agents
Clinical Strategies
Supportive Care
Long-Term Effects of Cancer Treatment
Conclusions
References
CHETK
CHFR
Synonyms
Definition
Characteristics
Activity in Cancer
Biomarker Potential
Conclusion
References
See Also
Childhood Adrenocortical Carcinoma
Definition
Characteristics
Incidence
Causes
Normal Physiology and Tumor Biology
Clinical Manifestations
Testing
Treatment
Prognosis
Concluding Remarks
References
Childhood Cancer
Definition
Characteristics
Incidence
Causes and Risk Factors
Socioeconomic and Ethnic Factors
Infections
Radiation Exposure
UV Light Exposure
Genetic and Chromosomal Syndromes
Clinical Presentation and Diagnosis
Treatment
Outcome
Cross-References
References
Childhood Cancer and Pediatric Cancer Predisposition Syndromes
Definition
Characteristics
Hereditary Cancer Predisposition Syndromes
Central Nervous System Cancer Predisposition Syndromes
Hereditary Gastrointestinal Malignancies
Genitourinary Cancer Predisposition Syndromes
Endocrine Cancer Predisposition Syndromes
Sarcoma Predisposition Syndromes
Genodermatoses (Inherited Genetic Skin Disorders) with Cancer Predisposition
Leukemia/Lymphoma Predisposition Syndromes
Bone Marrow Failure Syndromes
Primary Immunodeficiency Disorders
Numerical Chromosomal Abnormalities
Miscellaneous
Cross-References
References
Childhood Cancer and Treatment-Related Toxicities
Definition
Risk awareness
Risk prevention
Death
Secondary Malignant Neoplasms
Cardiovascular Disease
Endocrine Complications
Thyroid Disorders
Gonadal Dysfunction
Neurocognitive Impairment
Socioeconomic Burden and Health Status
Cross-References
References
Children´s Brain Tumors
Chimeric Antigen Receptor (CAR)
Synonyms
Definition
Characteristics
The Basic CAR Design
The Binding Domain
The Spacer Domain
The Transmembrane Domain
The Signaling Domains
The Four Generations of CARs
CAR T Cell Therapy: Challenges and Safety
Cross-References
References
See Also
Chimeric Antigen Receptor on T Cells
Synonyms
Definition
Characteristics
Background on Manipulating T-Cell Responses to Cancer
Tumor-Specific CAR Development
Clinical Application of CAR-Specific T Cells
Improved Therapeutic Potential of CAR T Cells
Future Challenges
Conclusion
References
Chimeric Genes
Chimeric Immune Receptor
Chimeric Oncogenes
Chinese Medicine
Chinese Versus Western Medicine
Synonyms
Definition
Characteristics
Developing History
Theories and Principles
Etiology and Pathogenesis
Diagnostic Methods
Treatment Modalities
References
2-Chloro-2-deoxyadenosine
2-Chlorodeoxyadenosine
ChoK
Cholangiocarcinoma
Synonyms
Definition
Characteristics
Etiology
Biology
Diagnosis
Treatment and Prognosis
Cross-References
References
Cholangiocellular Carcinoma
Choline Kinase
Synonyms
Definition
Characteristics
Choline Kinase Alpha and Human Tumorigenesis
References
Choline Phosphokinase
Choline-Ethanolamine Kinase
Chorioallantoic Membrane
Synonyms
Definition
Characteristics
Angiogenesis
Tumor-Induced Angiogenesis
Cancer Progression
Cross-References
References
Chorioallantois
Choriocarcinoma
Choroidal Melanoma
C-HRAS
Chromate
Chromatin Modification
Chromatin Remodeling
Synonyms
Definition
Characteristics
Mechanisms and Clinical Aspects
Chromatin-Remodeling Complex
Histone Modifications
Histone Variant
Epigenetic Therapy
Cross-References
References
Chromium Carcinogenesis
Synonyms
Definition
Characteristics
Chromium: The Chemical Element and Its Ionic Species and Chemical Compounds
Important Commercial Uses of Chromium and Chromium Compounds
Biological Aspects of the Essentiality of Chromium(III) in Mammalian Cells
Exposure of Humans to Chromium Compounds
Genotoxicity of Chromium Compounds
Chromium-Induced Cell Transformation
Chromium Carcinogenesis
Mechanisms of Chromium Carcinogenesis
Cross-References
References
See Also
Chromium Tumorigenesis
Chromium(VI)
Chromium-Induced Carcinogenesis
Chromium-Induced Cell Transformation
Chromophore-Assisted Laser Inactivation
Synonyms
Definition
Characteristics
Advantages
Limitations
Application
Developments
Conclusions
Cross-References
References
See Also
Chromosomal Fluorescence In Situ Hybridization
Synonyms
Definition
Interphase FISH
Metaphase FISH
Cross-References
See Also
Chromosomal Instability
Synonyms
Definition
Characteristics
Historical Background
Chromosome Segregational Defects Lead to Chromosomal Instability
A Defective Response to DNA Damage Leads to Chromosomal Instability
Telomere Dysfunction May Lead to Chromosomal Instability
Cell Cycle Disturbances Result in Chromosomal Instability
Cross-References
References
Chromosomal Translocation t(8;21)
Synonyms
Definition
Characteristics
Cytogenetics and Morphology
AML1/MTG8
AML1/MTG8 in Leukemogenesis
Clinical Relevance and Therapy
Cross-References
References
See Also
Chromosomal Translocations
Definition
Characteristics
Biology
Clinical Relevance
Cross-References
References
Chronic Granulocytic Leukemia
Chronic Idiopathic Myelofibrosis
Chronic Lymphocytic Leukemia
Synonyms
Definition
Characteristics
Diagnosis
Epidemiology
Etiology
Pathogenesis
Risk Prediction in CLL
Symptoms and Signs of Active CLL
Treatment Strategies in CLL
Allo-HCT in CLL
Cross-References
See Also
Chronic Myelogenous Leukemia
Chronic Myeloid Leukemia
Synonyms
Definition
Characteristics
Pathophysiology
Prognosis
Therapy of CML
Chemotherapy Agents and Interferon Alpha
Imatinib
Dasatinib and Nilotinib as Second-Line Tyrosine Kinase Inhibitors
Second-Generation Tyrosine Kinase Inhibitors for Newly Diagnosed Patients
Third-Line Inhibitors
Allogeneic Bone Marrow Transplantion
References
Chronic Obstructive Pulmonary Disease and Lung Cancer
Definition
Characteristics
COPD
Lung Cancer
Link to Decline in FEV1
COPD and Lung Cancer Associations
Possible Mechanisms of the COPD: Lung Cancer Risk
Summary
Cross-References
References
Ciliary Body Melanoma
CIN
Cip1
Cip-Interacting Zinc Finger Protein 1 Ciz1
Definition
Characteristics
Cross-References
References
See Also
Circadian Clock Induction
Definition
Characteristics
Relevance of Circadian Disruption for Cancer Processes
Circadian Clock Control of Cell Cycle
Circadian Disruption in Cancer Tissues
Physiologic Clock
Tumor Clock
Circadian Clock Induction
Cross-References
References
Circulating Cancer Cells (CCC)
Circulating Nucleic Acids
Synonyms
Definition
Characteristics
Basic Aspects
Clinical Aspects
Cross-References
References
Circulating Tumor Cells
Synonyms
Keywords
Definition
Characteristics
CTC Detection and Characterization
Clinical Impact of CTC Detection
Cross-References
References
See Also
cis-Diamminedichloroplatinum
cis-Dichlorodiammineplatinum(II)
Cisplatin
Synonyms
Definition
Characteristics
Mechanisms of Action
Cisplatin and Cancer Treatment
Cisplatin-Induced Resistance
Inhibition of Drug Uptake and Decreased Intracellular Accumulation
Increased DNA Repair
Development of Cisplatin Analogs
References
Cisplatin-Refractory Germ Cell Tumors
Cisplatin-Resistant Germ Cell Tumors
cis-Platinum II
c-Jun Activation Domain-Binding Protein-1
c-Jun N-Terminal Kinase
CK
CK2
Synonyms
Definition
Characteristics
Regulation of Expression and Activity
Role in Carcinogenesis
Role in Drug Resistance
Targeting CK2
Cross-References
References
CKII
c-Kit
C-KRAS
Cladribine
Synonyms
Definition
Characteristics
Administration and Pharmacokinetics
Clinical Activity
Toxicity and Adverse Effects
Cross-References
References
CLARP
Class II Tumor Suppressor Genes
Definition
Characteristics
Evidence for a Class II Tumor Suppressive Activity
Class II Tumor Suppressor Identification
Mechanisms of Inactivation
Clinical Relevance
Cross-References
References
Clastogenesis
Clathrin Assembly Lymphoid Myeloid Protein
Clathrin-Mediated Endocytosis
Clinical Cancer Biomarker
Clinical Cancer Biomarkers
Synonyms
Definition
Characteristics
Diagnostic Tumor Markers
Prognostic/Predictive/Markers
Surrogate/Monitoring Markers
Miscellaneous Markers
References
Clinical Trial
Definition
Phase I Trials
Phase II Trials
Phase III Trials
References
See Also
CLL
CLN
Clu
Cluster of Differentiation 44
Cluster of Differentiation Antigen 135 (CD135)
Cluster of Differentiation Antigen 66 a
Cluster of Differentiation Antigens
Clusterin
Synonyms
Definition
Characteristics
Discovery
mRNA Isoforms and Protein Forms
Structure
Clu and Neurological Diseases
Clu and Cancer
References
CML
CMM2
CNAs
CNC
CNDF
Coactivator ACTR
Coagulation Factor II Receptor
Coagulation Factor II Receptor-Like 1
Coagulation Factor II Receptor-Like 2
Coagulation Factor II Receptor-Like 3
Coagulopathy
Definition
Characteristics
References
COE
Coffee Consumption
Definition
Characteristics
Coffee Constituents
Coffee Association with Cancer
Cross-References
References
See Also
Cohesin
Cohesin Pathway Dysregulation
Cohesins
Synonyms
Definition
Characteristics
Noncanonical Role of Cohesin
Cohesin and Human Diseases
Cohesin and Cancer
Conclusions
Cross-References
References
Cohort Study
Definition
Cross-References
Coiled Bodies
Cold Surgery
Collapsin
Collier-Olf-EBF
Colloid Carcinoma
Colon Cancer
Colon Cancer Carcinogenesis in Human and in Experimental Animal Models
Definition
Characteristics
Precursor and Premalignant Lesions of CRC
Carcinogenic Pathways
Adenoma-Carcinoma Sequence Type
HNPCC Type Pathway
``De Novo´´ Type Pathway
Colitic Cancer Pathway (Ulcerative Colitis Dysplasia Carcinoma Sequence)
Experimental Colon Carcinogenesis
Colonic Carcinogens
AOM/Dextran Sodium Sulfate (DSS) Model for Colitis-Related Colon Carcinogenesis
Cross-References
References
Colon Cancer Classification
Definition
See Also
Colon Cancer Genomic Pathways
Synonyms
Definition
Characteristics
Histopathological Characteristics
Genomic Alterations Involved
Chromosomal Instability Pathway
APC
KRAS
SMAD2, SMAD4 and DCC
p53
Microsatellite Instability (MSI) Pathway
Animal Models for the Study of Colon Carcinogenesis
Carcinogen-Induced Models
AOM
DSS
Genetic Models
APCMin
APC Transgenics
APC1638N
APC(Delta14/+)
Perspective
Cross-References
References
See Also
Colon Cancer Molecular and Targeted Experimental Therapy
Definition
Characteristics
Molecularly Targeted Therapies
Targeting Cell Proliferation Signaling as an Anticancer Approach
Targeting EGF/EGFR Signaling
Monoclonal Antibodies Against EGFR
Tyrosine Kinase Inhibitors
Targeting PI3K/AKT/mTOR Signaling
Targeting MEK/MAPK Signaling
MET/Hepatocyte Growth Factor (HGF) Signaling Pathway
Targeting Angiogenesis as an Anticancer Approach
Monoclonal Antibodies Against VEGF
VEGF Trap
Tyrosine Kinase Inhibitor
Targeting Apoptosis Signaling as an Anticancer Approach
Targeting Bcl-2 Family
IAP Antagonists
Targeting the Extrinsic Apoptosis Pathway
Targeting CRC Stem Cells
Targeting Wnt Pathway Components
Targeting Notch Pathway Components
Colorectal Cancer Immunotherapy
Cancer Vaccines
Autologous Tumor Cell Vaccines
Peptide Vaccines
Dendritic Cell-Based Vaccines
Viral Vector-Based Vaccines
Adoptive Cell Therapy
Immunomodulatory Strategy
Acknowledgments
Cross-References
References
Colon Cancer Pathology of Hereditary Forms
Definition
Characteristics
Colorectal Cancer in Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer)
Colorectal Cancer in Familial Polyposis Syndromes
Hamartomatous Polyposis Syndromes
Peutz-Jeghers Syndrome
Juvenile Polyposis Coli Syndrome
Hamartomatous, Juvenile-Type Polyps
References
Colon Cancer Risk
Colorectal Cancer
Synonyms
Definition
Cross-References
See Also
Colorectal Cancer Chemoprevention
Synonyms
Definition
Characteristics
Aspirin
Sulindac
Coxibs
5-Aminosalicilate (5-ASA)
Metformin
Vitamin D
Selenium
The Future
Cross-References
References
See Also
Colorectal Cancer Clinical Oncology
Synonyms
Definition
Characteristics
Screening Strategies for the Average-Risk Population
FOBT (Fecal Occult Blood Test)
Sigmoidoscopy
Colonoscopy
Double-Contrast Barium Enema
Preoperative Diagnosis of Colon Cancer
Preoperative (Neoadjuvant) Therapy
Surgical Therapy (with the Aim to Cure)
Cancers of the Cecum and Ascending Colon
Cancers of the Right Flexure and the Proximal Transverse Colon
Cancers of the Transverse Colon
Tumors of the Left Colonic Flexure
Tumors of the Descending Colon and Proximal Sigmoid Colon
Tumors of the Middle and Distant Sigmoid Colon
Further Constellations Influencing Surgical Strategy
Intra- and Postoperative Histopathological Diagnosis
Classification of Colorectal Cancers
Adjuvant Therapy
Follow-Up
Cellular and Molecular Features
Perspective
References
Colorectal Cancer Nutritional Carcinogenesis
Synonyms
Keywords
Definition
Characteristics
Relation Between Nutrition and Colon Cancer: Evaluation by an International Expert Group
Nutritional Factors Increasing the Risk of Colon Cancer
Red Meat and Processed Meat
Alcoholic beverages
Body and Abdominal Fatness
Adult Attained Height
Nutritional Factors Decreasing the Risk of Colorectal Cancer
Physical Activity
Foods Containing Dietary Fibre
Other Factors
Conclusion
Cross-References
References
See Also
Colorectal Cancer Pathology
Definition
Characteristics
Localization
Macroscopy/Gross Morphology
Microscopy/Histomorphology
Histological Typing
Histologic Grading
Tumor Spread
Local Spread
Lymphatic Spread
Venous Spread
Perineural Spread
Extent of Resection and Lymph Node Dissection
Staging
Explanatory Notes Concerning the Application of the UICC TNM Classification
General Aspects
Primary Tumor
Regional Lymph Nodes
Distant Metastasis
Stage Grouping
Residual Tumor Classification
Tumor Regression Score
Prognosis
Histopathological Report
Cross-References
References
Colorectal Cancer Premalignant Lesions
Synonyms
Definition
Characteristics
Adenomas
Adenoma-Carcinoma Sequence
Hyperplastic Polyps
Hamartomatous Polyps
Aberrant Crypt Foci
Inflammatory Bowel Diseases
Cross-References
References
Colorectal Cancer Therapeutic Antibodies
Definition
Characteristics
Treatment of metastatic colorectal cancer
Cetuximab (Erbitux) and Panitumumab (Vectibix): Epidermal Growth Factor Receptor (EGFR) Inhibitors
Bevacizumab (Avastin): Vascular Endothelial Growth Factor (VEGF) Inhibitor
Ramucirumab (CYRAMZA): Vascular Endothelial Growth Factor- Receptor 2 (VEGF-R2) inhibitor
Other Antibodies with Anti-CRC Activity
Conclusions
Cross-References
References
Colorectal Cancer Vaccine Therapy
Definition
Characteristics
Whole-Cell Vaccines (WCV)
Heat Shock Proteins (HSP)
Peptide and Protein Vaccines
Anti-idiotype Vaccines
DNA Vaccines
Recombinant Virus Vaccines
Dendritic Cell-Based Vaccines
Outlook
References
Combinatorial Cancer Therapy
Synonyms
Definition
Characteristics
Introduction
Major Types of Combinatorial Cancer Therapy
Combinatorial Cancer Chemotherapy
Combined Modality Cancer Therapy
Conclusion
Cross-References
References
See Also
Combinatorial Selection Methods
Synonyms
Definition
Characteristics
CASTing
REPSA
SELEX
Cross-References
References
Combined Modality Treatment
Comet Assay
Synonyms
Definition
Characteristics
Modifications
Areas of Application
Genotoxicity Testing
DNA Repair
Biomonitoring
Cross-References
References
See Also
Comparative Oncology
Definition
Characteristics
History
Scientific Advancements
Need for New Models of Drug Development
Clinical Applications
Cross-References
References
Complement Cytolysis Inhibitor (Cli)
Complement-Associated Protein SP-40 (SP-40)
Complement-Dependent Cytotoxicity
Synonyms
Definition
Cross-References
See Also
Complement-Mediated Cytotoxicity
Compound Screen
Conditionally Replicating Adenovirus
Conditionally Replicative Adenovirus
Confocal Laser-Scanning Microscopy In Vivo
Definition
Characteristics
Clinical Relevance
Cross-References
References
Congenic Mice
Congenital Mesoblastic Nephroma
Congenital Telangiectatic Erythema
Conjugated Linolenic Acids
Synonyms
Definition
Characteristics
In Vitro Studies
In Vivo Studies
References
Connexins
Definition
Characteristics
Connexins as Tumor Suppressors
Connexins as a Therapeutic Target
Cross-References
References
Constitutive Androstane Receptor
Synonyms
Definition
Characteristics
Structure of CAR
Mechanism of Action
Regulators of CAR
CAR Targets
CAR and Carcinogenesis
Conclusions
Cross-References
References
See Also
Constitutive Photomorphogenic-9 (COP9) Signalosome 5 (CSN5)
Contact Normalization
Synonyms
Definition
Characteristics
Cross-References
References
Contralateral Breast Cancer
Definition
Cross-References
Convection-Enhanced Delivery
Synonyms
Definition
Characteristics
Clinical Application
Future Directions
Cross-References
References
See Also
Coordination Compound
COP9/CSN5
Core Binding Factor A2
Corin (TMPRSS10)
Cortactin
Synonyms
Definition
Characteristics
Structure and Binding Partners
Function
Regulation
Role in Cancer
References
Cowden Disease
Cowden Syndrome
Synonyms
Definition
Characteristics
Molecular Features
Clinical Aspects
References
COX
COX-2
COX-2 in Colorectal Cancer
Synonyms
Definition
Characteristics
COX-2 and Colorectal Cancer Prevention
Summary
Cross-References
References
See Also
COX-2 Inhibitors
CpG Islands
Synonyms
Definition
Characteristics
Preservation of CpG Islands
Clinical Relevance
Normal, Developmentally Regulated, CpG Island Methylation
Aberrant CpG Island Methylation in Cancer
Cross-References
References
CPT-11
Cr(VI)
Cr
cRaf
C-Raf
Cripto-1
Synonyms
Definition
Characteristics
Structure of Cripto-1, a Member of the EGF-CFC Protein Family
Cripto-1 During Embryonic Development and in Embryonic Stem (ES) Cells
Cripto-1 in Mammary Gland Development
Cripto-1 in Transformation, Tumorigenesis, and Angiogenesis
Transgenic Mouse Models Overexpressing CR-1 in the Mammary Gland
Expression of CR-1 in Human Carcinomas and Premalignant Lesions
Cross-References
References
c-Rmil
Crohn Colitis
Synonyms
Definition
Cross-References
Crohn Disease
Synonyms
Definition
Characteristics
Chemoprevention of IBD-Related CRC
Mechanistic Explanations
Concluding Remarks
Cross-References
References
Crow-Fukase Syndrome
CRP55
CRP-Ductin (Mouse)
CRT
Cryosurgery
Synonyms
Definition
Characteristics
Cryobiology
Tumors Suitable for Cryosurgical Treatment
Indications for Cryosurgery
Cryosurgical Technique
Results of Treatment
Complications
Wound Infection
Venous Gas Embolism
Fracture
Epiphyseal Damage
Degenerative Osteoarthritis
Damage to Nerves
Prospects to the Future
Cross-References
References
See Also
Cryptotanshinone
Synonyms
Definition
Characteristics
Antiangiogenesis
Antilymphangiogenesis
Anticancer Activities
References
Crypts
Definition
Cross-References
CSC
c-Src
CT4
CT6.1
CTAG
CTAG1
CTAG1A
CTAG1B
CTCF
CTCFL (Stands for CTCF-Like)
CTCF-T (Stands for CTCF-Testis Specific)
CTLA-8 in Rodents
CTLO
Cucurbitacin B
Definition
Characteristics
Chemical names
Activities on Cancer Cells
Mechanism of Action
Position of Cucurbitacin B
Adverse Effects
Conclusion
Cross-References
References
Cullin Ubiquitin E3 Ligases
Synonyms
Definition
Characteristics
Regulation
Conclusion
Cross-References
References
Cullin-Containing Ubiquitin E3 Ligases
Cullin-RING Ubiquitin E3 Ligases (CRLs)
3D Culture
Curcumin
Synonyms
Definition
Characteristics
Structure-Activity Relationships
Bioavailability
Modulation of Cancer Cell Signaling Pathways
Induction of Cancer Cell Death
Clinical Trials
Cross-References
References
Cushing Syndrome
Definition
Characteristics
Diagnostic Guidelines
References
Cutaneous Desmoplastic Melanoma
Synonyms
Definition
Characteristics
Epidemiology
Microscopic Features
Clinical Features
Management
Cross-References
References
See Also
Cutaneous Neoplasms
Cyclin D
Definition
Characteristics
Regulation of D Cyclins
CDK-Independent Activities of D-Type Cyclins
Clinical Relevance
References
Cyclin G-Associated Kinase
Synonyms
Definition
Characteristics
GAK and the Androgen Receptor
GAK and Prostate Cancer Progression
Cross-References
References
See Also
Cyclin-Dependent Kinase Inhibitor 1A
Cyclin-Dependent Kinase Inhibitor 1B (CDKI1B)
Cyclin-Dependent Kinase Inhibitor 2A
Cyclin-Dependent Kinases
Synonyms
Definition
Characteristics
The Discovery of CDKs
Members of the CDK Family
CDKs in Cancer and Other Human Diseases
Cross-References
References
Cyclins
Definition
Cross-References
Cyclooxygenase
Definition
Cross-References
See Also
Cyclooxygenase (Prostaglandin Endoperoxide Synthase 2)
Cyclophosphamide
Definition
Cross-References
Cylindromatosis
Synonyms
Definition
Cross-References
CYP 450arom
CYP450
Cystadenocarcinoma
Cystadenoma
Cystatins
Synonyms
Definition
Characteristics
Domain Structure
Gene Structure and Evolution
Function
Role in Cancer
Cross-References
References
See Also
Cystatins A, B
Cystic Fibrosis
Definition
Cross-References
Cystic Nephroma
Definition
Cross-References
Cytochalasin B
Definition
Cross-References
See Also
Cytochrome P450
Synonyms
Definition
Characteristics
Detoxification
Using CYPs to Improve Therapy
Genetic Polymorphisms
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4
CYP3A5
Cancer Incidence
Cross-References
References
See Also
Cytokine
Definition
Cross-References
Cytokine Receptor
Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy
Synonyms
Definition
Characteristics
Cytokine Receptor as the Target for Immunotherapy
Cytokine Receptor as the Target for Immunotoxin Therapy
Cross-References
References
See Also
Cytokine Toxin Fusions or Conjugates
Cytoplasmic Scaffolding Apoptotic Protease Activating Factor
Cytoskeleton
Definition
Characteristics
Microfilaments (Actin Filaments)
Microtubules
Intermediate Filaments
Cross-References
References
Cytosolic Transglutaminase
Cytotactin
Cytotoxic T Cells
Definition
Characteristics
Dendritic Cells Play a Major Role in the Induction of CD8 T Cell Responses
Activation of CD8 T cells
Cytotoxic Potential of Activated CD8 T Cells
Cross-References
References
Cytovillin
D
3-(1,3-Dihydro-3-oxo-2H-indol-2-ylidene)-1,3 dihydro-2H-indol-2-one
DAC
Dacogen
Dactinomycin
Definition
Cross-References
See Also
DAF
Damage Response
DANCE, EVEC, UP50, FBLN-5
Dap160 (Dynamin-Associated Protein of 160 kDa)
DAP6
DARC
Daxx
Synonyms
Definition
Characteristics
Basic Information
Subcellular Distribution of Daxx Protein
Daxx Mediates Stress-Induced Cell Death
In Unstressed Cells, Daxx Seems to Play an Antiapoptotic Role
Cross-References
References
DCIS
DcR3
DCX
DDP
DDS
Death
Death Receptors
Definition
Cross-References
See Also
Death-Associated Protein 6
Decatenation G2 Checkpoint
Definition
Characteristics
Molecular Biology
Clinical Implications
Cross-References
References
Decay-Accelerating Factor
Synonyms
Definition
Characteristics
Actions of DAF in Cancer
Perspectives in Cancer Therapy
Cross-References
References
See Also
Decay-Accelerating Factor for Complement
Decitabine
Decoy Receptor
Definition
Cross-References
Decoy Receptor 3
Synonyms
Definition
Characteristics
Tissue Expression and Decoy Function of DcR3
Decoy Unrelated Novel Actions of DcR3
New Action Mechanisms of DcR3
Cross-References
References
See Also
Deferasirox
Synonyms
Definition
Characteristics
Pharmacological Properties
Tolerability
Synthesis
Iron
Iron and Cancer
Iron Chelation in Cancer Therapy
Deferasirox in Cancer Therapy
Deferasirox and Mantle Cell Lymphoma
Deferasirox and the PI3K/AKT/mTOR Pathway
Deferasirox and the Wnt Signaling Pathway
Deferasirox and Nuclear Factor-kappaB (NF-kappaB)
Interesting Case Report
Conclusion
Cross-References
References
See Also
Deleted in Liver Cancer 1
Deleted in Malignant Brain Tumors 1
Synonyms
Definition
Characteristics
Functional Characteristics
DMBT1 and Innate Immunity/Mucosal Protection
DMBT1 and Epithelial/Stem Cell Differentiation and Regenerative Processes
DMBT1 and Tumorigenesis
References
Deleted in Pancreatic Carcinoma Locus 4
Synonyms
Definition
Characteristics
TGFbeta-Smad Signaling Cascade
Transcriptional Regulation Through Smads
What Makes DPC4/Smad4 Unique Among the Other Smad Family Members?
Which Human Tumors Show Alterations of the DPC4 Gene?
How Are Naturally Occurring DPC4 Mutations Interfering with the Smad Signaling Cascade
Does DPC4 Contribute to the Familial Risk for Pancreatic Cancer?
How Does DPC4 Contribute to Tumor Formation?
Cross-References
References
Dendritic Cells
Definition
Characteristics
Origin and Function
Dendritic Cell-Based Immunotherapy
Dendritic Cells in Cancers
Cross-References
References
See Also
Dental Pulp Neoplasms
Definition
Characteristics
History
Inflammatory Pulp Reactions
Radiotherapy
Animal Investigations
Clinical Relevance
Cross-References
References
See Also
2-Deoxy-5-azacytidine
Deoxyazacytidine
Dephosphorylating Enzyme
DES
Designer Foods
Desmoglein-2
Synonyms
Definition
Characteristics
Cell Junctions
Desmosome
Desmoglein and Cancer
Adherens Junctions
Cross-References
References
Desmoid Tumor
Synonyms
Definition
Characteristics
Pathology
Etiology
Clinical Features
Treatment
Cross-References
References
Desmoplasia
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Desmoplastic Melanoma
Desmoplastic Small Round Cell Tumor
Synonyms
Definition
Characteristics
Clinical and Pathological Features
Molecular Diagnosis
Molecular Genetics
Cross-References
References
See Also
Desmoplastic Tumor Microenvironment
Desmosomes
Synonyms
Definition
Characteristics
Desmosomal Cadherins
Armadillo Family
Plakin Family
Null Mutations in Mice
Clinical Relevance
Cross-References
References
See Also
Detachment-Induced Cell Death
Determination of Tumor Extent and Spread
Detoxication
Detoxification
Synonyms
Definition
Characteristics
Genetic Variation
Cellular Regulation
Clinical Relevance
References
Deubiquitinating Enzymes (DUBs)
Development Lymph Vessel
Development of New Lymphatic Vessels
Dezocitidine
D-factor
dFdC
DIA
Diabody
Synonyms
Definition
Characteristics
Structure
Pharmacokinetics and Distribution
Agonistic Activity
Application of Diabodies
Future Directions
Cross-References
References
See Also
Diagnostic Pathology
Dibasic Processing Enzyme
Diet
Dietary Carcinogens
Dietary Essential Minerals
Diethylstilbestrol
Synonyms
Definition
Characteristics
Pharmacology
Initial Use and Early Epidemiologic Studies
Animal Studies
Neoplastic Effects in Humans
Nonneoplastic Effects in Humans
Mechanism of Toxicity
Summary
Cross-References
References
See Also
Diferuloylmethane
Differentiation-Inducing Factor
Diffuse
Diffuse Astrocytoma
Diffuse Intrinsic Pontine Glioma
Diffuse Large B-Cell Lymphoma
Synonyms
Definition
Characteristics
Diagnosis
Therapy
Genetics
Subtypes of Diffuse Large B-Cell Lymphoma
Primary Mediastinal (Thymic) Large B-Cell Lymphoma
Intravascular Large B-Cell Lymphoma
Primary Effusion Lymphoma
Lymphomatoid Granulomatosis
Cross-References
References
See Also
Diffuse Large Cell
Diffuse Mixed Small and Large
Diffuse or Nodular
2,2-Difluoro-2-deoxycytidine
Dihydrogen Dioxide
3,4-Dihydro-3-methyl-4-oxoimidazo(5,1-d)-as-tetrazine-8-carboxamide
3,4-Dihydro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-8-carboxamide
3,4-Dihydro-3-methyl-4-oxoimidazo(5,1-d)-1,2,3,5-tetrazine-8-carboxamide
Dihydrotestosterone Receptor
Dimethylfumarate
Definition
Chemistry and Pharmacodynamics
Characteristics
Biological Effects of FAEs In vitro
Drug Formulations of Currently Used Fumaric Acid Esters (FAEs)
Biological Effects of FAEs In vivo
Clinical Aspects and Future Perspectives
References
Dioxin
Synonyms
Definition
Characteristics
Carcinogenic Activity
Mechanisms
Clinical Relevance
Cross-References
References
See Also
Dioxin Receptor
Dipeptidyl-Peptidase IV
Dipropylacetic Acid
Directed Migration
Directed Motility
Disintegrin Metalloproteases
Disordered Domain
Distal Intestinal Serine Protease (Mouse Only, TMPRSS8)
Distant Bystander Effect
Distant Bystander Effects
Distributed Stem Cells
DLC1
Synonyms
Definition
Characteristics
Functions of DLC1
Negative Regulation of Cytoskeleton
Tumor Suppressive Effect
Clinical Relevance
Genetic Alteration
Epigenetic Silencing
Cross-References
References
See Also
DMBT1
DNA Damage
Synonyms
Definition
Characteristics
Agents Causing Bulky, Structurally Distorting DNA Adducts
Nucleotide Base Damage
Single- and Double-Strand Breaks
Therapeutic Approaches
Cross-References
References
See Also
DNA Damage Response
Definition
Characteristics
Sources of Damage
Checkpoint Pathway
G1 Checkpoint
Intra-S-phase Checkpoint
G2/M Checkpoint
DNA Repair Responses
Excision Repair
Double-Strand Break Repair
Translesion Synthesis Repair
Interstrand DNA Cross-Link Repair
Arrest, Repair, or Die?
p53-Dependent Mechanisms
p53-Independent Mechanisms
Cancer Susceptibility
Cross-References
References
See Also
DNA Damage Response Genes
Definition
Characteristics
DNA Replication Polymerase Alterations in Cancer
DNA Repair Pathway Alterations in Cancer
DNA Damage Signaling Pathways in Cancer
References
See Also
DNA Damage Tolerance
Synonyms
Definition
Characteristics
Mechanism
Nonreplicative DNA Polymerases
Translesion DNA Synthesis
Association with Cancer
References
DNA Damage-Induced Apoptosis
Synonyms
Definition
Characteristics
Summary
Physiological Role of Apoptosis Induced by DNA Damage
Cross-References
References
See Also
DNA Damage-Triggered Death Signaling Pathways
DNA Demethylation
DNA Lesion
DNA Lesion Bypass
DNA Oxidation Damage
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
See Also
DNA Repair and Damage Processing
DNA Undermethylation
DNA Vaccination
Synonyms
Definition
Characteristics
Mechanisms of Action
Adjuvant Activity of DNA
Tailoring Immune Responses by DNA Vaccine Design
Formulation
Mixed Modality Vaccines
Results from Clinical Trials
References
DNA-Bound Carcinogens
DnaJ (Hsp40) Homolog Subfamily C Member 15
DNAJC15
DNAJD1
DNF15S2
Docetaxel
Synonyms
Definition
Characteristics
Pharmacokinetics
Toxicity
Dosage
Indications
Cross-References
References
See Also
Docking Proteins
Dormancy
Synonyms
Definition
Characteristics
Blocked Angiogenesis
Maintenance of Tumor Dormancy by Endogenous Angiogenesis Inhibitors
Escape from Tumor Dormancy by a Switch to the Angiogenic Phenotype
Experimental Analysis of the Angiogenic Switch in Human Dormant Cancers
Molecular Mechanisms of Angiogenic Switching in Dormant Cancers
Other Forms of Tumor Dormancy
Immune Surveillance as a Cause of Tumor Dormancy
Hormonal Depletion as a Cause of Tumor Dormancy
Dormancy of Single Metastatic Tumor Cells
Future Directions
Biomarkers to Detect Dormant Tumors
Cross-References
References
See Also
Doublecortex
Doublecortin
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
References
Doxorubicin
DPC4
DPPIV
DR4 Antibodies
DR5 Antibodies
Draining Lymph Node
DRF
Drug Carriers
Drug Delivery Systems for Cancer Treatment
Keywords
Definition
Characteristics
Characteristics of Vascular Walls of Cancer
Carriers for Drugs, Chemicals, and Nucleic Acids
Targeted Drug Delivery
Cross-References
References
See Also
Drug Delivery Vehicles
Drug Design
Synonyms
Definition
Characteristics
Identification and Validation of Molecular Targets
Characterization of Molecular Targets
Compound Screening and Lead Optimization
Preclinical Optimization
Clinical Evaluation
Cross-References
References
See Also
Drug Development
Drug Discovery
Definition
Characteristics
Cancer Targets
Fusion Genes, Oncogenes, and Tumor Suppressors
Pathway Activations
Drug Types
Design and Screen the Drug Candidates
Characterize the Drug Candidates in Preclinical Models
Conclusions
References
Drug Metabolism
Drug Resistance
Synonyms
Definition
Characteristics
De Novo Drug Resistance
Acquired Drug Resistance
Drug Metabolism
Drug Efflux
Intracellular Changes in Drug Resistance
Drug Penetration
References
Drug Responses
Drug Targeting
Dry Eye Syndrome
Dry Mouth Syndrome
Dsg2
DS-ML
Ductal Carcinoma In Situ
Synonyms
Definition
Characteristics
Diagnosis
Risk Factors
Classification
Treatment
DCIS in Men
Recurrence
References
Duffy Antigen Receptor for Chemokines
Synonyms
Definition
Characteristics
References
DUG (Rat Pdcd4)
Dynamic Contrast-Enhanced Magnetic Resonance Imaging
Definition
Characteristics
T1 and T2* DCE-MRI Techniques
Data Acquisition
Data Analysis
Clinical Findings
Evaluation of Angiogenesis Inhibitors
Conclusion
References
Dysgerminoma
Dysgerminomas - Dysgerminoma
Dysmyelopoietic Syndrome
Dysplasia
Definition
Cross-References
E
E Motif
ETV6
Synonyms
Definition
Characteristics
Discovery
Protein Domains
ETV6 Fusion Partners in Cancer
Protein Tyrosine Kinase Fusion Partners of ETV6
Transcription Factors and Other Fusion Partners of ETV6
The ETV6/RUNX1 Fusion
The MN1/ETV6 Fusion
The ETV6/ARNT Fusion
``Unproductive´´ ETV6 Fusions
Putative Tumor Suppressor Gene and Physiological Function
References
E100
E11 Antigen
E2A-PBX1
Definition
Characteristics
Clinical Aspects
Wild-Type E2A and PBX1 Gene Products
Structure and Function of E2A-PBX1
References
E3 Ubiquitin Protein Ligase
Eag
Eag1
EAP1
Early B-Cell Factors
Synonyms
Definition
Characteristics
Potential Roles in Cancer
References
Early Detection
Early Genes of Human Papillomaviruses
Definition
Characteristics
Clinical Relevance
References
Early-Stage Ovarian Cancer
Synonyms
Definition
Characteristics
Preoperative Workup of an Adnexal Mass
Surgical Staging
Non-epithelial Ovarian Cancer: Surgical Staging and Treatment
Germ Cell Tumors
Immature Teratoma
Endodermal Sinus Tumor
Sex Cord Tumors
Sertoli-Leydig Cell Tumors
Fertility Preservation
Conclusion
References
EBF
Ebnerin (RAt)
E-Box
Synonyms
Definition
Characteristics
Genes Containing E-Boxes and the bHLH Proteins That Control Them
Genes and Gene Products That Are Related to Growth Control and Cancer
How Do bHLH Proteins Bind to E-Boxes?
References
See Also
EBV
EC 2.7.11.1
E-Cadherin
Synonyms
Definition
Characteristics
E-Cadherin and the Formation of Adhesion Junctions
Signals Elicited by the Loss of Cell Adhesion
E-Cadherin in Animal Models of Cancer
E-Cadherin in Human Cancer
Clinical Relevance
Cross-References
References
See Also
ECMRIII
ECSA
Ecteinascidin 743
Ectonucleotide Pyrophosphatase/Phosphodiesterase 2
Eczema
EDF
Edible Salt
EDN
Efferocytosis
Keywords
Definition
Characteristics
The Efferocytic Process
Apoptotic Cell Death
Phagocyte Recruitment by Apoptotic Cells
Recognition of Apoptotic Cells by Phagocytes
Apoptotic Cell Engulfment
The Post-Phagocytic Immune Response
Diseases Caused by Malfunctioning Efferocytosis
Rheumatic Diseases
Atherosclerosis
Chronic Lung Diseases
Allograft Tolerance
Infection and Microbes
The Role of Efferocytosis in Cancer
Prospects
Cross-References
References
See Also
Eg5
EGF
EGF Family
EGF-Like Ligands
EGFR
EGP-2
EGP34
EHSH1 (EH Domain/SH3 Domain-Containing Protein)
Eicosanoid Signaling
Eicosanoids
Definition
Cross-References
ELA2
Elastase
ELAV1
Electrolytes
Electromagnetic Fields
Definition
Characteristics
Epidemiological and Clinical Evidence
Experimental Evidence
Clinical Relevance
Cross-References
References
Electropermeabilization
Electroporation
Synonyms
Definition
Characteristics
Physical Aspects
Experimental Electroporation for Nucleic Acid Delivery
Clinical Uses of Electroporation
Electrochemotherapy
Electrogenetherapy
Irreversible Electroporation
Cross-References
References
Eliminate Gynecomazia
ELISA
Definition
Cross-References
Elongin BC Complex
Definition
Characteristics
The VHL Tumor Suppressor Complex
SOCS-Box Proteins and the Elongin BC Complex
Clinical Relevance
Cross-References
References
Embryonal Carcinoma
Embryonal Serum Alpha-Globulin
Embryonal Serum α-Globulin
Embryonic Stem Cells
Definition
Cross-References
Embryo-Specific Alpha-Globulin
Embryo-Specific α-Globulin
Empirin
EMS1
Endocan
Synonyms
Definition
Characteristics
References
Endocannabinoids
Endocrine Neoplasms
Endocrine Oncology
Definition
Characteristics
Hereditary Syndromes Associated with Endocrine Tumors
Endocrine Tumor Markers
Endocrine Syndromes Secondary to Functioning Neuroendocrine Tumors
Endocrine Paraneoplastic Syndromes
Hormonal Influences on Tumor Growth
Hormone-Sensitive Tumors
Late Effects of Cancer Therapy on the Endocrine System
Conclusion
References
Endocrine Therapy
Synonyms
Definition
Cross-References
Endocrine Therapy in Breast Cancer
Synonyms
Definition
Characteristics
Estrogen Receptors
Therapeutic Strategies Based On ERα Targeting
Selective Estrogen Receptor Modulators (SERMs)
Selective Estrogen Receptor Downregulators (SERDs)
Therapeutic Strategies Based On Estrogen Deprivation
Ovarian Ablation/Suppression
Aromatase Inhibitors
Resistance to Endocrine Therapy
Cross-References
References
Endocrine-Related Cancers
Synonyms
Definition
Characteristics
Pathogenesis
Prevalence
Clinical Presentation and Pathology
Pituitary Tumors
Thyroid Tumors
Parathyroid Tumors
Endocrine Pancreatic Tumors
Adrenal Tumors
Ovarian Tumors
Testicular Tumors
Multiple Endocrine Gland Tumors
Carcinoids
Secondary Endocrine Malignancies
Ectopic Hormone Syndromes
Treatment
Prognosis
Cancers of Endocrine Target Tissues
Cross-References
References
See Also
Endocrine-Responsive Cancers
Endocurietherapy
Endocytosis
Synonyms
Definition
Characteristics
Endocytosis and Signaling
Molecular Mechanisms
Clinical Relevance
Cross-References
References
See Also
Endolysosomal Pathway
Endometrial Cancer
Definition
Characteristics
Risk Factors
Classification
Pathogenesis Mechanisms
Treatment
Surgery
Radiation Therapy
Hormonal Therapy
Progestogens
Aromatase Inhibitors
Cross-References
References
See Also
Endometrioma
Definition
See Also
Endoplasmic Reticulum Stress
Synonyms
Definition
Characteristics
Molecular Mechanisms
Signal Transduction by the Three Arms of the UPR
Clinical Relevance
Glossary
References
Endoplasmic Reticulum Stress Response
Endosomal Compartments
Synonyms
Definition
Characteristics
Clinical Aspects
Cross-References
References
Endosomal Protein Sorting
Endostatin
Definition
Characteristics
Discovery
Structure and Function
Mechanism of Action
Genetic Variations
Preclinical Studies
Clinical Trials
References
Endothelial Cell-Specific Molecule-1
Endothelial Transglutaminase
Endothelial-Derived Gene-1
Synonyms
Definition
Characteristics
Mechanisms
EG-1 in Human Cancer
Translational Aspects
Cross-References
References
See Also
Endothelial-Leukocyte Adhesion Molecule 1 (ELAM1)
Endothelin-2
Endothelins
Synonyms
Definition
Characteristics
Endothelin Expression in Cancer
Cross-References
References
See Also
Endotoxin-Induced Factor in Serum
Engineered Antibody
Enlarged Breast Male
ENPP2
Enteropeptidase (TMPRSS15)
Enzymes
Enzymic Mouth to Mouth Feeding
Ep
EpCAM
Synonyms
Definition
Characteristics
Structure and Function
Structure
Tissue Morphogenesis
Pattern of Tissue Expression
Normal Tissue
Abnormal Tissue
Paradox of Expression with Advance in Carcinomas
EpCAM-Targeted Immunotherapy
Mechanism of Tumor Inhibition
Clinical Trials (Table1)
Monospecific Murine Antibody
What Are the Solutions?
Humanized Antibody
Bispecific Antibodies
Structure and Rationale for Development (Fig.2)
In Vitro Cytotoxicity
Prolonged Antitumor Immunity In Vivo
Clinical Trials (Table2)
Cross-References
References
See Also
Eph Receptors
Definition
Characteristics
Normal Eph Signaling
Eph Signaling in Cancer
References
Ephelide
Epidemiology of Cancer
Synonyms
Definition
Characteristics
Study Design
Risk Factors
Cross-References
References
Epidermal Growth Factor Inhibitors
Definition
Characteristics
EGFR Activation and Downstream Signaling
Molecular Mechanisms of Targeted Therapies
EGFR Mutations
EGFR Inhibitors in GI Tumors
EGFR Inhibitors in Lung Cancer
References
Epidermal Growth Factor Receptor
Synonyms
Definition
Characteristics
EGFR Structure
EGFR Ligands
EGFR Signal Transduction
EGFR Transactivation
EGFR in Cancer
Increased EGFR Ligand Production
Increased EGFR Protein Levels
EGFR Mutations
Impairment of EGFR Downregulation
EGFR Cross Talk with Other Receptors
Therapies Targeting EGFR
Summary
Cross-References
References
Epidermal Growth Factor Receptor (EGFR) Inhibitors
Epidermal Growth Factor-like Ligands
Synonyms
Definition
EGF-like Ligands and Cancer
Brain Tumor
Carcinoma of the Lung
Head and Neck Squamous Cell Carcinoma (HNSCC)
Colorectal Cancer
Cross-References
References
See Also
Epidermoid Carcinoma
Epigallocatechin
Synonyms
Definition
Characteristics
Cellular and Molecular Targets of EGCG
General Anticancer Effects of Green Tea Polyphenols
Direct Molecular Targets of EGCG
Clinical Relevance
Cross-References
References
See Also
Epigenetic
Definition
Cross-References
Epigenetic Biomarker
Definition
Characteristics
Epigenetic Modifications
Epigenetic Biomarkers in Cancer
Prostate Cancer
GSTP1 DNA Methylation in Prostate Cancer
Conclusion
References
Epigenetic Gene Silencing
Definition
Characteristics
Epigenetics and Cancer
DNA Methylation
Histone Modifications and Chromatin Remodeling
Clinical Relevance
Cross-References
References
See Also
Epigenetic Modifications
Definition
Cross-References
Epigenetic Therapy
Definition
Characteristics
DNA Methylation and Histone Deacetylation
Epigenetic Abnormalities in Cancer
DNA Methyltransferase and Histone Deacetylase Inhibitors
Epigenetic Therapy in Cancer
Cross-References
References
See Also
Epigenomics
Definition
Characteristics
The Epigenome and Cancer
Technologies
Epigenomic Researches and Therapies
Cross-References
References
See Also
Epithelial Cadherin
Epithelial Carcinogenesis
Epithelial Cell Adhesion Molecule
Epithelial Tumorigenesis
Synonyms
Definition
Characteristics
Introduction
Predisposing Factors
The Multistage Pathogenesis Model of Epithelial Tumorigenesis
Example of Multistage Carcinogenesis in Colorectal Cancer
The Role of Stem Cells in Epithelial Tumorigenesis
Epigenetic Changes and Epithelial Tumorigenesis
The Interaction of Cellular Microenvironment in Epithelial Tumorigenesis
Epithelial Mesenchymal Transition (EMT)
Chronic Inflammation and Epithelial Cancers
Summary
Cross-References
References
See Also
Epithelial-to-Mesenchymal Transition
Definition
Characteristics
Mechanisms
Clinical Relevance
Cross-References
References
Epitheliasin (TMPRSS 2)
Epithelium
Definition
Cross-References
See Also
Epo
Epoetin
Epothilone B Analogue
Synonyms
Definition
Characteristics
Introduction
Preclinical Testing
Phase I Studies in Cancer Patients
Phase II Studies in Cancer Patients
Summary and Future Outlook
Cross-References
References
Epothilones
Definition
Cross-References
See Also
Epstein-Barr Virus
Synonyms
Definition
Characteristics
In vitro Immortalization of Primary B Cells by EBV: The Growth Transcription Program
EBV Biological Cycle In Vivo
Viral Productive Cycle
Clinical Relevance
Cross-References
References
See Also
Epstein-Barr Virus Latent Membrane Protein 1
Definition
Characteristics
Mechanisms
Cross-References
References
ER
ER Stress
ERBB
ErbB-1
ERBB2
Erlotinib
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Erlotinib (Tarceva)
ERM Proteins
Synonyms
Definition
Characteristics
History, Structure, and Sequence
Regulation
Function, Distribution, Localization
The Expression and Functions of ERM Proteins in Cancer
Cross-References
References
ERp60
Erythrocytosis
Erythroid Colony-Stimulating Activity
Erythroid Differentiation Factor
Erythroleukemia
Definition
Cross-References
Erythropoiesis-Stimulating Factor
Erythropoietin
Synonyms
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
References
ESA
ESE (EH Domain and SH3 Domain Regulator of Endocytosis)
E-Selectin-Mediated Adhesion and Extravasation in Cancer
Keywords
Synonyms
Definition
Characteristics
Structure
Expression
Function
Clinical Relevance of E-Selectin in Cancer
Targeting E-selectin and Its Ligands
Soluble E-selectin as a Diagnostic Marker
E-selectin-Mediated Capture
E-selectin as a Receptor for Targeted Delivery
Cross-References
References
See Also
ESF
ESM-1
ESO1
Esophageal Adenocarcinoma
Synonyms
Definition
Characteristics
Epidemiology, Risk Factors, and Clinical Treatment
Mechanisms of Esophageal Adenocarcinoma Pathogenesis
Mutational Landscape of Esophageal Adenocarcinoma
Cross-References
References
See Also
Esophageal Cancer
Definition
Characteristics
Cross-References
References
ESR1
ESR2
Estradiol
Definition
Characteristics
Mechanism of Action
Sources of Estrogens
Role of Estrogens in Human Breast Carcinogenesis
Receptor-Mediated Pathway
Oxidative Metabolism of Estrogen
Estrogens as Inducers of Aneuploidy
References
Estren-Dameshek Syndrome (= Variant Form)
Estrogen Receptor
Synonyms
Definition
Characteristics
History
Molecular Mechanisms of Action
What Have We Learned from ER Knockout Mice?
Involvement of Estrogen Receptors in Physiology and Cancer
Cross-References
References
See Also
Estrogen Receptor Alpha
Estrogen Receptor Beta
Estrogen Receptor, Progesterone Receptor, and HER-2 Negative
Estrogen Signaling
Definition
Characteristics
ER-Mediated Signaling
GPER-Mediated Signaling
Estrogen Signaling in Cancer
References
Estrogen Synthase
Estrogenic Hormones
Definition
Characteristics
Etiology
Breast Cancer
Uterine, Cervical, and Ovarian Cancer
Vaginal Adenocarcinoma
Hepatoma
Therapy
Breast Cancer
Uterine and Cervical Cancers
Cross-References
References
See Also
Estrogen-Replacement Therapy
ET
ET-2
ET-743
Eta-1
Ether à-go-go Potassium Channels
Synonyms
Definition
Characteristics
Oncogenic Potential of h-Eag1 Channels
Eag1 as a Diagnostic Marker
Eag1 as a Therapeutic Target and Prognostic Marker
Mechanisms of Oncogenicity
Outline
Cross-References
References
Etidronate
Definition
Cross-References
Etiology of Prostate Adenocarcinoma
Etoposide
Synonyms
Definition
Cross-References
See Also
Ets Transcription Factors
Definition
Characteristics
Ets Factors and Development
Regulation of Ets Protein Activities
Ets Proteins and Cancer
References
ETS Variant Gene 6
Everolimus
Synonyms
Definition
Characteristics
Mechanism of Action in Renal Cell Carcinoma
Clinical Activity in Renal Cell Carcinoma
Conclusion
Cross-References
References
See Also
Ewing Sarcoma
Synonyms
Definition
Characteristics
Cytogenetics and Gene Alterations
Etiology
Therapy
References
Ewing Sarcoma Family Tumors
Ewing Tumor
EWS-FLI (ets) Fusion Transcripts
Definition
Characteristics
Structure
Properties of the EWS-FLI1 Fusion Transcript and Protein
EWS and Other Specific Human Translocations
Genesis of the Translocation
The EWS-FLI1 Fusion and the Pathogenesis of Ewing Sarcoma
Clinical Relevance
EWS-FLI1 (ets) in Diagnosis
EWS-FLI1 (ets) Fusion Type and Prognosis
EWS-FLI1 (ets) and Detection of Minimal Residual Disease
EWS-FLI1 (ets) and Therapeutics
Cross-References
References
See Also
Exfoliation of Cells
Synonyms
Definition
Characteristics
Mechanisms
Cell Exfoliation in Neoplasia
Clinical Aspects
Cross-References
References
See Also
Exjade
Exobiotics
Exosomal MicroRNA
Exosomal miRNA
Synonyms
Definition
Characteristics
Biogenesis of Exosomes and Exosomal miRNA Packaging
Bio-functions of Exosomal miRNA in Cancer
Exosomal miRNAs in Cancer Diagnostic and Clinical Use
Cross-References
References
See Also
Exosomal Shuttle MicroRNA
Exosome
Synonyms
Definition
Characteristics
Mechanism
Cargo
Protein
Lipid
RNA
Trafficking
Exosomes in Cancer
Microenvironment
Immune System
Biomarkers
Cross-References
References
See Also
Experimental Carcinogenesis
Extracellular Matrix Remodeling
Definition
Characteristics
References
Extracellular Nucleic Acids
Extracellular Signal-Regulated Kinases 1 and 2
Synonyms
Definition
Characteristics
The ERK Activation Cascade
ERK Substrates
Termination of pERK Activity
Nuclear Translocation and Localization of ERK
Oncogene-Driven ERK Activation: A Therapeutic Target
Radiation Therapy and ERK Activation
Future Directions
Cross-References
References
See Also
Extracellular Vesicles
Extrahepatic Bile Duct Carcinoma
Extrahepatic Cholangiocarcinoma
Extrahepatic Cholangiocellular Carcinoma
Extrapulmonary Small Cell Cancer
Synonyms
Definition
Characteristics
Epidemiology
Pathology
Clinical Features
Diagnosis and Staging
Management
Prognosis
Genitourinary Tract
Urinary Bladder
Prostate
Gynecological Sites
Cervix
Gastrointestinal Tract
Esophagus
Colon and Rectum
Head and Neck Region
Larynx
References
Extreme Hypoxia
F
4F9
FADD-Like Antiapoptotic Molecule 1
FADD-Like ICE
Familial (Constitutional) Panmyelocytopathy (Type) Fanconi
Familial Adenomatous Polyposis
Familial Hypoplastic Anemia Fanconi
Familial Polyposis Coli
FAMP
Fanconi Anemia
Synonyms
Definition
Characteristics
Myelodysplastic Syndromes (MDSs)
Leukemia
Solid Tumors
Squamous Cell Carcinoma (SCC) of the Head and Neck (HNSCC)
SCC of the Vulva and Cervix
Liver Tumors
Other Tumors
FA Gene Involvement in Neoplastic Disease of Non-FA Individuals
Cross-References
References
See Also
Fanconi Pancytopenia
Fanconi Panmyelopathy
FAP
Farnesylation
Definition
See Also
Fas-Associated Death Domain
Faslodex
Fatty Acid Transport
Definition
Characteristics
Tumor
Mechanisms
Cross-References
References
See Also
Fbl1
FBLN-2
F-Box and Leucine-Rich Repeat Protein 1
Fbxl1
FDG-PET
Definition
Characteristics
Principle
Instrumentation
Acquisition
Image Analysis
Qualitative
Semiquantitative
Image Reconstruction
Applications
Safety
Limitations
Conclusions
References
Fecal Immunochemical Test
Synonyms
Definition
Characteristics
Fate of Hemoglobin in the Gastrointestinal Tract
Uses for FIT
Quantifiable FIT
Types of FIT Currently Available
Stool-Sampling Procedures
Biology of Bleeding from Colorectal Neoplasia
References
Fecal Occult Blood Test
Synonyms
Definition
Cross-References
Fenretinide
Synonyms
Definition
Characteristics
Use of Fenretinide in Clinical Trials
Activity of Fenretinide in Nonmalignant Diseases
Conclusion
References
Ferroptosis
Synonyms
Definition
Characteristics
Modulating Ferroptosis Under Pathological Conditions
Conclusion
Cross-References
References
See Also
Fetal Exposure to Carcinogenic Agents
Fetal Hamartoma of the Kidney
Fetal Liver Kinase-2 (FLK2)
α1-Fetoglobulin
α-Fetoprotein
α-Feto-protein
Feto-Specific Proteins
Fetuin
Fetuin-A
Fever of Obscure Origin
Fever of Unexplained Origin
Fever of Unknown Origin
Synonyms
Definition
Characteristics
Cancer and Fever of Unknown Origin
Diagnosis
Prognosis of Cancer Associated with Fever of Unknown Origin
Conclusion
References
FH
FHIT
Fibrinogen
Definition
Characteristics
References
Fibroblast Growth Factor Receptor (FGFR) Inhibitors
Fibroblast Growth Factors
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
References
Fibrofolliculomas with Trichodiscomas and Acrochordons
Fibromyoma
Fibronectin
Definition
Characteristics
Forms of Fibronectin
Functions
Structure
Fibronectin Polymorphism
Posttranslational Modifications
Alternative Splicing
Biological Significance of ED-B
Fibronectin Isoforms in Disease
Fibronectin Isoforms and Cancer
Cross-References
References
Fibrosarcoma
Definition
See Also
Fibrosis and Cancer
Definition
Characteristics
Tumor-Stromal Cell Interactions
Lysyl Oxidases, Fibrosis, and Cancer
Tumor Suppressor Activity of LOX
References
Fibulins
Synonyms
Definition
Characteristics
Fibulin-1
Fibulin-2
Fibulin-3
Fibulin-4
Fibulin-5
Fibulin-6
Conclusion and Perspective
References
Fine Needle Aspiration
Fine Needle Aspiration Biopsy
Synonyms
Definition
Characteristics
Background
What Is FNAB?
What Cellular Characteristics Do Cytologists Study in Order to Make a Microscopic Diagnosis of Cancer?
Method
Palpation-Guided Aspirates
Deep-Seated Aspirations
Complications and Contraindications
Diagnostic Accuracy and Yield
Diagnostic Utility of FNAB
Limitations of the FNAB Method of Study
References
See Also
Fine Needle Aspiration Cytology
Firestorm Pattern
Definition
Characteristics
Contrast with Other Forms of Gene Amplification
Mechanism of Firestorm Formation
Relation of Patterns to Clinical Outcome
Cross-References
References
See Also
First-Echelon Node
First-Tier Node
FISH
FIT
FKHL-16
FL
FLAME-1
Flavonoids
Definition
Cross-References
FLICE
FLICE-Inhibitory Protein
Synonyms
Definition
Characteristics
Function
Modulation of c-FLIP Level
Role in Carcinogenesis
Implications in Cancer Therapy
References
FLJ10796
FLJ33629
Flow Cytometry
Definition
Characteristics
The Technology
Applications
Immunophenotyping (IP)
Transplant Support
DNA Ploidy and S-Phase Fraction
Determining Treatment Efficacy
Newer and Future Applications
References
FLT3
Synonyms
Definition
Characteristics
Gene
Ligand
FLT3 Mutations in Leukemia
Signaling Through FLT3
FLT3 Inhibitors
References
Fludarabine
Synonyms
Definition
Characteristics
Clinical and Cellular Pharmacology
Mechanisms of Anticancer Activity
Clinical Applications
Cross-References
References
See Also
Fludarabine Phosphate
Fluid Phase Endocytosis
Fluorescence Diagnostics
Synonyms
Definition
Characteristics
Application
Detection of Early Lung Cancer by Fluorescence Bronchoscopy
Urinary Bladder
Other Organ Sites
References
Fluorescence in Situ Hybridization
Synonyms
Definition
Cross-References
See Also
Fluorescence Resonance Energy Transfer
Fluoro-Sorafenib
5-Fluorouracil
Fluorouracil
Synonyms
Definition
Characteristics
Mechanisms of Action
Determinants of Cell Sensitivity to 5-FU
Toxic Side Effects
Capecitabine, an Orally Administered Prodrug of Fluorouracil
References
Fluoxetine
Synonyms
Definition
Characteristics
Multiple Drug Resistance
MDR Reversal by Chemosensitization
Fluoxetine as a Multi-pump Chemosensitizer
In Vitro Studies
In Vivo Studies
Cross-References
References
See Also
Fms-Like Tyrosine-Kinase 3
FNA
FNAC
FOBT
Focal Adhesion Kinase
Keywords
Definition
Characteristics
FAK Phosphorylation and Regulation
Focal Contact
Regulation of Focal Contacts
Signaling Pathways in Cancer
Cell Migration
Invasion
Cell Survival
Growth and Proliferation
Tumor Progression
Clinical Impact
FAK Overexpression
Tumor Biomarker
Cancer Therapy
Cross-References
References
See Also
Foeto-Protein
Folate Hydrolase
Follicular Adenoma, Oncocytic Variant
Follicular Carcinoma, Oncocytic Variant
Follicular Lymphoma
Definition
Cross-References
See Also
Follicular Thyroid Adenomas
Follicular Thyroid Carcinoma
Follicular Thyroid Tumors
Synonyms
Definition
Characteristics
Classification
Etiology
Diagnosis
Treatment
Genetics
References
Follistatin
Synonyms
Definition
Characteristics
Follistatin Structure
Function and Expression
Follistatin-Like Proteins
Role of Follistatin and Follistatin-Like Proteins in Malignancy
Conclusion
Cross-References
References
Follistatin-Like 1, FSTL1
Follistatin-Like 3, FSTL3
Follistatin-Like 4, FSTL4
Follistatin-Like 5, FSTL5
Follistatin-Related Gene, FLRG
Follistatin-Related Protein, FRP
Food
Food for Specified Health Use
Food-Borne Carcinogens
Synonyms
Definition
Characteristics
N-Nitroso Compounds
Heterocyclic Aromatic Amines
Polycyclic Aromatic Hydrocarbons
Acrylamide
Other Carcinogens Present in Food
Cross-References
References
See Also
Foreign Substances
Forkhead Box M1
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Formation of New Blood Vessels
Fossil Tree
Found in Inflammatory Zone 3 (FIZZ3)
FPC
Fragile Histidine Triad
Synonyms
Definition
Characteristics
The FHIT Gene Product
Clinical Relevance
Tumor Suppressor
Genome Caretaker
Cross-References
References
See Also
Fragile Sites
Definition
Characteristics
Categories of Fragile Site
Rare Fragile Sites
Common Fragile Sites
Molecular Basis of Fragile Site Expression
Rare Fragile Sites
Common Fragile Sites
Cellular Regulation of Common Fragile Site Instability
Clinical Relevance
Rare Fragile Sites
Common Fragile Sites
Cross-References
References
See Also
Free Radical
Free Radicals
Freeze Surgery
FS
FST
5-FU
Fucosylation
Definition
Characteristics
Finding of Fucosylation-Deficient Cancer Cell Lines
Clinical Significance of GMDS Mutation
Cross-References
References
See Also
α1-6 Fucosyltransferase
Synonyms
Definition
Cross-References
Fucoxanthin
Definition
Characteristics
Effect on Cancer Cell Growth
Apoptosis
Mechanism
Combination with Troglitazone
Cross-References
References
See Also
Fulvestrant
Synonyms
Definition
Characteristics
Mode of Action
Current Utilization of Fulvestrant
Administration and Tolerability
Future Uses of Fulvestrant
Summary
Cross-References
References
See Also
Fumarase
Fumarate Hydratase
Synonyms
Definition
Characteristics
Clinical Features of HLRCC/MCUL
Mutations in the FH Gene
Cross-References
References
Functional Foods
Functional Vascular Stabilization
Funnel Factors
Definition
Characteristics
Background of Molecular Human Carcinogenesis
Cell Signaling in Human Tumors
Searching for Funnel Factors: eIF4E/4E-BP1
Other Essential Oncogenic Funnel Factors
Cross-References
References
See Also
Furin
Synonyms
Definition
Characteristics
References
Fusin
Fusion Genes
Synonyms
Definition
Characteristics
Formation of Fusion Genes
Clinical Relevance
Cross-References
References
Fusion Oncogenes
FUT8
G
G Antigen
G Proteins
Synonyms
Definition
Characteristics
Molecular and Cellular Regulation
Clinical Relevance
Cross-References
References
G2/M Transition
Definition
Characteristics
Structural Changes at the G2/M Transition
Triggering G2/M Transition
Cross-References
References
See Also
GA733-2
GAGE Proteins
Synonyms
Definition
Characteristics
References
Gain-of-Function p53
Definition
Characteristics
Molecular Mechanisms
Clinical Aspects
References
GAK
Gallbladder Cancer
Definition
Characteristics
Descriptive Epidemiology
Risk Factors
Conclusions and Perspectives
Cross-References
References
See Also
Gamma Ray-Induced Cancer
Gangliosides
Synonyms
Definition
Characteristics
Structure
Cellular Ganglioside Expression in Cancer
Ganglioside Shedding in Cancer
Functional Properties
Immunosuppression
Growth Factor-Induced Cell Signaling
Cell Proliferation and Angiogenesis
Conclusion
Cross-References
References
See Also
Gangrene
Gankyrin
Synonyms
Definition
Characteristics
pRB and Gankyrin
p53 and Gankyrin
Miscellaneous
Clinical Aspects
References
Gap Junctions
Definition
Characteristics
Clinical Relevance
Cross-References
References
Gardner Syndrome
Gastric Cancer
Synonyms
Definition
Characteristics
Epidemiology
Etiology
Molecular Biology of Gastric Cancer
Familial Clustering of Gastric Cancer
Hereditary Diffuse Gastric Cancer
Diagnosis and Detection
Management
References
Gastric Cancer Therapy
Definition
Characteristics
Therapy of Early Stage Gastric Cancer
Therapy of Advanced Gastric Cancer (AGC)
Antiangiogenic Therapy
Targeting Generic and Molecular Aberrations in AGC
HER2
MET
FGFR2
Novel Targets
References
Gastric Carcinoma
Gastrin
Definition
Characteristics
Processing and Distribution
Release
Receptors and Signalling Pathways
Biological Functions
Role in Disease
Cross-References
References
Gastrinoma
Synonyms
Definition
Characteristics
Diagnosis
Therapy
Genetics
Cross-References
References
See Also
Gastrin-Releasing Peptide
Synonyms
Definition
Characteristics
GRP Receptors
Physiological Functions of GRP
GRP and Cancer
GRP and Lung Carcinoma
GRP and Prostate Carcinoma
GRP and Breast Cancer
GRP and Gastric Carcinoma
GRP and Pancreatic Carcinoma
GRP and Colorectal Carcinoma
GRP and Gastrointestinal Carcinoid Tumors
Clinical Significance
Cross-References
References
See Also
Gastrointestinal Hormones
Gastrointestinal Stromal Tumor
Synonyms
Definition
Characteristics
Epidemiology and Clinical-Pathological Features of GIST
Diagnosis
Oncogenic Signaling Pathways in GIST
Cytogenetics and Molecular Cytogenetic Alterations
Therapy
References
GBM Therapy
GBP28
GDEPT
GDF15
Gefitinib
Definition
Characteristics
Gefitinib in Cancer Therapy
Gefitinib Sensitivity
Mechanism of Action
Cancer Resistance to Gefitinib
Conquering Cancer Resistance
List of Strategies to Conquer Gefitinib Resistance
Cross-References
References
See Also
Gefitinib (Iressa)
Gelatin-Binding Protein 28
Geldanamycin
Gelsolin
Definition
Characteristics
Gelsolin and Cancer
Gelsolin Considered as a Tumor Suppressor
Cross-References
References
See Also
Gemcitabine
Synonyms
Definition
Characteristics
Mechanism of Action and Resistance
Clinical Profile
Toxicity
Indications
Clinical Pharmacology
Future Perspective
Cross-References
References
GEMZAR
Gene Mutations
Gene Therapy
Definition
Characteristics
Transfer of Genes
Clinical Relevance
References
Gene-Directed Enzyme Prodrug Therapy
Genetic Association Study
Genetic Immunization
Genetic Instability
Genetic Polymorphisms
Genetic Predisposition
Genetic Toxicology
Synonyms
Definition
Characteristics
Genetic Toxicology
Chemical, Viral, and Radiation Carcinogens
Chemical Carcinogenesis
Chemically Induced Morphological and Neoplastic Transformation and the Molecular Biology of Carcinogenesis/Cell Transformation and Human Cancer
Cross-References
References
See Also
Genetic Variations
Genetically Engineered Mice
Genistein
Synonyms
Definition
Characteristics
Soy and Cancer
Cancer Prevention by Genistein
Mechanisms of Action of Genistein
Bioavailability and Safety
Other Indications
References
Genomic Imbalance
Definition
Characteristics
Genomic Imbalance Concept
Cancer as a Genetic Disease
Cause of Genomic Imbalance in Cancer
Cross-References
References
See Also
Genomic Imprinting
Definition
Characteristics
Imprinted Genes and Their Regulation
Imprinting and DNA Methylation
Loss of Imprinting (LOI) in Cancer
Cross-References
References
See Also
Genomic Instability
Synonyms
Definition
Cross-References
See Also
Germinoma
Definition
Characteristics
Diagnosis
Treatment
Surgery
Radiotherapy
Chemotherapy
Genetics
Cross-References
References
See Also
Giant Cell Tumor
Definition
Characteristics
Epidemiology and Pathophysiology of Giant Cell Tumor
Tumorigenesis of GCT
Clinical Management and Future Direction
References
Ginkgo biloba
Synonyms
Definition
Characteristics
G. biloba Seeds
G. biloba Extract
Cross-References
References
Ginkgo Tree
GIST
Gleason Grading
Definition
Characteristics
Clinical Aspects
References
Gleevec
GLI Proteins
Synonyms
Definition
Characteristics
Regulation of GLI Activity in Response to HH Signaling
GLI Proteins in Cancer
Cross-References
References
See Also
Glial/Mesenchymal Extracellular Matrix Protein
GLI-Kruppel Family Member
Glioblastoma
Glioblastoma Multiforme Therapy
Glioblastoma Therapy
Synonyms
Definition
Characteristics
Current Treatment Regimen
Novel Therapies for GBM
Antiangiogenesis Therapy
Targeted Immunotoxins
Tyrosine Kinase Inhibitors
Proteasome and HDAC Inhibitors
Novel Drug Delivery Strategies for GBM
Gliadel Wafer
Brachytherapy
Stem Cell Therapeutics
Viral Therapeutics
Immunotherapy
Future Directions
References
Glioma Retinae
Glioma-Associated Oncogene Homolog 1
Gliomas
Glivic
Glucose Metabolism
Definition
Characteristics
Glucose Transport
Fate of Glucose upon Entry into the Cell
Glycolysis
Citric Acid Cycle
Pentose Phosphate Pathway
Glycogenesis (Glycogen Synthesis)
Energy Yield and Advantage of the Metabolism of Glucose in Cancer Cells
Summary
Cross-References
References
See Also
Glutamate Carboxypeptidase II
Glutathione Conjugate Transporter RLIP76
Synonyms
Definition
Characteristics
Discovery of RLIP76
Clinical Significance
Drug Resistance
Heat Shock and Oxidative Stress
Radiation Toxicity
References
Glutathione S-Transferase
Synonyms
Definition
Characteristics
Catalytic Function
Signaling Regulation
Cancer Related
References
Glutathione Transferases
Glycobiology and Cancer
Definition
Characteristics
Glycan Structural Diversity Is Theoretically Immense but Genetically Restricted
Glycans Evoke Antibody Responses
Glycans Are Clinically Useful as Cancer Markers
TACAs Are Recognized by Lectins on Antigen-Presenting Dendritic Cells
Human Retrovirus HTLV-1 (Human T-Lymphotropic Virus) Induces Specific Glycan Biosynthesis
Selectin-Glycan Interactions Initiate Adhesion of Malignant Cells to Vascular Endothelium
Drugs May Inhibit Metastases by Blocking Selectin-Glycan Interactions
Concluding Sweet Remarks
References
Glycolysis
Glycolytic Inhibitors
Glycoprotein-340 (gp-340 Human)
Glycosphingolipids (Sialic Acid Containing)
Glycosylation
Synonyms
Definition
Characteristics
Glycosylation Alterations and Functions in Cancer
Glycans Used as Serological Markers in Cancers
Therapeutic Applications of Glycans in Cancers
References
GM
GMEM
GnRH, Gonadotropin-Releasing Hormone
Gonadal Neoplasms
Gonadotropin-Releasing Hormone
Synonyms
Definition
Characteristics
References
Good Laboratory Practices
Definition
Characteristics
Principles
References
Good Manufacturing Practices
Definition
Characteristics
Quality Assurance
Documentation and SOPs
FDA Quality Initiatives
Pharmaceutical Quality for the Twenty-First Century
Process Analytical Technology (PAT)
ICH Q8 Pharmaceutical Development
ICH Q9 Quality Risk Management
Quality System Approach to Pharmaceutical Good Manufacturing Practices
ICH Q10 Pharmaceutical Quality Systems
References
Gordon-Kosswig Melanoma System
Gorlin Syndrome
gp140 (Glycoprotein 140)
gp170
gp36
gp38
gp90
gp-Fy
G-Protein-Coupled Receptor 11
Grading of Tumors
Synonyms
Definition
Characteristics
Basic Principles
Methods of Grading
Future Perspectives
Cross-References
References
See Also
Graft Acceptance and Rejection
Definition
Characteristics
Cellular and Molecular Mechanisms
Clinical Relevance
Cross-References
References
Granulocyte Elastase
Granulocytopenia
Granulomatous Colitis
Granulosa Cell Tumors
Definition
Characteristics
Cross-References
References
See Also
Grape Seed Extract
Definition
Characteristics
Biological Activity
Health Beneficial Effects
Cardiovascular Diseases
Other Health Conditions
Anticancer and Cancer Preventive Efficacy
Clinical Studies
Conclusions
Cross-References
References
See Also
Graves Disease
Definition
Cross-References
Green Tea Cancer Prevention
Keywords
Definition
Characteristics
Green Tea Catechins
Green Tea Extract
Delayed Cancer Onset with Green Tea
Significance of Multiple Drinks per Day
Effective Cancer Preventive Amount per Day
Prevention of Cancer Recurrence
Green Tea as Synergist with Anticancer Drugs
Cross-References
References
Green Tea Polyphenol
GRIP1
Growth Differentiation Factor 15
Growth Factor Receptors
Growth Factor Toxin Fusions or Conjugates
Growth Factors
Growth Guidance Cues
Growth Hormone-Releasing Factor (GHRF)
Growth Hormone-Releasing Hormone
Synonyms
Definition
Characteristics
Pituitary-Type GHRH Receptor
GHRH Expression
GHRH and Cancer
Splice Variants of GHRH Receptor (SVs) and Cancer
GHRH Antagonists
GHRH Agonists
Conclusion
References
Growth of New Lymphatic Vessels
GS
GTPase
Synonyms
Definition
Characteristics
The Ras Family
The Rho Family
The Rab Family
The Arf Family
The Ran Family
Regulation of GTPases
Clinical Implications
Signaling Pathways
Cross-References
References
GTPases
GTP-Binding Proteins
Guanine-Rich Tandem DNA Repeats of Chromosomal Ends
Gut Microbiota
Definition
Characteristics
Dynamics of Gut Microbiota
Composition of Gut Microbiota
Tools for Evaluating Microbiota Diversity
Functions of Gut Microbiota
Immune Modulation
Obesity
Energy Uptake
Bile Acid Metabolism
Choline Metabolism
Protection Against Pathogenic Bacteria
Drug Metabolism
Alteration of Gut Microbiota as a Therapeutic Approach
Gut Microbiota in Colorectal Cancer
References
Gut Peptides
Synonyms
Definition
Characteristics
Overview
Gastrin
Gastrin-Releasing Peptide
Neurotensin
Somatostatin
Cross-References
References
Gynecomastia
Synonyms
Definition
Characteristics
References
Gynecomazia
H
[HOC6H3(OCH3)CH:CHCO]2CH2
[1-Hydroxy-2-(imidazo [1,2-a] pyridin-3-yl)ethylidene]-bisphosphonate
1,7-bis [4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione
(2-hydroxy-3-phosphonooxy-propyl) (Z)-octadec-9-enoate
H Antigens
gammaH2AX
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
References
H3 (Rhesus Monkey)
H731 (Human PDCD4 Variant 2, Cancer Related)
HAAs
HACBP
Hairy Cell Leukemia
Synonyms
Definition
See Also
Hamartin
Synonyms
Definition
Characteristics
References
HAMSV
Haploinsufficiency
Definition
Characteristics
Mechanism of Disease Initiation Involving Haploinsufficiency
Cross-References
References
Ha-ras
HARP
Harvey Murine Sarcoma Virus Oncogene
HATs
HAUSP De-ubiquitinase
Hay Fever
HBBM
HB-EGF
Definition
Characteristics
Clinical Relevance
References
HB-GAM
HBGF-8
HBGFs
HBNF
HBNPF
HBV
H-CAM
HCC
HCL
HCV
HDAC Inhibitors
Definition
Cross-References
See Also
HEA125
Head and Neck Cancer
Definition
Characteristics
Epidemiology
Anatomic and Histopathologic Classification
Risk factors
Molecular Pathogenesis
Workup
Staging
Treatment of HNSCC
Prognosis and Survival
Acknowledgments
Cross-References
References
Heat-Shock Protein 90
Hedgehog Signaling
Definition
Characteristics
Hedgehog Signaling and Cancer
Hedgehog, Stem Cell Renewal, and Cancer
Targeting Hedgehog Signaling for Therapeutics
Cross-References
References
See Also
Helicobacter Pylori in the Pathogenesis of Gastric Cancer
Definition
Epidemiology and the Pathogenic Role of H. pylori
Characteristics
Mechanisms of H. pylori-Induced Gastric Carcinogenesis
H. pylori-Induced Inflammation in the Gastric Epithelium
H. pylori-Induced Oxidative Stress and DNA Damage in the Gastric Epithelium
H. pylori-Induced Methylation and Loss of Function of Key Genes in Gastric Epithelium
H. pylori Infection Induces Aberrant Activities of the Signaling Pathways in the Gastric Epithelium
Detection and Treatment for H. pylori Infection
References
Hematological Malignancies, Leukemias, and Lymphomas
Definition
Characteristics
The Hematopoietic System
The WHO Classification
Major Myeloid Subclasses Within the WHO Classification
Major Lymphoid Subclasses Within the WHO Classification
Diagnosis
Treatment
Cross-References
References
See Also
Hemicentin, him4, FIBL6, HMCN1, FBLN-6
Henle-Koch Postulates
Hensin (Rabbit)
Heparanase
Definition
Characteristics
Heparan Sulfate Proteoglycans (HSPGs)
Molecular Properties
Preferential Expression in Human Tumors
Involvement in Tumor Metastasis
Involvement in Tumor Angiogenesis
Clinical Relevance
References
Heparanase Inhibitors
Definition
Characteristics
Inhibitors Targeting the Heparanase Substrate
Heparin, Chemically Modified Derivative and Nonanticoagulant Heparin
Sulfated Phosphomannopentaose
Oligomannurarate Sulfate (JG3)
Suramin
Noncarbohydrate Substrate Mimetic Polymers
Inhibitors Targeting Heparanase
Small Molecules
Peptides Competing with the Heparin/HS-Binding Domains of Heparanase
Antiheparanase Antibodies
Conclusions and Perspectives
References
Heparin-Affin Regulatory Peptide
Heparin-Binding Brain Mitogen
Heparin-Binding Growth Factor 8
Heparin-Binding Growth Factors
Heparin-Binding Growth-Associated Molecule
Heparin-Binding Neurite-Promoting Factor
Heparin-Binding Neurotropic Factor
Hepatic Carcinoma
Hepatic Epithelioid Hemangioendothelioma
Synonyms
Definition
Characteristics
Etiology
Diagnosis
Therapy
Clinical Outcome
Cross-References
References
See Also
Hepatic Ethanol Metabolism
Definition
Characteristics
References
Hepatic-Fixed Macrophages
Hepatitis B Virus
Synonyms
Definition
Cross-References
Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma
Keywords
Definition
Characteristics
References
Hepatitis C Virus
Synonyms
Definition
Characteristics
Untranslated Regions
Structural Proteins
Nonstructural Proteins
Cellular and Molecular Regulation
Clinical Relevance
Cross-References
References
See Also
Hepatoblastoma
Definition
Characteristics
Pathological Classification
Staging
Therapy and Outcome
Predictive Factors
Genetic and Molecular Characteristics
Cellular Characteristics
References
Hepatocarcinoma
Hepatocellular Carcinoma
Synonyms
Definition
Characteristics
Epidemiology
Risk Factors
Diagnosis
Prognosis
Prognostic Staging System
Treatment
Prevention
Cross-References
References
See Also
Hepatocellular Carcinoma Molecular Biology
Definition
Characteristics
Epidemiology
Etiology
Role of Viral Factors
Direct Mutagenic Role
Oncogenic Potential of the HBx Transactivator
Immunopathogenesis
Genetic and Epigenetic Alterations
Oncogenic Signaling Pathways
Wnt/beta-Catenin Signaling
Cell Cycle Control Pathways
PI3K/Akt/mTOR Pathway
Ras/Raf/MAPK Pathway
JAK/STAT Pathway
Conclusions
Cross-References
See Also
Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention
Synonyms
Definition
Characteristics
Etiology
Oncogenic and Environmental Factors
High-Risk Nonviral Liver Diseases
Viral Hepatitis-Induced HCC
Molecular Aspects of Hepatocarcinogenesis
Diagnosis
Treatment
Prognosis and Prevention
References
Hepatocyte Growth Factor
Hepatocyte Growth Factor-Like Protein
Hepatocyte-Stimulating Factor
Hepatocyte-Stimulating Factor-III
Hepatoma
HER1
HER-2/neu
Synonyms
Definition
Characteristics
Cross-References
References
Herceptin
Synonyms
Definition
Characteristics
Clinical Activity
Mechanisms of Activity
Molecular Level
Cellular Level
Mechanisms of Resistance
Insulin-like Growth Factor-I Signaling
Akt Signaling
Extracellular Domain and Truncated Forms of HER-2
New Directions
Cross-References
References
See Also
Hereditary Hamartosis Peutz-Jeghers
Hereditary Nonpolyposis Colorectal Cancer
Herpesvirus-Associated Ubiquitin-Specific Protease De-ubiquitinase
Synonyms
Definition
Characteristics
p53-Mdm2 Pathway
Implications in Cancer Therapy
References
Heterochromatin
Definition
Characteristics
Constitutive and Facultative Heterochromatin
Properties of Heterochromatin
Components Required for Heterochromatinization
Functions of Heterochromatin
Cross-References
References
Heterocyclic Amines
Synonyms
Definition
Characteristics
Classes of HAAs
Metabolism of HAAs
Health Concerns of Food-Derived HAAs - Mutagenicity and Carcinogenicity
Modulation of HCA Formation and HCA-Mediated Damage
Conclusions
Cross-References
References
Heterocyclic Aromatic Amines
Heterologous Growth Control
Heterotrimeric GTP-Binding Proteins
Heterotrimeric Guanine Nucleotide-Binding Proteins
Hexabrachion
1,2,6,7,8,9-hexahydro-1,6,6-trimethyl[1,2-b]furan-10,11-dione
(R)-1,2,6,7,8,9-hexahydro-1,6,6-trimethyl-phenanthro(1,2-b)furan-10,11-dione
1,2,6,7,8,9-hexahydro-1,6,6-trimethyl-(R)-phenanthro(1,2-b)furan-10,11-dione
Hexavalent Chromium
Synonyms
Definition
Characteristics
Uses and Sources
Epidemiology
Animal Studies
Cell Culture Studies
Role of Valence State, Uptake, and Metabolism
Role of Solubility
Mechanism of Carcinogenicity
Cross-References
References
Hexavalent Chromium-Induced Carcinogenesis
Hexokinase 2
Synonyms
Definition
Characteristics
Tissue Expression of Hexokinase II
Functional Properties of Hexokinase II
Regulation of Hexokinase II
Clinical Perspectives and Therapeutic Implications
Conclusions
Cross-References
References
HFH-11B
HGF
HGFL
HHV4
hIAP3
hIAP-3
HIES
HIF-1
High Affinity Ca-Binding Protein
High-Dose (Myeloablative) Chemotherapy
High-Flow Microinfusion
High-Frequency Ultrasound Imaging
High-Grade/Undifferentiated MEC
High-Throughput Screening
Hilar Cholangiocarcinoma
HILDA
Histiocytosis X
Histo-Blood Group Lewis Antigens
Histocompatibility
Definition
Histocompatibility Antigens
Synonyms
Definition
Cross-References
Histologic Grading
Histone Acetyltransferases
Synonyms
Definition
Characteristics
HAT Families
HATs Functions and Complexes
Mutations of HATs in Cancer
Histone Acetyltransferases Inhibitors
Cross-References
References
Histone H2AX Phosphorylated at Serine 139
Histone Modification
Definition
Characteristics
Histones and Chromatin
Histone Modifications
Histone Modifications and Cancer Prognosis
Epigenetic Therapy: HDAC Inhibitors
Cross-References
References
HIV/AIDS-Associated Cancers
HIV-Associated Cancers
HIV-Associated Malignancies
HIV-Related Cancers
HKII
HLA Class I
Definition
Characteristics
Bioactivity
Altered HLA Class I Phenotypes in Tumors
Functional Significance of HLA Class I Loss: Implications for Immune Evasion
Cross-References
References
hMAWD: Human MAPK Activator with WD Repeats
HNPCC
Hodgkin and Non-Hodgkin Lymphomas
Hodgkin and Reed/Sternberg Cell
Definition
Characteristics
Associated Pathologies
Cellular Origin
Immunophenotype
Cell Differentiation and Function
Transforming Mechanisms of HRS Cells
Cross-References
References
See Also
Hodgkin Disease
Definition
Characteristics
Cross-References
References
Hodgkin Lymphoma, Clinical Oncology
Synonyms
Definition
Characteristics
Pathology
Basic
Clinical
Cross-References
References
See Also
Hollow Fiber Assay
Definition
Characteristics
Methodology
References
Homeobox Genes
Definition
Characteristics
Hox Genes and Leukemia
Homeobox Genes and Solid Tumors
References
Homing Peptides and Vascular Zip Codes
Definition
Characteristics
Biological Significance
Clinical Significance
Cross-References
References
Homing Receptor
Homo Sapiens Mitotic Arrest Deficient-Like 1 Protein
Homo Sapiens Nuclear Protein 1
Homogenous Assays
Homogenous Time-Resolved Fluorescence
Homologous Recombination Repair
Definition
Characteristics
Cellular Regulation
Clinical Relevance
References
Homologue of Synaptopodin
HOP-1
Hormonal Carcinogenesis
Synonyms
Definition
Characteristics
Mechanisms
Cross-References
References
Hormonal Therapy
Synonyms
Definition
Breast Cancer
Ovarian Cancer
Cross-References
Hormone Carcinogenesis
Hormone Replacement Therapy
Synonyms
Definition
Cross-References
See Also
Hormone Therapy
Hormone-Induced Cancers
Hormone-Refractory Prostate Cancer
Synonyms
Definition
Cross-References
Hormone-Related Cancers
Hormones
Definition
Characteristics
Mechanisms
The Receptor Concept
Endogenous Metabolic Activation of Steroids
Epidemiology and Animal Studies
Steroid Hormones
Prolactin
Insulin
Gastrointestinal Hormones
Thyroid Hormones
Hormonal Management of Cancer
Cross-References
References
See Also
Hornstein-Knickenberg Syndrome
HpaII Tiny Fragments Islands
4-HPR
hPTTG1
HRAS
Synonyms
Definition
Characteristics
Localization (in Homo sapiens)
Pseudogenes
HRAS Gene Structure
HRAS Regulatory Elements
p21 Protein Structure and Subcellular Localization
Biochemical Properties of p21 Protein
Functional Properties of p21 Protein
Tissue Expression of p21 Protein
HRAS and Cancer
HRAS and Familial Syndromes
HRAS and Other Pathological Conditions
References
HRAS1
HRAS1DX
HRPC
HRT
HSD18
HSF
HSF-III
hSNF5
HSNF5/INI1/SMARCB1 Tumor Suppressor Gene
Synonyms
Definition
Characteristics
The Chromatin Remodeling and the SWI/SNF Complex
hSNF5/INI1 Gene and Protein Structures
Biological Roles of hSNF5/INI1
Genetic Alterations of hSNF5/INI1 in Human Malignancies
Pathological and Clinical Aspects of hSNF5/INI-Deficient Tumors
Conclusion
References
Hsp90
Synonyms
Definition
Characteristics
Mechanism
Clinical Aspects
As a Single Modality
In Combination with Radiotherapy
Cross-References
References
See Also
HSV-TK-/Ganciclovir-Mediated Toxicity
Synonyms
Definition
Characteristics
Toxicity to HSV-TK-Expressing Cells
Toxicity to Non-HSV-TK-Expressing Cells (Bystander Effect)
Characteristics In Vivo
Experimental Tumors in Animals
Mechanism of the Bystander Effect In Vivo
Clinical Relevance
Improving Cancer Therapy with HSV-TK/GCV
Cross-References
References
See Also
HTF Islands
Hu Antigen R
Human
Human ESP1-Associated Protein 1
Human Herpesvirus 4
Human Herpesvirus 6
Definition
Characteristics
Clinical Aspects
References
Human Kallikrein 3
Human Non-Sucrose Fermenting 5
Human Papillomaviruses in Head and Neck Cancer
Definition
Characteristics
Epidemiology
The Role of p16
References
197/15a (Human PDCD4 Variant 1)
Human Pituitary Tumor-Transforming Gene 1
Human Stem Cell Kinase-1 (STK1)
Human T-Lymphotropic Virus
Synonyms
Definition
Characteristics
Leukemia
Clinical Presentation
Pathology
Neurologic Disease
Clinical Presentation
Pathology
Cross-References
References
Human T-lymphotropic Virus (HTLV)
HUMARA
Huntingtin Interacting Protein 1
Definition
Characteristics
HIP1
HIP1´s Original Link to Cancer
How Might HIP1 and HIP1r Use Clathrin-Mediated Trafficking to Promote Cancer?
Targeted Mutations of Murine Hip1 and Hip1r
HIP1 and HIP1r Have Restricted Patterns of Expression and Are Not Housekeeping Genes
HIP1 and Solid Tumors
Humoral Response to HIP1 Over-Expression
Over-Expression of HIP1 Is Sufficient to Transform Fibroblasts
Future
Significance
Cross-References
References
See Also
HuR
Synonyms
Definition
Characteristics
General HuR Biology
The RNA-Binding Protein HuR (ELAV1)
HuR Cancer-Associated Targets (Pro-survival Network)
HuR-Mediated Response to Stress and Cancer Therapy
HuR as an Oncogene in Cancer
Conclusions
References
Hurthle Cell Adenoma and Carcinoma
Synonyms
Definition
Characteristics
Current and Past Controversies
References
Hutchinson-Weber-Peutz Syndrome
HXK2
Hyaluronan Receptor
Hyaluronan Synthases
Synonyms
Definition
Characteristics
Hyaluronan Synthases and Hyaluronan
Hyaluronan Synthases and Hyaluronan in Cancer
Proliferation and Tumor Growth
Invasion and Metastasis
Multidrug Resistance
Cancer Stem Cells
Cross-References
References
See Also
Hyaluronic Acid (HA)
Hyaluronic Acid Synthases (HASs)
Hyaluronidase
Definition
Characteristics
HAase Expression as a Cancer Biomarker
Regulation of HAase Activity
HAase Functions in Cancer
HAase-Induced Signaling in Cancer Cells
HAase for Cancer Therapy
Summary
Cross-References
References
See Also
Hybrid Genes
Hybrid Positron Emission Tomography/Computed Tomography
Hydrogen Dioxide
Hydrogen Peroxide
Synonyms
Definition
Characteristics
Carcinogenic Effects
Clinical Relevance
Cross-References
References
See Also
14-Hydroxydaunorubicin
2-Hydroxyoleic Acid
Synonyms
Definition
Characteristics
Structure
Mechanism of Action
Clinical Trials
Cross-References
References
7-Hydroxystaurosporine
Hydroxysteroid Dehydrogenases
Hypercalcemic Type
Hypericin
Synonyms
Definition
Characteristics
Physical Properties
Clinical Applications
PDD of Cancers
PDT of Cancers
Preclinical Investigations
Cross-References
References
See Also
Hypericum Extract
Hypericum Red
Hyperplasia
Definition
Cross-References
Hypersensitivity
Hyperthermia
Definition
Characteristics
Cytotoxic Effects
Physiologic Effects
Immunological Effects
Hyperthermia Physics
Delivery Modalities
Thermometry
Thermal Dosimetry
Clinical Hyperthermia
Radiation Therapy and Hyperthermia
Chemotherapy and Hyperthermia
Clinical Trials
References
Hypertrophy of Male Breast
Hypomethylation of DNA
Synonyms
Definition
Characteristics
References
Hypoxia
Synonyms
Definition
Characteristics
Phenotypes of Hypoxic Cells
Gene Regulation in Hypoxic Cells: Implications for Tumorigenesis
Cross-References
References
Hypoxia Sensing
Hypoxia-Inducible Factor-1
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
References
I
IA4
IAP-3
IARC TP53 Database
Definition
Characteristics
TP53 Gene Mutations in Human Cancers
Database Structure and Content
Web Site and New Search Tools
Recommendation to Users
Cross-References
References
See Also
ICF Syndrome
Definition
Characteristics
Clinical and Cytological Features
Immunodeficiency
Centromeric Instability
Facial Anomalies
Other Clinicopathological Features
Causative Genes and Molecular Background
Type-1 ICF Syndrome (ICF1)
Type-2 ICF Syndrome (ICF2)
Type-3 ICF syndrome (ICF3)
Type-4 ICF syndrome (ICF4)
Others (Type X)
Clinical Management
Similarity Between ICF Syndrome and Human Cancers
Cross-References
References
ICL670A
Idiopathic Myelofibrosis
Idiotype Vaccination
Synonyms
Definition
Characteristics
Field of Application
Formulation
Production
Clinical Aspects
Open Questions
Cross-References
References
See Also
Idiotypic Vaccination
IDO
I-FLICE
iFOBT
IgE-Mediated Hypersensitivity
IHC
IL-6
ILP1
Imatinib
Synonyms
Definition
Characteristics
BCR-ABL1 and CML as a Target for Imatinib
Activity of Imatinib in CML
Mechanisms of Relapse/Resistance to Imatinib
Activity of Imatinib in Other Diseases
Conclusion
Cross-References
References
See Also
Imatinib (Gleevec)
Imatinib Mesylate
Imidazo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, 3, 4-dihydro-3-methyl-4-oxo-
Immediate Early Stress Response
Immune Escape
Definition
Characteristics
Host Ignorance
Tumor Cell Adaptation
References
Immune Responses to Autoantigens
Immune-Mediated Suppression
Immunochemical Fecal Occult Blood Test
Immunocytochemistry
Immunoediting
Definition
Characteristics
History
The Role of Interferons and Other Immune Components
The Role of Induced Immune Pressure
Evidence of Immunoediting in Human Cancer
Cross-References
References
See Also
Immunoglobulin Rearrangements
Immunohistochemistry
Synonyms
Definition
Characteristics
History
Techniques
Uses
Use in Research
Clinical Uses (Examples)
Diagnosis and Tumor Classification
Intermediate Filaments
Nuclear Transcription Factors
Cell Surface Membrane Markers
Carcinoma
Melanoma
Sarcoma
Lymphoma
Infectious Agents
Tumor Stage and Occult Metastases
Prognosis
Treatment Response: Companion Diagnostics
Targeted Therapy
Summary
Cross-References
References
See Also
Immunohistology
Immunoliposomes
Definition
Characteristics
Immunoliposome Types and Antibody Formats
Drugs and Targets
References
Immunological Fecal Occult Blood Test
Immunophenotypic Determinants
Immunoprevention
Synonyms
Definition
Characteristics
Immunoprevention of Viral Tumors
Immunoprevention of Tumors Unrelated to Infectious Agents
Immune Mechanisms of Cancer Prevention
Target Antigens of Cancer Immunoprevention
Clinical Developments
The Risks of Cancer Immunoprevention
Cross-References
References
See Also
Immunoprophylaxis of Cancer
Immunoproteasome
Immunoreceptor
Immunosuppression and Cancer
Synonyms
Definition
Characteristics
Communicative Barriers Inhibiting Anticancer Immunity
Checkpoint Inhibition of Anticancer Immune Responses
Cytotoxic T-Lymphocyte-Associated Antigen-4 (CTLA-4)
Programmed Death-1 (PD-1)
T-Cell Immunoglobulin and Mucin Domain-Containing Protein-3 (TIM-3)
Lymphocyte-Activating Gene 3 (LAG3)
Regulatory Immune Cells that Contribute to Tumor Escape
T Regulatory Cells
Myeloid-Derived Suppressor Cells (MDSCs)
Tumor-Associated Macrophages (TAMs)
Conclusion
Cross-References
References
See Also
Immunosurveillance of Tumors
Definition
Characteristics
Development of the Cancer Immunosurveillance Theory
Basic Mechanism
Elimination
Equilibrium
Escape
Clinical Impact
References
Immunotherapy
Synonyms
Definition
Characteristics
Nonspecific Immunotherapy
Specific Immunotherapy
Cross-References
References
See Also
Immunotoxins
Synonyms
Definition
Characteristics
Catalytic Activity
Developments
Cross-References
References
See Also
Imprinting
Definition
Characteristics
Cellular and Molecular Aspects
Clinical Relevance
References
In Situ Breast Cancer
In Situ Cancer
In Situ Carcinoma
In Utero Exposure to Carcinogenic Agents
In Vitro Bystander Effect
In Vitro Genetics
In Vivo Bystander Effect
Indirect Effect
Indirubin and Indirubin Derivatives
Synonyms
Definition
Characteristics
History
Mechanism
Structure/Activity
References
Individualized Treatment
Indoleamine 2,3-Dioxygenase
Synonyms
Definition
Characteristics
Enzymatic Properties, Tissue Distribution, and Regulation of Expression
IDO and Immune Suppression
IDO and Cancer
Clinical Relevance
IDO as a Prognostic Indicator for Human Cancer
Targeting IDO as a Therapeutic Strategy for Cancer Treatment
Implication of IDO for DC-Based Cancer Immunotherapy
References
Induced Pluripotent Stem Cells
Induction Chemotherapy
Synonyms
Definition
Characteristics
Solid Tumors
Leukemia
Cross-References
References
See Also
Infiltration
Inflammation
Definition
Characteristics
Cross-References
References
See Also
Inflammatory Bowel Disease (also includes Ulcerative Colitis)-Related Cancer
Inflammatory Bowel Disease-Associated Cancer
Definition
Characteristics
IBD-Associated CRC
Small Bowel Adenocarcinoma in Crohn Disease
Cancer Risk Associated to Immunosuppressive Therapy in IBD
Cross-References
References
Inflammatory Breast Cancer
Definition
Characteristics
Incidence of Inflammatory Breast Cancer (IBC)
Screening
Multimodality Treatment
Molecular Biology of IBC
Cross-References
References
See Also
Inflammatory Response and Immunity
Definition
Characteristics
An Overview of Inflammation
An Overview of Cancer
Interrelationship Between Inflammation and Cancer
Examples of Inflammation-Associated Cancers
Therapeutic Strategies Against Cancer
References
Inflammosome
Definition
List of Abbreviations
Inflammasome Assembly
Inflammasome Core Proteins and Their Activation Signals
Inflammatory Caspases and ASC Adaptor
Possible Mechanisms for NLRP3 Co-activation
Expression of Inflammasomes in Many Cell Types
Inflammasome-Associated Diseases
References
Infusion of T Cells
INHBA
INHBB
INHBC
INHBE
Inherited Human Polycystic Kidney Disease
Inhibin-beta Chain
Inhibitor of FLICE
INK4A
Definition
Keyword Definitions
Characteristics
Structure of INK4b/ARF/INK4a Locus and Molecular Functions of INK4s/ARF
Roles in Tumorigenesis
Roles in Senescence
Activators of the p15INK4b/ARF/p16INK4a
Ras-Raf-p38MAPK Pathway
E2F
c-Myc
Suppressors of the p15INK4b/ARF/p16INK4a
AML1/ETO
p53
Polycomb Proteins
Long Noncoding RNA in INK4b-ARF-INK4a Regulation
Cross-References
References
See Also
Innate Immunity
Synonyms
Definition
Characteristics
Barriers
Anatomical Barriers
Physiological and Chemical Barriers
Biological Barriers
Cellular Components of Innate Immunity
Phagocytes
Cell-Based Innate Immune Recognition
Cell-Based Innate Immune Effectors
Humoral Components of Innate Immunity
Humoral-Based Innate Immune Recognition
Humoral-Based Innate Immune Effectors
Conclusion
References
Inositol Lipids
Definition
Characteristics
Hydrolysis of PtdIns(4,5)P2 to Generate Second Messenger Molecules
Binding of PtdIns(4,5)P2 to Specific Proteins
Generation of PtdIns(3,4,5)P3 and Other 3-Phosphorylated Inositol Lipids and Their Binding to Specific Intracellular Targets
Clinical Relevance
Cross-References
References
See Also
Insulin Receptor
Definition
Characteristics
Regulation
Clinical Relevance
Cross-References
References
See Also
Insulin Signaling
Synonyms
Definition
Characteristics
Insulin Signaling and Cell Growth
Insulin Signaling and Glucose Metabolism
Insulin Signaling and Aging
References
Insulin-Like Growth Factor (IGF) Signaling
Insulin-Like Growth Factors
Definition
Characteristics
IGFs
IGF Receptors
IGFBPs
Mechanisms
Altered Expression of the IGF System Components in Tumor Cells
Syndrome of Hypoglycemia
Clinical Aspects
Cross-References
References
See Also
Insulinoma
Synonyms
Definition
Characteristics
Diagnosis
Differential Diagnosis
Treatment
Malignant Insulinoma
Cross-References
See Also
Integrated Positron Emission Tomography/Computed Tomography
Integrin Signaling
Definition
Characteristics
Signaling
Growth and Differentiation
Motility and Invasion
Survival
References
Integrin-Mediated Death
Interferon-Alpha
Synonyms
Definition
Characteristics
Malignant Melanoma
Pancreas Carcinoma
Renal Cell Carcinoma/Leukemia/Multiple Myeloma
Hairy Cell Leukemia
Direct Effects of IFN-α
Radio- and Chemosensitizing Effects
Antiangiogenic Effects
Immunogenic Effects
Immunomodulation
References
Interleukin-17
Synonyms
Definition
Characteristics
Binding of IL-17 to IL-17 Receptors
Gene Expression of IL-17
Role of IL-17 in Cancer
IL-17 and Its Pathway as a Therapeutic Target
Conclusion
References
Interleukin-4
Definition
Characteristics
Bioactivity
Clinical Relevance
IL-4 and Allergic and Inflammatory Diseases
Human Severe Combined Immunodeficiency (SCID)
References
Interleukin-6
Synonyms
Definition
Characteristics
Genes
Clinical Relevance
Cross-References
References
See Also
Intermediate Endpoint
Intermediate Junction
Intermediately or Poorly Differentiated Lymphocytic Lymphoma
Internalization
Interphase Cytogenetics
Definition
Characteristics
Cross-References
References
See Also
Intersectin
Synonyms
Definition
Characteristics
Structure and Function
ITSN and Cancer
Future Studies
Cross-References
References
See Also
Interstitial Microinfusion
Intestinal Alkaline Phosphatase
Definition
Characteristics
Structure
Gene Regulation
Biology
Clinical Implications
Cross-References
References
See Also
Intestinal Metaplasia
Definition
Characteristics
Gastric Intestinal Metaplasia
Epidemiology
Etiology Related to Gastric Cancer
Molecular Biology
Management
Barrett Esophagus
Epidemiology
Etiology Related to Esophageal Cancer
Molecular Biology
Management
Cross-References
References
Intestinal Organoids
Synonyms
Definition
Characteristics
Identification of Intestinal Stem Cell Markers
Intestinal Stem Cell Cultures
The Intestinal Stem Cell Niche
Mouse Colonic Organoids
Intestinal Cancer Organoids
Human Colonic Organoids
Research and Clinical Applications of Intestinal Organoids
Organoids from Other Organ Systems
Conclusion
Cross-References
References
See Also
Intestinal Sphere
Intracerebral Clysis
Intracerebral Microinfusion
Intraductal Carcinoma
Intraductal Papillary Mucinous Tumor
Intraocular Melanoma
Intrinsically Disordered Proteins
Intrinsically Unfolded Proteins
Intrinsically Unstructured Proteins
Synonyms
Definition
Characteristics
Identification of Unstructured Proteins
Unstructured Proteins and Cancer
Other Roles in Cancer Biology
Cross-References
References
See Also
3D In Vitro Culture
Invadosome
Synonyms
Definition
Characteristics
Invadosome Formation and Signaling
Biological Functions of Invadosomes
Conclusions
Cross-References
References
See Also
Invasion
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
Ion Channels
Definition
Characteristics
Structural Features and Biophysical Properties of Ion Channels Involved in Cancer
Ion Channels in Pathogenesis of Cancer
Potential Roles of Ion Channels as Biomarkers and Targets in Cancer
Conclusion and Perspectives
References
Ionizing Radiation (IR)-Induced Protein-8 (XIP-8)
Ionizing Radiation Therapy
Synonyms
Definition
Characteristics
References
Ionizing Radiation-Induced Cancer
IPL1
iPS Cells
Synonyms
Definition
Characteristics
Reprogramming and iPS Cells
iPS Cell Technology
Conclusions
Cross-References
References
See Also
iPSCs
IQ Motif-Containing GTPase-Activating Protein
IQGAP1 Protein
Synonyms
Definition
Characteristics
Functions
IQGAP1 and Cancer
Cross-References
References
Irinotecan
Synonyms
Definition
Characteristics
Mechanism of Action
Position of Irinotecan
Adverse Effects
Metabolism and Excretion
Dosage Individualization
Role of Genetic Screening
Cross-References
References
See Also
Iron-Regulated Necrosis
Islet Cell Tumor
Definition
Cross-References
See Also
Islet Cell Tumors
Islet Cell Tumors (Pancreatic NET)
Isoflavones
Definition
Characteristics
History
Examples
Metabolism
Mechanisms
Diseases
References
(-)-1-Isothiocyanato-4(R)-(methylsulfinyl) Butane
Ixabepilone
J
J1 220/200
JAB1
Synonyms
Definition
Characteristics
Structure
Function
Jab1/CSN5 Works as a Coactivator of Gene Transcription
Jab1/CSN5 as a Modulator of Intracellular Signaling
Jab1/CSN5 Regulation of Cell Cycle and Apoptosis
Jab1/CSN5 Act as an Inhibitor of DNA Checkpoint and Damage Repair
Jab1/CSN5 Regulation of Angiogenesis Pathway
Regulation of Jab1/CSN5 Activity
Jab1/CSN5 in Cancer
References
Japanese Silver Apricot
Jasmonates in Cancer Therapy
Definition
Characteristics
Anticancer Activities of Jasmonates In Vitro and In Vivo
The Mechanisms of Action of Jasmonates in Cancer Cells
Biochemical Outcomes Shared by Jasmonate-Treated Plant and Animal Cells
Conclusions
Cross-References
References
Jelly Belly
JFY1
JmjC Domain-Containing Protein 3
JMJD3
JNK Subfamily
Synonyms
Definition
Characteristics
Cross-References
References
See Also
JNK1
JNK2
JNK3
Jumonji Domain-Containing Protein 3
Juvenile Type
K
K Cells
K Lymphocyte
KABUK2
Kahler Disease
KAI1
Kallikrein-Related Peptidases
Kallikreins
Synonyms
Definition
Characteristics
The Kallikrein Genes
The Role of Kallikreins in Tumor Progression
Kallikreins as Diagnostic and Prognostic Markers
References
Kaplan-Meier Product Limit Estimator
Kaplan-Meier Survival Analysis
Synonyms
Definition
Characteristics
Censoring
Parametric, Semi-parametric, and Nonparametric Survival
Kaplan-Meier Product Limit Estimator
Comparing Survival Functions
Cross-References
References
See Also
Kaposi Sarcoma
Definition
Characteristics
Histology
Virus Involvement
Viral Strains
KSHV Latent Proteins
KSVH and Immunity
Anti-Herpesviral Drugs
Treatment for KS
Cross-References
References
See Also
KCNH1
KCS
KDM6
Synonyms
Definition
Characteristics
Cross-References
References
KDM6A
KDM6B
KDM6C
Kennedy Disease (KD)
Keratoconjunctivitis Sicca
Ket
Kew Tree
Ki-1
Ki1 Lymphoma
KIAA0413
Kidney Cancer Diagnosis
Kidney Cancer Therapy
Killer Cell Lectin-Like Receptor Subfamily K, Member 1
Killer Cells
Kinesin Spindle Protein
Kinesin-5
Kinesin-Like Protein KIF11
Kinetochore-Microtubule Interactions
Definition
Characteristics
Kinetochore-Microtubule Attachments and Cancer
Cross-References
References
Kinome_766
KIP1
KI-RAS
Kirsten Rat Sarcoma Viral Oncogene Homologue
KISS1
Definition
Characteristics
How Does KISS1 Suppress Metastasis?
Is KISS1 a Marker of Metastasis?
Clinical Relevance
Cross-References
References
Kit/SCF-R
Kit/Stem Cell Factor Receptor in Oncogenesis
Synonyms
Definition
Characteristics
Molecular and Cellular Regulation
Clinical Relevance
Mastocytosis/Mast Cell Leukemia
Gastrointestinal Tumors
Acute and Chronic Myeloid Leukemias
Germ Cell Tumors
Malignant Melanoma
Other Neoplastic/Malignant Lesions
Gene Transfer, Immunotherapy, and Vaccination
Cross-References
References
Klatskin Tumors
Synonyms
Definition
Characteristics
History and Classification
Epidemiology
Etiology and Risk Factors
Biology
Diagnosis
Treatment
Survival
Cross-References
References
KLK3
Klotho Tumor Suppressor
Definition
Characteristics
Klotho and Cancer
Klotho Expression in Cancer
Reduced Expression of Klotho in Cancer
Mechanisms Mediating Klotho Silencing
Klotho Expression as a Prognostic Factor
Klotho Blood Levels and Cancer
Klotho Functional Variants in Cancer
Cellular Targets of Klotho in Cancer Cells
IGF-1 Pathway
FGF Pathway
Wnt/beta-Catenin
TGF-beta1
Klotho: A Potent Inhibitor of Cancer Development and Progression
Klotho Structure-Function Relationships in Cancer
Cross-References
References
See Also
KLRK1
Knockout Mice
Knudson Hypothesis
Definition
Cross-References
See Also
Koch´s Postulates
Synonyms
Definition
Characteristics
References
kpm-Mammalian LATS2
KRAS
Synonyms
Definition
Characteristics
Structure of the KRAS Gene
KRAS Protein
KRAS in Cancer
Cancer Treatments Based on KRAS
Cross-References
References
KRAS in Colorectal Cancer Therapy
Definition
Cross-References
See Also
KRAS1
KRAS2
KRAS2A
KRAS2B
KRAS4A
KRAS4B, NS
KSA
KSP
KSP Mitotic Spindle Motor Protein
Synonyms
Definition
Characteristics
Regulation
Relevance to Cancer
Cross-References
References
Ku70-Binding Protein 1
Kulchitsky Cell Carcinoma
Kupffer Cells
Synonyms
Definition
Characteristics
Kupffer Cells and Liver Disease
Relationship of Kupffer Cells to Primary and Secondary Liver Cancers
Kupffer Cell Cancers
References
Kupffer Cells
Kv10.1
L
L Antigen Family Member 2
Lactoferricin Antiangiogenesis Inhibitor
Definition
Characteristics
Clinical Perspectives
Cross-References
References
LAGE-2
LAGE2B
LAK
LAMB
Laminin Signaling
Definition
Characteristics
Laminin Signaling
Laminin Receptors
Integrins
Nonintegrin Receptors
67kDa Laminin Receptor and Integrins
Dystroglycan
References
LANA-1 (Latency-Associated Nuclear Antigen-1)
Langerhans Cell Histiocytosis
Synonyms
Definition
Characteristics
Epidemiology
Etiology
Pathophysiology
Clinical Manifestations
Pathology and Diagnosis
Prognosis
Treatment
Late Sequelae
References
Langerhans Cells
Lapatinib (Tyverb)
Large Cell Immunoblastic
Large Cell Neuroendocrine Carcinoma
Definition
Cross-References
See Also
Large Granular Lymphocyte
Large Tumor Suppressor
Laryngeal Carcinoma
Definition
Characteristics
Genetic Changes in Laryngeal Carcinomas
Chromosome Abnormalities in Laryngeal Carcinomas
Fluorescence In Situ Hybridization (FISH)
Molecular Genetic Findings
References
Laser-Induced Fluorescence Diagnosis
Laser-Induced Hyperthermia
Latency-Associated Nuclear Antigen (LANA)
Synonyms
Definition
Characteristics
Structure
Functions
Episome Persistence
Transcription and Chromatin-Associated Factors Interaction
Apoptosis and Cell Cycle
LANA and RTA Regulation
Remarks and Overview
Cross-References
References
See Also
Lats in Organ Size Regulation and Cancer
Synonyms
Characteristics
The Lats/Hippo Pathway
Clinical Significance
Cross-References
References
See Also
LCH
LD
Lead Exposure
Definition
Characteristics
Sources of Lead Exposure
Biological Markers of Lead Exposure
Lead Carcinogenesis: Possible Etiologic Mechanisms
Lead Exposure and Cancer Epidemiology
Cross-References
References
See Also
Lead Optimization
Definition
Characteristics
Physicochemical Optimization
Pharmacokinetic Optimization
Toxicological Optimization
References
Leiomyoma
Leiomyomatous Hamartoma of the Kidney
Leiomyosarcoma
Definition
Cross-References
Lentigineses
Lentiginosis polyposa Peutz
Lentivector
Lentiviral Vectors
Synonyms
Definition
Characteristics
Composition of LVs
LV Infection of Various Mammalian Cell Types
Application of LV in Cancer Therapy
Tumor Vaccines
DC Vaccines
T Cell Vaccines
Conclusions
References
Leptomeningeal Disease
Leptomeningeal Dissemination
Synonyms
Definition
Characteristics
Symptoms
Diagnosis
Neuroimaging
Laboratory Findings
Treatment
References
Leptomeningeal Metastasis
Leptomeningeal Seeding
Lesion on Tongue
Leukemia Inhibitory Factor
Synonyms
Definition
Characteristics
LIF
LIF Receptor
LIF Overexpression and LIF Knockouts
Pleiotropic Effects of LIF
LIF and Oncogenesis
Clinical Aspects
References
Leukemia Initiating Cells
Leukemia Stem Cells
Synonyms
Definition
Characteristics
Surface Phenotype of LSCs
Altered Self-Renewal Capacity
Prolonged Survival and Evasion of Apoptosis
Clonal Evolution of LSCs
LSCs and Their Niche
Clinical Relevance and Molecular Targeting of LSCs
Cross-References
References
See Also
Leukemic Stem Cells
Leukocyte Elastase
Leukocyte Interferon
Leukocyte-Endothelial Cell Adhesion Molecule 2 (LECAM2)
Leukopenia
Leukotrienes
Definition
Characteristics
References
Leustatin
Lewis Antigens
Synonyms
Definition
Characteristics
Lewis Antigens in Cancer
References
Lexatumumab
Leydig Cell Tumor
Definition
See Also
LHRH, LH-Releasing Hormone
LIF
LIFD
Life Table Estimates
Li-Fraumeni Syndrome
Definition
Characteristics
Diagnostic Criteria
Genetics
TP53 Germline Mutations
Type and Origin of TP53 Mutations
Tumor Spectrum in TP53 Mutation Carriers
Age Distribution in Relation to Tumor Type
Gender Distribution of Patients with TP53 Germline Mutations
Functional Consequences and Phenotype of Germline Mutations
Mutation Detection Methods and Results Interpretation
Cross-References
References
LIM Domain Containing Preferred Translocation Partner in Lipoma
LINE-1 Elements
Definition
Characteristics
Structure
L1 Retrotransposition in Healthy Cells
Genomic Impact
Role in Human Cancer
Cross-References
References
See Also
Lingual Cancer
Linkage Disequilibrium
Synonyms
Definition
Characteristics
Introduction to Linkage Disequilibrium
Measures of Linkage Disequilibrium
Estimation of Linkage Disequilibrium
Haplotype Blocks
Linkage Disequilibrium Versus Linkage
Origins of Linkage Disequilibrium
Linkage Disequilibrium in Human Populations
Using Linkage Disequilibrium to Select Informative Markers
Utility of Linkage Disequilibrium to Genome-Wide Association Studies
Other Uses of Linkage Disequilibrium in Cancer Research
Cross-References
References
See Also
Linoleic Acid
Definition
Characteristics
Cross-References
References
See Also
Lip
Lipid Mediators
Synonyms
Definition
Characteristics
References
Lipid Peroxidation
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
Lipid Raft
Synonyms
Definition
Characteristics
Isolation Techniques
Serving as Signaling Platforms
In Regulation of Apoptosis
As Cancer Drug Targets
Conclusion
References
Lipid Replacement Therapy
Lipid Second Messengers
Lipid Therapy
Lipocalin
Definition
Characteristics
Cross-References
References
Lipoma-Preferred Partner
Synonyms
Definition
Characteristics
LPP and Tumors
Cell Biological Characteristics of the LPP Protein
LPP as a Transcriptional Coactivator
LPP as a Smooth Muscle Marker
References
Lipomas
Lipomatous Tumors
Liposarcomas
Liposomal Chemotherapy
Synonyms
Definition
Characteristics
The Magic Bullet Concept
Drug Delivery Technology
Sterically Stabilized (Stealth) Liposomes
Ligand-Targeted Stealth Liposomes
Future Perspectives for Liposomal Chemotherapy
References
5-Lipoxygenase
Synonyms
Definition
Characteristics
Pharmacology
Localization
5-LOX and Tumorigenesis
5-LOX-Mediated Mechanisms of Tumorigenesis
5-Lox and Hypoxia
Epigenetic Regulation of 5-LOX
Perspectives
Cross-References
References
Lipoxygenase
Live Cell Imaging
Definition
Characteristics
Microscopic Techniques
Methods in Live-Cell Imaging
Fluorescent Lifetime Imaging Microscopy
Fluorescence Resonance Energy Transfer
Fluorescence Recovery After Photobleaching
TIRF
STED
The Future Is Super-Resolution
Cross-References
References
Liver Cancer
Liver Cell Carcinoma
Liver Transglutaminase
LNA (Latency Nuclear Antigen)
5-LO
Lobular Involution of the Breast
Synonyms
Definition
Characteristics
Anatomy of the Human Breast
Involution
Age-Related Lobular Involution
Factors Affecting Involution
Measurement of Involution
Failure to Involute
Acknowledgments
Cross-References
References
Local Ablation Therapy
Locoregional Therapy
Synonyms
Definition
Characteristics
Transarterial Chemoembolization (TACE)
Percutaneous Ethanol Injection (PEI)
Percutaneous Acetic Acid Injection (PAI)
Radiofrequency Ablation (RFA)
Microwave Coagulation Therapy (MCT)
Comments
References
Log-Kill Hypothesis
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Low Grade Appendiceal Mucinous Neoplasm (LAMN)
Low Molecular Weight G Proteins
Low-Grade/Well-Differentiated MEC
5-LOX
LOX
18:1 LPA
LPA
Synonyms
Definition
Characteristics
Molecular Species of LPA
Synthesis and Metabolism of LPA
Wound Healing
Reproductive System
Nervous System
Hair Growth
Cancer
Atherosclerosis
Envenomation by Loxosceles Spiders
References
LPC
LPP
LRF, Luteinizing Hormone (LH)-Releasing Factor
LRP
LS-80558
LSCs
Luciferase Reporter Gene Assays
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Lugol-Unstained Lesion
Definition
Characteristics
Carcinogenesis (Precursor of ESCC)
Multiple LULs
References
Lunasin
Definition
Characteristics
Cross-References
References
See Also
Lung Cancer
Synonyms
Definition
Characteristics
Pathology
Etiology
Biology and Molecular Pathogenesis
Activation of Dominant Oncogenes
Inactivation of Tumor Suppressor Genes
Autocrine Growth Factors
Clinical Presentation
Treatment
Prevention
Cross-References
References
See Also
Lung Cancer and Smoking Behavior
Definition
Characteristics
Incidence and Mortality
Prognosis
Cigarette Smoke
Active Smoking
The Trends in Smoking Prevalence in the UK, the USA, and Japan as an Example
Exposure to Environmental Tobacco Smoke
Appendix
Dietary Factors
Air Pollution and Indoor Pollution
Radon Exposure
Occupational Exposure Other Than Radon Exposure
Genetic Polymorphisms Involved in Smoking Behavior
Genetic Polymorphisms Involved in the Metabolism of Cigarette Smoke
References
Lung Cancer Clinical Oncology
Synonyms
Definition
Characteristics
Screening Strategies for Lung Cancer
Staging Workup of Lung Cancer
Staging of Lung Cancers
Non-small Cell Lung Cancer (NSCLC)
Early-Stage NSCLC (Stages I-II)
Stage I
Stage II
Locally Advanced Resectable NSCLC (Stage IIIA)
Locally Advanced Unresectable NSCLC (Stage IIIB)
Metastatic NSCLC (Stage IV)
Genetics of Non-small Cell Lung Cancer
Small Cell Lung Cancer (SCLC)
Mesothelioma
Perspective
Cross-References
References
Lung Cancer Epidemiology
Definition
Characteristics
Types of Lung Cancer
Lung Cancer Incidence and Mortality
Lung Cancer Survival Rates
Age, Sex, and Race Patterns of Lung Cancer
Etiology of Lung Cancer
Overview of Causes of Lung Cancer
Tobacco Exposure
Radon Gas and Asbestos
Future Trends in Lung Cancer Incidence
Cross-References
References
Lung Cancer Pharmacogenomics
Synonyms
Definition
Characteristics
EGFR as a Target for Lung Adenocarcinoma
ALK as a Target for Lung Adenocarcinoma
DNA Damage Response (DDR): A Biological Rationale for Predictive Models in NSCLC
Conclusion
Cross-References
References
See Also
Lung Cancer Staging
Definition
Characteristics
Noninvasive Staging
CT of the Thorax
Magnetic Resonance Imaging of the Thorax
Positron Emission Tomography Scan
Imaging for Distant Metastases
Invasive Staging
Bronchoscopy
Endobronchial Ultrasound
Transthoracic Needle Aspiration
Endoscopic Ultrasound
Mediastinoscopy
Extended Mediastinoscopy
Anterior Mediastinotomy
Cervical Lymph Node Staging
Video-Assisted Thoracic Surgery
Tissue for Staging: What and How Much Is Enough?
Molecular Staging: The Final Frontier?
Conclusion
References
Lung Cancer Targeted Therapy
Definition
Characteristics
EGFR TKIs for NSCLC with EGFR Mutation
EGFR TKIs for NSCLC with Unknown EGFR Mutation Status
EGFR TKIs in Maintenance Treatment for Advanced NSCLC
Targeted Agents in Combination with Chemotherapy
Resistance to EGFR TKIs
Target Therapy with Monoclonal Antibodies for NSCLC
Future Direction
References
Lung Carcinoma
Lung Resistance-Related Protein
Luteotropic Hormone
Luteotropin
LV
Lymphadenectomy
Definition
Characteristics
Lymphangiogenesis
Synonyms
Definition
Characteristics
Molecular Regulation
Clinical Relevance
Cross-References
References
See Also
Lymphatic Vessels
Definition
Characteristics
Clinical Relevance
References
Lymphoepithelioma
Lymphokine Activated Killer
Lynch Syndrome
Synonyms
Definition
Characteristics
History
Management
References
Lysine (K) Acetyltransferases (KATs)
Lysine (K)-Specific Demethylase 6A
Lysine (K)-Specific Demethylase 6B
Lysolecithin
Lysophosphatidic Acid
Lysophosphatidylcholine
Synonyms
Definition
Characteristics
Signaling Mechanisms
Clinical Aspects
Cross-References
References
See Also
Lysophospholipase D
M
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide
M&B 39831
MA-3, TIS (Mouse Pdcd4)
MabThera (Non-US Countries)
Mach
Machine Learning
MACH-Related Inducer of Toxicity
Macro- and Microminerals
Macroautophagy
Macropain
Macrophage Inhibitory Cytokine 1
Macrophage Stimulating 1 Receptor
Macrophages
Synonyms
Definition
Characteristics
Cross-References
References
See also
Macrophage-Stimulating Protein
Synonyms
Definition
Characteristics
Structure
Biological Activities and Targets
Cellular and Molecular Regulation
MSP Receptor Signaling
Clinical Relevance
Cross-References
References
Macula Adherens
Maculae Adherentes
MAD1
MAD1L1
MADH4
MAGE-A11
Synonyms
Definition
Characteristics
MAGE-11 Protein
Regulated Expression
Steroid Hormone Receptor Coregulator
Target for Prostate Cancer Therapy
Cross-References
References
See Also
MAGEA11 Gene
Magicin
Maidenhair Tree
Maintenance Chemotherapy
Definition
Cross-References
Major Histocompatibility Complex (MHC) Class I-Related Chain Molecules
Major Microtubule Organizing Center
Major Vault Protein
Synonyms
Definition
Characteristics
The Vault Complex
Major Vault Protein (MVP)
Drug Resistance
Prognostic Significance
Cross-References
References
Male Breast Cancer
Definition
Characteristics
Risk Factors
Treatment
Molecular Characterization
References
Malignancy of Small Round Blue Cell Type
Malignancy-Associated Changes
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
See Also
Malignant Adrenocortical Tumor
Malignant Breast Carcinoma
Malignant Endocrine Tumors
Malignant Lymphoma: Hallmarks and Concepts
Definition
Characteristics
Basic Clinical Features of Malignant Lymphoma
Etiology
Classification
The Molecular Genetic Basis of NHL
Antigen Receptors in NHL
The Immunoglobulin B Cell Receptor:
T Cell Receptors in Peripheral T Cell Lymphomas
Gene Expression Profiling
Cell Biological Features of Hodgkin Lymphoma
Cross-References
References
See Also
Malignant Mesenchymal Nephroma of Infancy
Malignant Mesothelioma
Malignant Neoplasias Originating in Endocrine Tissues or Their Target Organs
Malignant Neoplastic Changes of the Colon
Malignant Neoplastic Changes of the Lung or Bronchus
Malignant Pleural Effusion
Malignant Prostate Carcinoma
Malignant Salivary Gland Cancer
Malignant Skin Tumors
Malignant Tumors of the Endocrine Glands
MALT Lymphoma
Definition
Cross-References
See Also
Mammalian Target of Rapamycin
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
References
MammaPrint Test
Definition
Who is Eligible for the MammaPrint Test?
How Does the MammaPrint Test Work?
Cross-References
See Also
Mammographic Breast Density and Cancer Risk
Definition
Characteristics
Effect of Mammography Screening
Benefits and Harms
Public Health Screening Recommendations
Cross-References
References
Mammographic Density
Definition
Characteristics
Mammographic Density and Risk of Breast Cancer
Factors Associated with Variations in Mammographic Density
Heritability of Mammographic Density
Summary and Conclusions
Cross-References
References
Mammotropic Hormone
Mammotropin
Mantle Cell Lymphoma
Synonyms
Definition
Characteristics
Incidence
Epidemiology
Pathogenesis
Pathology
Immunophenotype
Symptoms and Clinical Findings
Paraclinical Findings
Diagnosis and Differential Diagnosis
Disease Staging
Prognostic Factors
Course of Disease
Treatment
Stem-Cell Harvest and Purging
High-Dose Therapy with Autologous Stem-Cell Support (ASCT)
ASCT Conditioning Regimens
Maintenance
Second-Line Treatment
Summary
References
MAP Kinase
Definition
Characteristics
Members of the MAP Kinase Family
Structure of MAP Kinase Cascades
Signaling Via the MAP Kinase Cascades
The ERK1/2 Cascade
The p38 and JNK Cascades
The ERK5 Cascade
Regulation and Specificity Determination of MAP Kinases
Involvement of MAP Kinases in Cancer
Cross-References
References
See Also
MAP Kinase Kinase Kinases
MAP3K
Mapatumumab
MAPK10
MAPK8
MAPK9
MAPs
MAR
MAR Syndrome
Marginal Zone B-Cell Lymphoma
Definition
Characteristics
Histology
Pathogenesis
Genetics
Deregulation of NF-kappaB: The Unifying Concept for MALT Lymphomagenesis?
Clinical Features, Prognosis, and Therapy
References
Marijuana
Maspin
Definition
Characteristics
The Role of Maspin in Epithelial Pathophysiology
The Molecular Mechanisms of Maspin
Summary
Cross-References
References
See Also
Mass Recovery
Mast Cell Growth Factor
Mast Cells
Synonyms
Definition
Characteristics
MC and Tumor Growth
MC and Tumor Angiogenesis
Conclusions
Cross-References
References
Mastocytosis
Definition
Characteristics
Diagnosis
Classification
Therapy
References
Mastzellen
Matriptase: Epithin, MT-SP1, Suppression of Tumorigenicity 14
Matriptase-2 (TMPRSS6)
Matriptase-3 (TMPRSS7)
Matrix Metalloproteinase Inhibitor
Matrix Metalloproteinase-3
Matrix Metalloproteinases
Synonyms
Definition
Characteristics
Domain Structure
Zymogen Activation
Inhibition
Substrate Cleavage
Control of MMP Gene Expression
Clinical Relevance
Cross-References
References
Matrixins
Maturation-Promoting Factor
Mature/Immature
MBP1, UPH1, H411, EFEMP2, FBLN-4
Mch5
MCJ
MCL
Mcl Family
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
Cross-References
References
See Also
Mcl-1
Definition
Characteristics
Clinical Aspects
Cross-References
References
MCPH1
MCT-1 Oncogene
Definition
Characteristics
Regulation
Clinical Relevance
References
See Also
mda-6
MDC
MDM Genes
Synonyms
Definition
Characteristics
MDM2
MDM2 and Human Tumors
MDM4
MDM4 and Human Tumors
Cross-References
References
See Also
MDM2
Definition
Characteristics
Discovery and Significance
Function
Structure
MDM4 (Also Known as MDMX)
Structure/Function Relationships
The N-Terminal p53-Binding Pocket
Nuclear Import and Export Sequences
The Acidic Domain
The Zinc Finger
The RING Finger
Regulation
Autoregulatory Feedback Loop
Protein Turnover
Regulation by Protein-Protein Interactions
Regulation by Posttranslational Modification
DNA Damage Signaling
Survival Signaling
Interaction with Other Proteins: Substrates and Regulators
MDM2 in Cancer
Drug Target
Further Information
References
Mdr1 Protein
2ME
2ME2
MEC
Med28
Medical Foods
Medullary Thyroid Cancer Targeted Therapy
Definition
Characteristics
Molecular Therapeutic Targets in MTC
Ret and Angiogenesis-Related RTKs
Other Targets
RTK Inhibitors
Perspectives
References
Megapain/UCDEN (Ubiquitin Conjugate Degrading Endopeptidase)
MEK Kinases
Melanocytic Nevus
Melanocytic Tumors
Synonyms
Definition
Characteristics
Cross-References
References
Melanocytoma
Melanoma
Melanoma Antigen Family A-11
Melanoma Antigen Gene Protein-11
Melanoma Antigen-A11
Melanoma Antigens
Synonyms
Definition
Characteristics
Background
Classification
Tumor-Specific Antigens
Tumor-Specific Shared Antigens (CTA)
Antigens Encoded by Genes Overexpressed in Tumor Cells
Antigens Encoded by Differentiation Genes
Clinical Implications
References
Melanoma Drug Treatment
Synonyms
Definition
Characteristics
Chemotherapeutics for Melanoma Treatment
Dacarbazine
Fotemustine
Temozolomide
Nab-Paclitaxel
Immunotherapeutics for Melanoma Treatment
IL-2
Interferon Alfa
Ipilimumab
PD-1 Inhibitors
Target-Based Therapeutics for Melanoma Treatment
Drugs Targeting Melanoma Cells Carrying Mutation in the B-RAF Gene
Vemurafenib
Dabrafenib
Drug Targeting MEK Protein
Trametinib
Drugs Targeting Melanoma Cells Carrying Mutations in the C-KIT Gene
Newer Treatments
Cross-References
References
Melanoma Therapy
Melanoma Vaccines
Synonyms
Definition
Characteristics
Historical Background
Tumor Immune Defense Against Transplantable Tumors in Mouse Models
Culture of Melanoma-Specific Human T Cells and Identification of Melanoma Antigens
Rational Development of Melanoma Vaccine Strategies in Animal Models
Efficacy of Melanoma Vaccines in Clinical Trials
Barriers for the Successful Implementation of Therapeutic Melanoma Vaccines
Summary and Future Perspective
References
Melanoma-Associated Antigen-A11
Melanoma-Associated Antigens
Melanoma-Associated Retinopathy
Synonyms
Definition
Characteristics
Pathogenesis
Clinical Aspects
Diagnostic Procedure
Therapy
Prognostic Relevance
Cross-References
References
Melatonin
Definition
Characteristics
Melatonin Anticancer Action and Mechanisms
Nocturnal Melatonin Suppression by Light at Night and Human Breast Cancer Risk
Clinical Cancer Trials with Melatonin
Summary and Conclusions
References
Membrane Microdomain
Membrane Raft
Membrane Transport Proteins
Membrane Transporters
Synonyms
Definition
Characteristics
General Features
Pharmacological Relevance of Membrane Transporters
Role of Membrane Transporters in Drug Resistance
Cross-References
References
See Also
Membrane-Linked Docking Protein
Definition
Characteristics
Mechanism of Signal Transduction by MLDP
Roles of Individual MLDP Family in Normal and Cancer Cells
FRS2/SNT
Dok
Transmembrane Adaptor Proteins (TRAPs)
Gab
IRS
Cross-References
References
See Also
Membrane-Lipid Therapy
Synonyms
Definition
Characteristics
Diversity of Membrane-Lipid Composition and Structure
Mechanisms Involved in Membrane-Lipid Therapy
MLT Drugs that Target the Cell Plasma Membrane
Destabilization of Membrane Structure, as Occurs with Anticancer Drugs Like Anthracyclines and Hexamethylene Bisacetamide (HMBA)
Hyperfluidization of the Lipid Membrane
Lipid-Raft Microdomain Remodeling
Changes in Membrane-Lipid Composition
Regulation of Enzyme Activities that Modulate Lipids
Lipid Replacement Therapy Is Used to Restore Damaged Lipids Through Supplementation with Membrane Phospholipids and Antioxidan
MLT Drugs that Target the Nucleus
Lipid-Mediated Regulation of Nucleic Acid Function
The Regulation of Gene Expression to Alter Membrane-Lipid Composition
MLT Drugs Target Protein-Lipid Interactions
Development of Membrane-Lipid Therapy Drugs and Ongoing Studies
Cross-References
References
MEN1
MEN1B
MEN4
Menopausal Symptoms After Breast Cancer Therapy
Definition
Characteristics
Introduction
Treatment Options
Synthetic Hormonal Compound
Nonhormonal Therapies
Treatment of Urogenital Atrophy and Related Symptoms
Treatment of Bone Loss
Cardiovascular Complications
Conclusion
References
Merlin
Synonyms
Definition
Characteristics
References
Mesenchymal Stem Cells
Synonyms
Definition
Cross-References
Mesenteric Fibromatosis
Mesoblastic Nephroma
Synonyms
Definition
Characteristics
Diagnosis
Histology and Genetics
Treatment
Prognosis
Cross-References
References
See Also
Mesothelin
Synonyms
Definition
Characteristics
Tissue Expression
Regulation
Function
Diagnostic Marker
Therapeutic Applications
Cross-References
References
See Also
Mesothelioma
Synonyms
Definition
Characteristics
Pathogenesis
Asbestos
Chromosome Changes
Tumor Suppressor Genes
Diagnosis
Clinical Approaches
Cross-References
References
See Also
MET
Definition
Characteristics
Cellular and Molecular Features
Clinical Relevance
Cross-References
References
See Also
Meta-Analysis
Definition
Cross-References
Metabolic Polymorphisms and Cancer Susceptibility
Definition
Characteristics
History
Important Genetic Factors
Relevance of Pharmacogenetics
Ethnic Differences
Future
References
Metabolic Reprogramming
Definition
Characteristics
Introduction
Metabolic Reprogramming: Some Basic Changes
Aerobic Glycolysis
Upregulation of Components of Aerobic Glycolysis
Upregulation of ``Upstream´´ Components in Aerobic Glycolysis
Upregulation of ``Downstream´´ Components in Aerobic Glycolysis
Lactate as a Signaling Molecule
Aerobic Glycolysis and Anabolic Side Pathways
Glutamine (Gln) and the Switch of the TCA Cycle to Subserving De Novo Lipogenesis
Cross-References
References
See Also
Metal Complex
Metal Compound
Metal Drugs
Synonyms
Definition
Characteristics
History of Metals in Anticancer Medicine
Platinum(II) Drugs: Best Sellers in Cancer Therapy
Platinum(IV) Drugs: A Prodrug Strategy
Arsenic Trioxide: Rediscovery of an Old Drug
Ruthenium: Anticancer Agents with Reduced Side Effects
Conclusions
Cross-References
References
Metal-Based Drug
Metallodrug
Metalloprotease Disintegrin Cysteine Rich
Metaplasia
Metastasis
Synonyms
Definition
Characteristics
Mechanisms
Cross-References
References
Metastasis Suppressor Gene
Definition
Characteristics
Clinical Relevance
Cross-References
References
See Also
Metastasis Suppressor KAI1/CD82
Synonyms
Definition
Characteristics
Mechanism
Cross-References
References
See Also
Metastatic Breast Cancer Experimental Therapeutics
Synonyms
Definition
Characteristics
Gene Therapy
Vaccine Therapy
Chemotherapeutic and Biological Agents
Histone Deacetylase (HDAC) Inhibitors
Conclusion
Cross-References
References
See Also
Metastatic Cancer
Metastatic Colonization
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Metastatic Pancreatic Cancer
Metastatic PDAC
Metastatic Prostate Cancer
Methazolastone
2-Methoxyestradiol
Methoxyestradiol
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
References
Methylase
Methylation
Definition
Characteristics
Cellular and Molecular Aspects
Aberrant DNA Methylation in Cancer
Hypomethylation
Hypermethylation
References
Methylation-Controlled J Protein
Synonyms
Definition
Characteristics
Cross-References
References
4-Methylsulfinylbutyl Isothiocyanate
Methyltransferases
Synonyms
Definition
Characteristics
DNMTases
DNA Methylation Patterns in Normal and Cancer Cells
Types of Mammalian DNMTases
Structure and Function of Mammalian DNMTases
RNA MTases
Histone Methyltransferases (HMTases)
References
Metronomic Chemotherapy
Keywords
Definition
Characteristics
Origin
Mechanisms of Action
Clinical Settings
Advantages
Drawbacks
Conclusions
Cross-References
References
See Also
Metronomic Scheduling
MGC126245
MGC126246
MGC126806
MGC138284
MGC34538
MGF Receptor
Mib-2
MIC Molecules
Synonyms
Definition
Characteristics
Clinical Aspects of MIC Expression by Tumors
Clinical Aspects of Soluble MIC Molecules in Blood
Soluble MIC in Diagnosis of Cancer Disease
Soluble MIC in Staging of Cancer Disease
Soluble MIC in Cancer Prognosis and Monitoring of Cancer Disease
Cross-References
References
MIC-1
Synonyms
Definition
Characteristics
Regulation of MIC-1/GDF15 Gene Expression
Serum MIC-1/GDF15 Measurement as a Clinical Tool
Role of MIC-1/GDF15 in Tumor Biology
Conclusion
Cross-References
References
See Also
Microadenomas
Microarray
Microarray (cDNA) Technology
Synonyms
Definition
Characteristics
cDNA Microarray Construction
Hybridization with Labeled cDNA
Scanning the cDNA Microarray Using a ``Reader´´
Image Analysis and Generation of cDNA Microarray Data
Analysis of Large-Scale cDNA Microarray Data
Application
Clinical Relevance
References
Microcell-Mediated Chromosome Transfer
Definition
Characteristics
Cross-References
References
See Also
Microcephalin
Microcystin-LR
Definition
Characteristics
Tumor-Promoting Activity in Rat Liver
Inhibition of Specific [H]okadaic Acid Binding
Inhibition of Protein Phosphatases 1 and 2A
Cellular Biochemical Response
Structure-Function Relationship of Microcystins
Crystal Structure of PP1-Microcystin-LR Complex
Cross-References
References
Microcytoma
Microglia
Micrometastasis
Definition
Characteristics
Detection Techniques
Immunocytochemistry
Molecular Techniques
EPISPOT Assay
Cellular and Molecular Biology
Clinical Relevance
Bone Marrow
Peripheral Blood
Lymph Nodes
References
Micronucleus Assay
Definition
Characteristics
Clinical Aspects
Cross-References
References
MicroRNA
Definition
Characteristics
miRNAs and Cancer
miRNAs as Tumor Suppressors and Oncogenes
miRNA Profiling and Cancer Diagnosis
Cross-References
References
See Also
Microsatellite Instability
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
Cross-References
References
See Also
Microtubule-Associated Proteins
Synonyms
Definition
Characteristics
MAPs and Cancer
Cancer Predisposition
Cancer Development
Cancer Diagnosis and Prognosis
Cancer Sensitivity/Resistance to Microtubule Targeting Drugs
References
Microtubule-Binding Proteins
Microtubule-Stabilizing/Microtubule-Destabilizing Proteins
Micro-Ultrasound Imaging
MIG-6
Migration
Synonyms
Definition
Characteristics
Mechanisms of Cell Migration: Rho-GTPases Are Key Players
Rac
Cdc42
Rho
Summary
Acknowledgments
Cross-References
References
See Also
MIHA
Milky Spots
Mind Bomb Homologue 2
Mineral Nutrients
Synonyms
Definition
Characteristics
Mineral Nutrients and Cancer
References
Minerval
Mini-gut
Mini-intestine
Minimal Residual Disease
Synonyms
Definition
Characteristics
Methods of Detecting MRD
Clinical Relevance
Cross-References
References
Minodronate
Synonyms
Definition
Characteristics
Effects of Minodronate on Endothelial Cells (ECs)
Effects of Minodronate on Tumor Cells In Vitro
Effects of Minodronate on Tumor Cells In Vivo
Cross-References
See Also
Minodronic acid
Mismatch Repair in Genetic Instability
Definition
Characteristics
Biological Consequences
Clinical Relevance
References
Mitochondrial DNA
Definition
Characteristics
Cross-References
References
See Also
Mitochondrial Membrane Permeabilization in Apoptosis
Synonym
Definition
Characteristics
Mechanisms of MMP
Regulation of MMP
MMP and Cancer
Therapeutic Implications
References
Mitogen-Activated Protein Kinase Kinase Kinases
Synonyms
Definition
Characteristics
Functional Role in Cancers
BRAF
MEKK1
Other MAP3K Linked to Cancer Biology
Conclusions and Future Directions
Cross-References
References
Further Reading
Mitogen-Activated Protein Kinases p42 and p44
Mitogen-Inducible Gene 6 in Cancer
Synonyms
Definition
Characteristics
Signaling Regulation
Clinical Relevance
References
Mitomycin C
Synonyms
Definition
Characteristics
Form
Bioreductive Activation
Enzymatic Bioreductive Activation of MMC
NQO1
NQO1 and Mitomycin C In Vitro
NQO1 and Mitomycin C in Anaerobic Conditions
NQO1 and Mitomycin C In Vivo
Xanthine Oxidase/Dehydrogenase
NADPH-Cytochrome C P450 Reductase
Other Reductases
Analogues of MMC
Pharmacokinetics
Clinical Use of MMC
Side Effects of MMC
References
Mitotic Arrest-Deficient Protein 1
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
References
Mitotic Catastrophe
Synonyms
Definition
Characteristics
Cellular and Molecular Aspects
Clinical Relevance
References
Mitotic Cell Death
Mixed Gliomas
MK-0683
MK-1
MKKK
MLPLI
MLS
MM
MMC6-amino-1,1a,2,8,8a,8b-hexahydro-8-(hydroxymethyl)-8a-methoxy-5-methyl-azirino[2, 3:3, 4] pyrrolo[1, 2-a]indo
MMP
MMP-3
MMPs
MOAB HER2
Modifier Loci
Synonyms
Definition
Characteristics
Rodent Models
Humans
Mechanisms
Perspectives
References
Modular Nanotransporters
Modular Recombinant Transporters
Modular Transporters
Synonyms
Definition
Characteristics
Objectives
Principles
Features
Cross-References
References
See Also
Modulation of Colon Carcinogenesis
Mole
Molecular Chaperones
Definition
Characteristics
HSP90 as an Anticancer Drug Target
Cross-References
References
See Also
Molecular Imaging
Definition
Characteristics
Molecular Imaging Probes
Imaging Modalities
Magnetic Resonance Imaging and Spectroscopy
Ultrasound
Optical Imaging
Positron Emission Tomography and Single-Photon Emission Computed Tomography
Computed Tomography
Hybrid Techniques
Cross-References
References
Molecular Morphology
Molecular Pathology
Synonyms
Definition
Characteristics
Historical and Clinical Background
Immunohistochemistry
In Situ Hybridization
Polymerase Chain Reaction
Future Developments
Cross-References
References
Molecular Therapy
Definition
Characteristics
Gene Therapy
Nucleic Acid Constructs
Small Molecule Drugs
Antibody Therapy
Cross-References
References
See Also
Monoclonal Antibodies for Cancer Therapy
Definition
Characteristics
Background
Applications of Mabs for Cancer Therapy
``Naked´´ (Unmodified) Mabs Which Interfere with Growth-Controlling Mechanisms
Tumor Killing via Complement-Dependent Cytotoxicity (CDC) and Antibody-Dependent Cellular Cytotoxicity (ADCC)
Interference with Tumor Vasculature
Antibodies to Lymphocyte Antigens Applied to Modify Antitumor Immunity via Check-Point Inhibition
Drug Immunoconjugates
Toxin Mab Constructs
Radioimmunotherapy
Bispecific Antibodies
Induction of an Immune Response by Mabs to Tumor Antigens
Anti-idiotypic Antibodies
Therapeutic Vaccination with Tumor Cells Expressing Mab-Derived scFv Fragments at Their Surface
Combination Therapy
Complications of Mab-Based Therapy
Cross-References
References
See Also
Monoclonal Antibody c-erb-2
Monoclonal Antibody HER2
Monoclonal Gammopathy of Undetermined Significance (MGUS)
Synonyms
Definition
Characteristics
Epidemiology
Etiology
Pathogenesis
Diagnosis
MGUS Variants
Association of MGUS with Other Diseases
Antibody Activity of mIg and Associated Diseases
Aggregation and Deposition of mIg and Associated Diseases
Predictors of Tumor Progression
Management of MGUS and Development of Early Treatment Strategies
Cross-References
References
See Also
Mononeuropathy
Mononuclear Phagocytes
Mother Against Decapentaplegic, Drosophila, Homolog of 4
Motility
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
Mouse Double Minute Gene
Mouse Inbred Lines
Mouse Models
Synonyms
Definition
Characteristics
History
Biological Relevance
Genetically Engineered Mice
Transgenic Animals
Conditional Transgenics
Inducible Systems
Spontaneous Recombination
Genetic Susceptibility
Inbred Strains
Mapping of Susceptibility Genes
Congenics
Recombinant Inbred Lines and Other Types of Inbred Mice
Outbred Strains
Therapeutics
Xenografts
Molecular Imaging
Cross-References
References
See Also
Mouse Xenograft Models
MPF
MPP2
MRD
MRIT
mRNA Translation
Synonyms
Definition
Characteristics
mRNA Synthesis
Protein Synthesis
Initiation of Translation (Fig.2)
Polypeptide Elongation
Termination of Translation
Some mRNAs Code More than One Protein
mRNAs Contain Regulatory Elements
Other mRNA Features: Internal Ribosome Entry Sites (IRES)
Future Prospects
Cross-References
References
MRP
MSAT - Metronomic Scheduling of Anticancer Treatment
MSCs
MSLN
MSP
MST1
MST1R
MTase
MTOC
mTOR
MTS1
Mucinous Cystadenocarcinoma
Mucins
Definition
Characteristics
References
Muclin (Mouse)
Mucocele of the Appendix
Mucocutaneous Myxoma
Mucoepidermoid Cancer
Synonyms
Definition
Characteristics
Historical Data
Histological Features
Epidemiology
Etiology
Staging Parameters
Treatment
References
Multidrug Resistance Transporters
Multidrug Resistance-Associated Proteins
Multidrug Transporter
Multigene Array
Definition
Cross-References
See Also
Multimeric Antibody Fragments
Multimodality Cancer Therapy
Multiple Endocrine Adenopathy Type 1
Multiple Endocrine Neoplasia Type 1
Synonyms
Definition
Characteristics
Genetics
MEN1 Mutations in Hereditary Endocrine Disorders
MEN1 Mutations in Sporadic Non-MEN1 Endocrine Tumors
Function of the MEN1 Protein (Menin)
Mouse Models for MEN1
Clinical Application
Acknowledgments
Cross-References
References
Multiple Hamartoma Syndrome
Multiple Myeloma
Synonyms
Definition
Characteristics
Immunobiology of MM
Molecular Biology of MM
Gene Expression Profiles (GEP) in MM
Clinical Treatment of MM
Cross-References
See Also
Multistep Development
Definition
Characteristics
History
Participating Genes
Minimum Requirements for Tumor Development
Clinical Relevance
Cross-References
References
See Also
Mutagen Sensitivity
Definition
Characteristics
Overview of Human DNA Repair System
Inter-Individual Variations in DNA Repair Capacity Within General Population
Development of Mutagen Sensitivity Assay
Heritability of Mutagen Sensitivity
High Mutagen Sensitivity Is a Genetic Susceptibility Factor for Cancer Development
References
Mutation Rate
Definition
Characteristics
Summary
References
Mutator Phenotype
Definition
Characteristics
Mutations and Cancer
Chromosomal Alterations in Human Cancers
Point Mutations in Human Cancer
Rarity of Spontaneous Mutations in Normal Cells
Historical Perspective
Tumor Evolution
Implications of a Mutator Phenotype
Cross-References
References
MUTYH-Associated Colorectal Polyposis
Definition
Characteristics
Inherited Predisposition to Colorectal Cancer
Identification of an Unusual (G:CT:A) Mutator Phenotype in Family N
Inherited Mutations in MUTYH Predispose to Colorectal Tumors
The Pathway of MAP Tumorigenesis
The Phenotype of MAP
MUTYH Mutation Spectrum
Genetic Testing and Clinical Management of MAP
References
MYB
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Aspects
References
MYC Oncogene
Definition
Characteristics
Myc Structure and Function
Posttranslational Modifications and Regulation of Myc Turnover
Other Members of the Max-Interacting Network
Effects of Myc Activation
Consequences of Myc Deregulation
Tumors Associated with Myc
Targeting Myc as an Approach to Treat Cancer
Conclusion
Cross-References
References
See Also
Mycobacterium bovis BCG
Myeloablative Megatherapy
Synonyms
Definition
Characteristics
Therapeutic Concept
Timing of Megatherapy Within a Treatment Strategy
Which Drugs Can Be Used for Megatherapy?
References
Myelodysplastic Syndromes
Synonyms
Definition
Characteristics
Epidemiology
Causative Factors
Clinical Presentation
Diagnosis and Classification
Cytogenetic Findings
Prognosis
Treatment Options
Supportive Care
Epigenetic Agents (Epigenetic Therapy)
Immunosuppressive Agents (Immunotherapy)
Intensive Chemotherapy and Allogeneic Hematopoietic Stem Cell Transplantation (Chemotherapy)
Cross-References
References
Myelofibrosis with Myeloid Metaplasia
Myeloid Cell Leukemia Sequence 1
Myeloid Leukemia of Down Syndrome
Myeloma
Myelosuppression
Definition
Characteristics
Hematopoietic Growth Factor Support
Regulation of Neutrophil Cell Death
Conclusion
Cross-References
References
See Also
Myofibroma
Myoma
Myopodin
Synonyms
Definition
Characteristics
Myopodin Gene Expressed in Prostate but Deleted or Downregulated in Prostate Cancer
Myopodin Expression and Relapses of Prostate Cancer
Mechanism of Myopodin Mediated Tumor Suppression
Myopodin Expressed in Muscles
References
Myotendinous Antigen
Myxoid Liposarcoma
Synonyms
Definition
Characteristics
Genetics
Etiology
Diagnostics
Treatment
Prognosis
References
Myxoid Neurofibroma
N
N-(4-hydroxyphenyl)retinamide
NA1/NA2
NAALADase
NAB
NaCHOleate
NAD-Dependent Deacetylase Sirtuin-1
Naevoid Basal Cell Carcinoma Syndrome
Synonyms
Definition
Cross-References
See Also
NAG-1
NAME
Nanocapsules
Definition
Cross-References
Nanoformulations
Nanomedicine
Nanoparticles
Nanoparticles in Cancer Therapy
Definition
Characteristics
An Example of Cancer Cell-Specific Nanocapsule Design
Conclusion
Acknowledgments
References
Nanoparticles in Diagnosis and Treatment
Definition
Characteristics
Types of Nanoparticles
Nanoparticles in Tumor Diagnostics
Tumor Detection
Profiling of Tumor Biomarkers
FISH
Nanoparticles in Antitumor Therapy
Liposomal-Based Drug Delivery
Gene Delivery
References
Nanospheres
Synonyms
Definition
Characteristics
Types of Nanospherical Formulations
Polymeric Nanoparticles
Solid Lipid Nanoparticles
Liposomes
Nanoemulsions
Micelles
Inorganic Nanoparticles
Multistage Particles
Viral Nanoparticles
Advantages of Nanosphere Formulations over Conventional Drug Delivery Strategies 2
Conclusions
References
Nanotechnology
Definition
Characteristics
Nanoparticles
Nanoscale Cancer Imaging Contrast Agents
Nanoscale Cancer Therapeutics
Laboratory Nanoscale Cancer Diagnostics
Current Applications of Nanotechnology in Clinical Medicine
References
Nasopharyngeal Carcinoma
Synonyms
Definition
Characteristics
Diagnosis
Treatment
Cross-References
References
See Also
NAT
Natural Immunity
Natural Killer Cell Activation
Synonyms
Definition
Characteristics
Cytokine Activation of NK Cells
Activation of NK Cytotoxicity by Tumor Cells
Activated NK Cells in Cancer Therapy
Acknowledgments
Cross-References
References
See Also
Natural Killer Group 2D
Natural Products
Synonyms
Definition
Characteristics
Parameters of Natural Products
Natural Product Drug Discovery
Natural Product Anticancer Drugs
Cross-References
References
See Also
Natural Yellow 3
Naturally Disordered Proteins
Naturally Unfolded Proteins
Naturally Unstructured Proteins
NBCCS
NBK (Natural Born Killer)
NCA-160
NCI60_003316
NCoA3
ncRNA
Necroptosis
Synonyms
Definition
Characteristics
History of Discovery
Cellular Features of Necroptosis
Necroptosis-Inducing Factors
Necroptosis Activation Mechanisms
TNFα-Induced Necroptosis
Genotoxic Stress-Induced Necroptosis
TLR-Mediated Necroptosis
Necroptosis Induced by Other Stimulations
Necroptosis in Health and Disease
Necroptosis in Health
Necroptosis in Disease
Concluding Remarks
Cross-References
References
See Also
Necrosis
Synonyms
Definition
Characteristics
Causes
Pathogenesis
Necrosis: Classification
Regulation of Necrosis: Necroptosis
Necrosis in Cancer
Cytotoxic Drugs and Necrosis
Conclusion
Cross-References
References
See Also
Needle Aspiration Biopsy
Needle Biopsy
Nek2
Definition
Characteristics
Cross-References
References
See Also
Nemosis
Definition
Characteristics
Possible Significance of Nemosis
References
Neoadjuvant Chemotherapy (Neoadjuvant Therapy)
Neoadjuvant Therapy
Synonyms
Definition
Characteristics
Cross-References
See Also
Neoplastic Meningitis
Neovascularization
Nephroblastoma
Synonyms
Definition
Characteristics
Genetics
Clinical Aspects
Treatment
Cross-References
References
See Also
Nestin
Definition
Characteristics
Structure
Distribution and Roles in Normal Cells
Expression and Roles in Nonneoplastic Disease
Expression and Roles in Tumor Growth
Nestin as a Cancer Stem Cell (CSC) Marker
Expression and Roles in Angiogenesis
References
NET
Neuroblastoma
Definition
Characteristics
Cross-References
References
Neurocytoma
Synonyms
Definition
Characteristics
Diagnosis
Therapy
Cross-References
References
Neuroectodermal Tumor
Neuroendocrine Carcinoma
Synonyms
Definition
Characteristics
Classification
Epidemiology
Natural History
Carcinoids
Neuroendocrine Tumors of the Pancreas (Islet Cell Tumors)
Diagnosis of Neuroendocrine Carcinomas
Pathological Diagnosis
Biochemical Diagnosis
Imaging Diagnosis
Treatment of Neuroendocrine Carcinomas
Surgery
Radiofrequency Ablation
Liver Embolization
Irradiation Therapy
Systemic Treatment
New Agents
Cross-References
References
See Also
Neuroendocrine Neoplasms
Synonyms
Definition
Characteristics
Diagnostic Criteria for Entering the NEN Category
Prognostic Classification Concepts of GEP-NENs
Neuroendocrine Tumor (NET)
Neuroendocrine Carcinoma (NEC)
Mixed Adeno-Neuroendocrine Carcinoma (MANEC)
Genetics
NENs of the Pancreas (PanNEN)
NENs of the Stomach and the Esophagus
NENs of the Small Intestine, Colon, and Rectum
Neuroendocrine Tumors of the Lung
Carcinoid Tumors
Pulmonary Small Cell Lung Carcinoma (SCLC) and Large Cell Neuroendocrine Carcinoma (LCNEC)
Thymic Neuroendocrine Tumors
Medullary Thyroid Carcinoma (MTC)
NEC of the Skin (Merkel Cell Carcinoma)
Cross-References
References
See Also
Neuroendocrine Tumors of the Pancreas
Definition
Cross-References
See Also
Neuroepithelioma
Neurofibromatosis 1
Synonyms
Definition
Characteristics
Diagnostic Criteria and Clinical Aspects
Genetics
Neurofibromin
Gene Mutations in NF1-Related Tumors
NF1 Gene Mutations in Cancer
Cross-References
References
See Also
Neurofibromatosis 2
Synonyms
Definition
Characteristics
Diagnostic Criteria and Clinical Aspects
Genetics
Merlin Protein
References
Neurokinins
Neuromedin
Definition
Characteristics
NMB in Human Cancer
NMU in Human Cancer
Clinical Relevance
Cross-References
References
See Also
Neuronectin
Neuron-Restrictive Silencer Factor (NRSF)
Neuro-oncology: Primary CNS Tumors
Synonyms
Definition
Characteristics
Epidemiology
Classification
Clinical and Radiological Presentation
Etiology
Molecular Pathogenesis
Current Management
Future Directions
Molecular Therapy: Molecular-Based Diagnostics-Prognosis-Targeted Therapy
Adjuvant Therapy: Novel Biologicals and Therapeutic Delivery
Biological Imaging
Conclusions
Cross-References
References
See Also
Neuropathy
Neuropeptides
Neuropilin Ligands
Neuroprotection
Definition
Characteristics
Cross-References
References
Neurotrophic Factors
Neurotrophins
Synonyms
Definition
Characteristics
Clinical Relevance
References
Neutropenia
Synonyms
Definition
Characteristics
Hematopoiesis
Neutrophil Disorders
Neutrophil Growth Factors
References
Neutrophil Elastase
Synonyms
Definition
Characteristics
NE Gene and Its Variations
NE and Cancer Development
NE and Cancer Progression and Prognosis
NE as Cancer Therapeutic Target
Possible Mechanisms of NE in Cancer Development and Progression
Carcinogen Exposure Enhancer
Apoptosis Inhibitor
Invasion and Metastasis Promoter
Cancer-Promoting Gene Derepressor
NE Suppresses Granulopoiesis of Hematopoietic Cells
Cross-References
References
See Also
Neutrophil Elastase 2
Neutrophil Expressed (ELANE as the official gene symbol)
Nevi
Nevocellular Nevus
Nevus
New York Esophageal Squamous Cell Carcinoma 1
Nexavar
NF1
NF2
NF2 Gene Product
NF-kappaB
NGF Family
NGRD2
Synonyms
Definition
Characteristics
References
Nickel Carcinogenesis
Synonyms
Definition
Characteristics
Nickel, the Chemical Element, and Its Ionic Species and Chemical Compounds
Important Commercial Uses of Nickel and Nickel Compounds
Exposure of Humans to Nickel Compounds
Genotoxicity of Nickel Compounds
Nickel-Induced Cell Transformation
Nickel Carcinogenesis
Mechanisms of Nickel Carcinogenesis
References
Nickel Tumorigenesis
Nickel-Induced Cell Transformation
Nicotine Addiction
Synonyms
Definition
Characteristics
The Nicotine Withdrawal Syndrome
Drug Tolerance
Early Onset of Addiction
References
Nicotine Dependence
Nijmegen Breakage Syndrome
Synonyms
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
References
Nilotinib
Synonyms
Definition
Characteristics
Implications of Nilotinib in Treatment of Imatinib-Resistant CML
Development and Preclinical Studies with Nilotinib
Nilotinib Resistance
Clinical Assessment of Nilotinib and FDA Approval
Future Prospects
Cross-References
References
See Also
Nitric Oxide
Definition
Characteristics
Chemistry and Biological Synthesis of NO
Biochemical Basis of NO Action
Cancer and NO
Role of NO in Initiating Cancer
Role of NO in Cancer Cell Proliferation and Tumor Growth
NO and Tumoricidal Activity
Action of NO on Cellular Respiration
Clinical Relevance
References
Nitrogen-Containing Bisphosphonate
NK Cell Induction
NK Cell Stimulation
NKG2D
NKG2D Receptor
Synonyms
Definition
Characteristics
The NKG2D Receptor
NKG2D Ligands
NKG2D and Cancer Immunosurveillance
Tumor Escape
Cross-References
References
See Also
N-myc Downstream-Regulated Gene
Synonyms
Definition
Characteristics
References
N-myc Downstream-Regulated Gene, NDRG1
Nodularin
Definition
Characteristics
Tumor-Promoting Activity in Rat Liver
Inhibition of Protein Phosphatase 1 and 2A
Expression of Early Response and TNF-α Genes
Nodularin-Related Agent Motuporin
References
Nonclassical Apoptosis
Noncoding RNA
Synonyms
Definition
Characteristics
Types of Noncoding RNAs
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
Small Nuclear RNA (snRNA)
RNA Interference (RNAi)
siRNA
microRNA (miRNA)
rasiRNA
Long Noncoding RNAs (lncRNAs)
Noncoding RNAs and Cancer
Noncoding RNAs and therapy
References
Noninvasive breast Cancer
Nonprimary APC Mutation Colorectal Cancer
Non-Rhabdomyosarcoma Soft Tissue Sarcomas
Synonyms
Definition
Characteristics
Clinical Diagnosis and Staging
Pathology and Biology
Treatment and Outcome
Particular Histotypes
Future Issues
Cross-References
References
See Also
Non-Seminomatous Germ Cell Tumor
Non-Small-Cell Lung Cancer
Definition
Characteristics
Survival Rates
KRAS and EGFR Mutations
BRCA1 Expression
BRCA1 mRNA Levels and EGFR Mutations in Patients with Advanced Lung Cancer
Median Survival
Progression-Free Survival
EGFR-Addicted Lung Cancers
Screening of EGFR Mutations for Individualizing Treatment with Erlotinib
Acquired Drug Resistance by Mutation of the T790 Residue in EGFR (T790M)
Conclusion
References
Nonspecific Cross-Reacting Antigen with a Mw of 160kD
Nonsteroidal Anti-inflammatory Drugs
Definition
Characteristics
Types of NSAIDs
Mechanism of Action
Cancer Chemoprevention
Colorectal Cancer
Breast Cancer
Prostate Cancer
Bladder Cancer
Lung Cancer
Conclusions
References
Nonsteroidal Anti-inflammatory Drugs (NSAIDs)
Nontargeted Effect
Non-Targeted Effects
Nonviral Vector for Cancer Therapy
Definition
Characteristics
Liposomes
Polymers
Virosomes
Fusion Liposomes
HVJ Envelope Vector
Cross-References
References
See Also
Norton-Simon Hypothesis
Definition
Characteristics
The Log-Kill Model
Gompertzian Growth Kinetics
Norton-Simon Hypothesis
References
Notch/Jagged Signaling
Definition
Characteristics
Bidirectional, Jagged1-Mediated Signaling
Clinical Relevance
T-Cell Leukemia
Cervical Carcinoma
B-Cell Leukemia
Mammary Carcinoma
Prostate Carcinoma
Feline Leukemia Virus (FeLV)-Induced T-Cell Leukemia
Cross Talk Between Signaling Pathways
Notch as a Potential Therapeutic Target
Cross-References
References
NPC
NPP2
NR1C
NR1I3
NR3C4
NRSTS
NS3
NSC-10514-F
NSC-312887
NSC362856
NSC-638850
NSC-684766
NSC-710428
Nuclear Factor-KappaB
Nuclear Factor-kappaB
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
References
Nuclear Receptor Coactivator 3
Nuclear Receptor Subfamily 1, Group I, Member 2 (NR1I2)
Nuclear Receptors
Nucleic Acid-Based Biomarkers
Definition
Characteristics
Types of Cancer Biomarkers
DNA-Based Cancer Biomarkers
RNA-Based Cancer Biomarkers
Conclusion
Cross-References
References
Nucleolin
Definition
Characteristics
Regulation of Nucleolin Function as a Phosphoprotein
Nucleolin Function in the Cell Cycle and Ribosomal Biogenesis
The Role for Nucleolin in Cancer
Role of Nucleolin in B-Cell Lymphomas
Nucleolin´s Interaction with Cellular Proteins: p53 and Rb
Nucleolin´s Interaction with Other Factors
References
Nucleoporin
Definition
Characteristics
The Nucleocytoplasmic Transport
Nucleoporins
Nucleoporins and Cancer
References
Nucleosome Remodeling
Nucleotide Excision Repair
Definition
Characteristics
Cellular Regulation
Global Genome Repair
Transcription-Coupled Repair
Excision and Gap Filling
Clinical Relevance
Cross-References
References
See Also
NUP98-HOXA9 Fusion
Definition
Characteristics
Mouse Models of Leukemogenesis
Human Models of NUP98-HOXA9 Leukemogenesis
Proposed Mechanism of Leukemic Transformation by the NUP98-NUP98 Fusion
Aberrant Transcriptional Activity
Stabilization of HOXA9 Protein Expression
Depletion of NUP98 from the NPC in Cells Expressing NUP98-HOXA9
References
NUPR1
Nutraceuticals
Synonyms
Definition
Characteristics
Nutraceuticals and Cancer
Cross-References
References
See Also
Nutrition Status
Definition
Characteristics
Mechanisms
Assessment of Nutrition Status
Nutrition Intervention
Benefit of Maintenance of Nutrition Status
Cross-References
References
See Also
Nutritional Factors
Nutritional Prevention of Colon Cancer
NY ESO 1
NY-ESO-1
Synonyms
Definition
Characteristics
Immunogenicity
NY-ESO-1-Derived Epitopes
Clinical Trials
Acknowledgments
Cross-References
References
O
1-(9Z-octadecenoyl)-2-sn-glycero-3-phosphate
42-O-(2-hydroxyethyl) Rapamycin
O/E
Oat Cell Carcinoma
Obesity and Cancer Risk
Definition
Characteristics
Trends in Obesity
Obesity and Cancer Risk
Obesity-Related Cancers
Cancers Likely Related to Obesity
Other Cancers
Mechanisms Linking Obesity to Cancer Risk
References
Occult Cancer
Oesophageal Adenocarcinoma
2OHOA
2-OHOA
Okadaic Acid
Definition
Characteristics
Tumor-Promoting Activity on Mouse Skin
Tumor-Promoting Activity in Rat Glandular Stomach
Crystal Structure of PP1-Okadaic Acid Complex
Biochemical Significance
Okadaic Acid Relatives
References
Further Reading
1-oleoyl lysophosphatidic acid
Olf
Olfactory Neuronal Transcription Factor
Olfactory/Early B-cell Factors
Oligoastrocytomas
Synonyms
Definition
Characteristics
References
Oligodendroglioma
Definition
Characteristics
History
Epidemiology
Clinical Presentation
Radiologic Appearance
Pathologic Features
Tumor Grading
Mixed Gliomas
Genetics
Therapy
Cross-References
References
See Also
o-LPA
Omental Immune Aggregates
Synonyms
Definition
Characteristics
Structure and Cellular Composition
Vascularization
Function
Tumor Cell Adherence and Metastases
References
Omentum-Associated Lymphoid Tissue
OncoArray
Definition
Cross-References
See Also
Oncodevelopmental Proteins
Oncofetal Antigen
Definition
Characteristics
Alpha-Fetoprotein: Role as an Oncofetal Antigen
Carcinoembryonic Antigen: Role as an Oncofetal Antigen
5T4 Oncofetal Antigen
Cross-References
References
Oncofetal Antigens
Oncofetal Stroma
Oncogene
Synonyms
Definition
Characteristics
Oncogene Cooperation
Cross-References
References
See Also
Oncogene Addiction
Synonyms
Definition
Characteristics
Multistage Carcinogenesis and Oncogene Addiction
Evidence for Oncogene Addiction
Mechanisms of Oncogene Addiction
Future Directions and Clinical Applications
Identification of the Critical Oncogene or ``Achilles´ Heel´´ in Specific Human Cancers
Combination Therapy
Conclusion
References
Oncogene Transduction
Oncologic Surgical Pathology
Oncology Models in Mice
Definition
Characteristics
Radiation and Chemically Induced Cancer Models
Genetically Engineered Mouse Models
Transgenic Mice
Gene Targeting
Conditional and Inducible Systems
Humanized Mice
Xenograft Models
Subcutaneous
Orthotopic
Sub-renal Capsule
Cross-References
References
Oncolytic Adenovirus
Synonyms
Definition
Characteristics
General Description
Mechanism
Clinical Applications
Future Directions
References
Oncolytic Virotherapy
Oncolytic Virus
Synonyms
Definition
Characteristics
Selectivity and Cancer-Targeting Mechanisms
Efficacy and Anticancer Mechanisms
Virus Species
Cross-References
References
Oncopeptidomics
Definition
Characteristics
Peptides
Peptide Biomarker
Peptide Biomarker Discovery
Challenges and Limitations in Oncopeptidomics
Conclusion
Cross-References
References
Oncoplastic Surgery
Definition
Characteristics
Historical Background
Clinical Impact of Oncoplastic Surgery
Summary
References
Oncostatin M
Synonyms
Definition
Characteristics
Oncostatin M Protein Structure
Oncostatin M Ligand-Receptor Interactions
Oncostatin M-Mediated Signal Transduction
Regulation of Oncostatin M and Oncostatin M Receptor Expression
Clinical Relevance of Oncostatin M in Cancer
Cross-References
References
See Also
Oncostatin M Precursor
Oncotype DX
Definition
Cross-References
See Also
ONO-5920
OPN
Optimal Treatment for Women with Ovarian Cancer
Definition
Characteristics
Screening and Early Diagnosis
Primary Surgery
Tumor Boards and Multidisciplinary Care
Neoadjuvant Chemotherapy
Initial Chemotherapy
Early-Stage Disease
Advanced-Stage Disease
Surveillance and Survivorship Issues
Treatment at Time of Recurrence
Palliative Care
Cross-References
References
See Also
Oral Cancer
Synonyms
Definition
Characteristics
Epidemiology and Etiology
Progression
Genetics
Treatment
Cross-References
References
See Also
Organometallic Drug
Orphan Nuclear Receptors
Synonyms
Definition
Characteristics
Mechanism
Clinical Aspects
Cross-References
References
See Also
OSF-1
Osf2
OSM
OSRC
Osteoblastic Bone Disease
Osteoblastic Bone Lesion
Definition
Cross-References
Osteoblastic Lesions of the Bone
Osteoblast-Specific Factor
Osteoblast-Specific Factor-2
Osteochondroma
Definition
Cross-References
Osteoclast
Definition
Cross-References
Osteogenic Sarcoma
Osteolysis
Definition
Cross-References
Osteolytic Bone Disease
Osteolytic Lesions of the Bone
Osteonectin
Osteopontin
Synonyms
Definition
Characteristics
OPN Gene Structure and Regulation of Gene Expression
OPN Protein Structure and Interaction with Other Proteins
Functions of OPN in the Context of Cancer
OPN-Mediated Cell Adhesion
OPN-Mediated Cell Migration and Invasion
OPN-Mediated Cell Survival/Resistance to Apoptosis
OPN and Angiogenesis
OPN and Metastasis
OPN Expression in Human Tumors and Prognostic Significance
Cross-References
References
See Also
Osteoporosis
Definition
Cross-References
Osteosarcoma
Synonyms
Definition
Characteristics
Epidemiology
Clinical Course
Treatment
Metastases
Recurrence
Genetics
Future
References
OTS-8
Out-of-Field Effect
Ovarian Cancer
Definition
Characteristics
Pathology
Staging
Epidemiology
Markers
Etiology
Molecular Mechanisms
Prognostic Indicators
Cross-References
References
See Also
Ovarian Cancer Chemoresistance
Definition
Characteristics
Symptoms and Treatment of Ovarian Cancer
Chemoresistant Disease
Mechanisms of Platinum Resistance
Mechanisms of Taxane Resistance
Circumventing Resistance
Cancer Stem Cells in Chemoresistance
Conclusions
Cross-References
References
See Also
Ovarian Cancer Chemotherapy
Definition
Characteristics
First-Line Chemotherapy
Taxane and Platinum-Based Chemotherapy
Intraperitoneal Chemotherapy
Novel Taxane Approaches
Neoadjuvant Chemotherapy
Maintenance Chemotherapy
Second-Line Therapy and Recurrent Disease
Platinum-Sensitive Recurrence
Platinum-Resistant Recurrence
Conclusions
Cross-References
References
See Also
Ovarian Cancer Clinical Oncology
Definition
Characteristics
Natural History
Screening and Early Diagnosis
Prevention
Primary Surgery of Ovarian Cancer
Primary Chemotherapy of Advanced Ovarian Cancer
Primary Chemotherapy of ``Early-Stage´´ Ovarian Cancer
Second-Line Chemotherapy in Ovarian Cancer
Future Directions in the Management of Ovarian Cancer
References
Ovarian Cancer Drug Resistance
Synonyms
Definition
Characteristics
Resistance to Platinum Drugs
Translational Research Bridges Cellular Drug Resistance and Clinical Outcome
References
Ovarian Cancer Epidemiology
Definition
Characteristics
References
Ovarian Cancer Hormonal Therapy
Definition
Characteristics
Hormones and Ovarian Cancer Etiology
Hormone Replacement Therapy and Ovarian Cancer Risk
Estrogens as Causative Factors
Progesterone as a Protective Factor
Progesterone and Estrogen Receptors as Prognostic Factors
Androgens as Causative Factors
Gonadotropins and GnRH as Risk Factors
Follicle-Stimulating Hormone, Luteinizing Hormone, and Their Receptors in Ovarian Cancer
GnRHs and Their Receptors in Ovarian Cancer
Hormonal Treatment
Aromatase Inhibitors
Antiandrogenic Drugs
GnRH Agonists
Selective ER Modulators
Anti-progestin Mifepristone in Ovarian Cancer
Progesterone in Recurrent Ovarian Cancer
Combination Hormonal Therapy
Tamoxifen Combined with Chemotherapy in Ovarian Cancer
Tamoxifen Combined with a GnRH Agonist in Recurrent Ovarian Cancer
GnRH Agonists Combined with Chemotherapy as First-Line Therapy in Ovarian Cancer
Combination of Progesterone and Other Hormonal Therapies in Recurrent Ovarian Cancer
Progesterone Combined with Chemotherapy in Ovarian Cancer
Contraindications to Hormonal Agents
Conclusions and Future Directions
Cross-References
References
See Also
Ovarian Cancer Immunotherapy
Definition
Characteristics
MAbs for the Immunotherapy of Ovarian Cancer
Cancer Antigen 125 (CA125, MUC16)
Folate Receptor Alpha (FRα)
Vascular Endothelial Growth Factor (VEGF)
Vaccines for the Immunotherapy of Ovarian Cancer
Cellular Vaccines
Vaccines Based on Defined Antigenic Substrates
Conclusion
Cross-References
References
See Also
Ovarian Cancer Pathology
Synonyms
Characteristics
General Classification of Ovarian Cancers
Epithelial Ovarian Cancer
Serous Carcinoma
Endometrioid Carcinoma
Clear Cell Carcinoma
Mucinous Carcinoma
Transitional Cell Carcinoma
Mixed Carcinoma
Undifferentiated Carcinoma
Malignant Mixed Müllerian Tumor
Sex Cord Stromal Tumors
Granulosa Cell Tumor
Sertoli-Leydig Cell Tumor
Steroid Cell Tumors Not Otherwise Specified
Sex Cord Tumor with Annular Tubules
Germ Cell Tumors
Teratoma
Dysgerminoma
Yolk Sac Tumor
Embryonal Carcinoma
Choriocarcinoma
Polyembryoma
Metastatic Tumors to the Ovary
Selected Diagnostic Markers
Staging of Ovarian Cancer
References
Ovarian Cancer Stem Cells
Synonyms
Definition
Characteristics
Classification of Ovarian Cancers
Isolation, Identification, and Characterization of Ovarian CSCs
Biology of Ovarian Cancer Stem Cells
Clinical Significance of Ovarian Cancer Stem Cells
Therapeutic Targeting of Ovarian Cancer Stem Cells
Controversies
Cross-References
References
See Also
Ovarian Cancer Stem-Like Cells
Ovarian Cancer-Initiating Cells
Ovarian Carcinoma
Ovarian Germ Cell Tumors
Definition
Classification of Ovarian Germ Cell Tumors
Stages
Markers
Treatments
Cross-References
See Also
Ovarian Mucinous Carcinoma
Definition
Cross-References
See Also
Ovarian Serous Carcinoma
Definition
Cross-References
See Also
Ovarian Small Cell Carcinoma
Ovarian Small Cell Carcinoma Hypercalcemic Type
Definition
Cross-References
Ovarian Stromal and Germ Cell Tumors
Definition
Characteristics
Epidemiology
Pathology
Clinical Presentation
Tumor Markers
Isolated Adnexal Mass
Hormonal Symptoms
Abdominal Pain
Advanced Disease
Surgery
Postoperative Treatment
Stromal Tumors
Germ Cell Tumors
Non-dysgerminoma Germ Cell Tumor Chemotherapy
Dysgerminoma Chemotherapy
Resection of Residual Masses
Posttreatment Prognosis and Follow-Up
Recurrent and Refractory Disease
Conclusion
Cross-References
References
See Also
Ovarian Tumors During Childhood and Adolescence
Synonyms
Definition
Characteristics
Epidemiology
Histology and Biology
Clinical Diagnosis
Surgical Therapy
Adjuvant Therapy
Cross-References
References
See Also
Oxidative Necrosis
Oxidative Stress
Definition
Characteristics
Oxidative Stress is Involved in Oncogenesis
Increased Oxidative Stress in Cancer Cells
Therapeutic Perspectives of Oxidative Stress Modulation
Differential Effects of Oxidative Stress in Normal and Cancer Cells
Use of Antioxidant Agents in Cancer Patients
Anticancer Agents Increase Oxidative Stress in Cancer Cells
Using Oxidative Stress Modulators as Anticancer Agents
Increasing the Therapeutic Index of Anticancer Agents by SOD Mimics
Cross-References
References
See Also
Oxygen Partial Pressure Distribution in Tumor
Oxygen Sensing
Synonyms
Definition
Characteristics
Prolyl Hydroxylase Domain-Containing Oxygenases (PHD)
Factor-Inhibiting HIF (FIH)
Histone Demethylases
DNA/RNA Demethylases
O2-Utilizing Enzymes in Other Biological Reactions
Concluding Remarks
References
Oxygen Tension Distribution in Tumor
Oxygen Toxicity
Oxygenation of Tumors
Synonyms
Definition
Characteristics
Pathomechanisms of Tumor Hypoxia
Detection of Tumor Hypoxia
Clinical Relevance
Tumor Oxygenation as a Prognostic Parameter
Modulation of Tumor Oxygenation
Cross-References
References
Oxygenation Status and Tumor Oxygen Level
Oxytosis
P
p/CIP
p14
p16
p16
p16INK4A
p16
p16
p185neu
p19
p21
Synonyms
Definition
Characteristics
Cell Cycle Inhibition by p21
Positive Regulation of Proliferation by p21
Binding of p21 to CDK and PCNA
Regulation of p21 Expression and Activity
Role of p21 in p53 Pathway
Role of p21 in Differentiation
Role of p21 in Senescence
Role of p21 in Apoptosis
Role of p21 in Cell Migration
Clinical Relevance
Tumor-Suppressing Function of p21
Tumor-Promoting Function of p21
Summary
Cross-References
References
p21(RAS)
p27
Synonyms
Definition
Characteristics
p27 Is a Strong Tumor Suppressor
Regulatory Mechanism for Degradation of p27
Phosphorylation Modifies the Functions of p27
p27 and Nuclear Export Molecules
Cytoplasmic Displacement of p27
p27 and Cell Migration
Conclusion
References
p27KIP1
p300/CBP-Interacting Protein
p40AIS
p51
p53
p53 Family
Synonyms
Definition
Characteristics
Molecular Structure and Function
Roles in Development
Role in Cancer: Tumor Suppressor or Oncogene?
Clinical Relevance of p63 and p73: Prognosis and Chemosensitivity
Concluding Remarks
References
p63
p73
p8
P8 Protein
Synonyms
Definition
Characteristics
Tumor Establishment and Progression
p8 and Cell Cycle Regulation
p8 and Apoptosis
Conclusion
Cross-References
References
See Also
p94
PA2.26
PA700
PACE
Paclitaxel
Definition
Characteristics
Mechanism of Action
Mechanism of Resistance
Pharmacokinetics
References
PADIs
PADs
PAF
Paired Basic Amino Acid-Cleaving Enzyme
Palladium Antitumor Agents
Palladium Antitumor Compounds
Palladium Drugs
Palladium-Based Anti-Cancer Therapeutics
Synonyms
Definition
Characteristics
Palladium
Palladium Complexes
The Interaction of Palladium Complexes with DNA
Palladium Complexes in Clinical Trials
Palladium-103 Brachytherapy
The Use of Palladium in Complementary Medicine
Conclusion
Cross-References
References
See Also
Palladium-Based Drugs
Palliative Care
Palliative Therapy
Definition
Characteristics
Palliative Radiotherapy
Palliative Chemotherapy
Hormonal Therapy
Conclusion
References
Pancreas Cancer
Pancreatic Cancer
Synonyms
Definition
Characteristics
Anatomy
Etiology (Cause of the Disease)
Pathology
Symptoms
Making the Diagnosis
Staging of the Disease
Prognosis
Treatment
Familial Pancreatic Cancer
Cross-References
References
Pancreatic Cancer Basic and Clinical Parameters
Definition
Characteristics
Pathology
Staging
Genetics
Cytogenetics
Oncogenes
Tumor Suppressor Genes
Epigenetic Changes
Hereditary Pancreatic Cancer
Diagnosis of Pancreatic Cancer
Abdominal Ultrasound
Computed Tomography (CT)
Magnetic Resonance Imaging (MRI)
Magnetic Resonance Cholangiopancreatography (MRCP)
Endoscopic Retrograde Cholangiopancreatography (ERCP)
Therapy of Pancreatic Cancer
Cross-References
References
Pancreatic Cancer Biology and Management
Definition
Characteristics
Human Pancreas
Symptoms of Pancreatic Cancer
Causes of Pancreatic Cancer
Diagnosis of Pancreatic Cancer
Treatment of Pancreatic Cancer
Self-Defense Against Cancer in Our Body
Expectation of Pancreatic Cancer Treatment (Prognosis)
Conclusion
Acknowledgments
References
Pancreatic Cancer Biomarkers
Definition
Characteristics
Tumoral/Peri-tumoral Biomarkers
Core Biopsy-Based Analysis
Aspiration-Based Analysis
Pancreatic Juice
Serum/Plasma
Cellular
Other Body Fluids
Importance of an Optimal Testing Population
Conclusion
Cross-References
References
Pancreatic Cancer Desmoplastic Reaction and Metastasis
Definition
Characteristics
Desmoplastic Stroma and Pancreatic Cancer Progression
Desmoplastic Stroma and Metastasis of Pancreatic Cancer
Conclusion
References
Pancreatic Cancer Development and Regeneration
Definition
Characteristics
Functional Morphology of the Pancreas
Endocrine Component
Exocrine Component
Epithelial Duct Cells
Cellular Events During Embryonic Development of the Pancreas
Signaling Pathways During Development
Hedgehog (Hh) Pathway: Gli TFs
Transforming Growth Factor-beta (TGF-b) Pathway: Smad TFs
Notch-Rbpj
Wnt Pathway: TCF/LEF TFs
Receptor Tyrosine Kinase (RTK) Pathway: FGF, EGF, IGF, NEGF, PDGF, and Eph TFs
Retinoic Acid (RA) Pathway and RAR TFs
Regeneration in the Mature Pancreas
References
Pancreatic Cancer Metastases
Pancreatic Cancer Metastasis
Synonyms
Definition
Characteristics
Clinical Management
Surgical Treatment
Palliative Treatment
Genetic and Molecular Background
Conclusion
Cross-References
References
Pancreatic Cancer Onset and Course
Pancreatic Cancer Pathogenesis
Synonyms
Definition
Characteristics
Pancreatic Cancer Epidemiology and Histological Classification
Progression of Pancreatic Cancer
Oncogenes
Tumor Suppressors
Growth Factors and Other Signaling Pathways
References
Pancreatic Cancer Stem Cells
Definition
Characteristics
References
Pancreatic Cancer Molecular Targets for Therapy
Definition
Characteristics
Oncogenes
KRAS Oncogene
Signaling Pathways of Ras
Raf-MAPK
Phosphoinositide-3-Kinase (PI3K) Pathway
Nuclear Factor kappaB
Ras Family GTPases
Tumor Suppressor Genes
CDKN2A/INK4A/p16
SMAD4 and TGF-beta
p53
Growth Factors
Epidermal Growth Factor Receptor (EGFR)
Vascular Endothelial Growth Factor Receptor (VEGFR)
Cytoplasmic Tyrosine Kinases
Matrix Metalloproteinases
References
Pancreatic Ductal Adenocarcinoma Metastasis
Pancreatitis
Definition
Characteristics
Causes and Diagnosis
Types of Pancreatitis
Acute Pancreatitis
Chronic Pancreatitis
Autoimmune Pancreatitis
Genetic Basis of Pancreatitis
PRSS1
SPNIK1
CFTR
CTRC
CASR
Experimentally Induced Pancreatitis
Pancreatic Cancer and Pancreatitis
Cross-References
References
See Also
Pannexins
Definition
Characteristics
References
Panzem
Papanicolaou Test
Definition
Cross-References
Papillary Thyroid Carcinoma
Definition
Cross-References
See Also
PAR
PAR1
PAR2
PAR3
PARAHOX
Definition
Parallel Gene Expression Analysis
Parathyroid Hormone-Related Protein
Synonyms
Definition
Characteristics
Protein Structure
Regulation of PTHrP Expression
Normal Functions
PTHrP and Cancer
References
PARP Inhibitors
Definition
Cross-References
Particle-Induced Cancer
Definition
Characteristics
Particle Size and Form
Deposition and Clearance of Particles in the Lungs
Mechanisms
Cross-References
References
See Also
Pathological Tumor Cell-Platelet Interaction
Pathology
Synonyms
Definition
Characteristics
Pathologic Factors Involved in the Prognostication of Human Neoplasms
Techniques in Diagnostic Pathology
References
Pathology of Malignant Ovarian Tumors
Pathway Addiction
Paxillin
Synonyms
Definition
Characteristics
Structure and Binding Partners
Regulation and Function
Other Paxillin Isoforms and Paxillin-Related Proteins
Cross-References
References
Paxillin α
PC Pathogenesis
PCA3
Definition
Characteristics
PCA3 gene
PCA3 as a Biomarker for Prostate Cancer
Conclusion
Cross-References
References
PCSK3
PDCD4
PD-ECGF
PDF
PDT
PDT-Induced Apoptosis
Synonyms
Definition
Characteristics
Role of Mitochondria in PDT-Induced Apoptosis
Lysosomes
Endoplasmic Reticulum (ER)
Golgi Complex
Role of Intracellular Signaling Pathways
Single Versus Two-Photon PDT
Translational Relevance
References
See Also
PEBP2αB
Pediatric Brain Cancers
Pediatric Brain Tumors
Synonyms
Definition
Characteristics
Introduction
Classification of Brain Tumors in Children
Biology of Pediatric Brain Tumors
Gliomas/Astrocytomas
Ependymomas
Medulloblastomas
PNETs
Atypical Teratoid-Rhabdoid Tumors (AT/RT)
Germ Cell Tumors
Craniopharyngiomas
Choroid Plexus Tumors
Meningiomas
Pediatric Brain Tumors Associated with Heritable Genetic Conditions
Clinical Presentation
Clinical Workup and Staging of Pediatric Brain Tumors
Staging of Pediatric Brain Tumors
Surgical Resection
Studies Required in Workup of Pediatric Brain Tumors
Special Considerations
Therapeutic Approaches
Surgical Resection
Radiation Therapy
Chemotherapy
Approach to Therapy in Specific Tumor Types
Malignant Brain Tumors in Infants and Young Children
Gliomas
Ependymomas
Medulloblastomas
PNETs
Atypical Teratoid-Rhabdoid Tumors (AT/RT)
Germ Cell Tumors
Craniopharyngiomas
Choroid Plexus Tumors
Meningiomas
References
Pediatric Central Nervous System (CNS) Tumors
Pediatric HIV/AIDS
Keywords
Definition
Characteristics
Determinants of Mother-to-Child Transmission of HIV
Determinants of Disease Progression in Pediatric HIV/AIDS
Epidemiological Data of Malignancies in HIV-Infected Children
Risk Factors of Malignancies in HIV-Infected Children
Conclusions
Cross-References
References
Further Reading
Pemetrexed
Definition
Cross-References
See Also
Pentakisphosphate
Definition
Characteristics
Inositol Phosphate Family
Inositol Pentakisphosphate (IP5)
IP5 Intracellular Functions
IP5 Anticancer Properties
Mechanism of Action of IP5
Clinical Perspective of IP5
References
Peptide Biomarkers
Definition
Characteristics
Peptide Biomarker Discovery
Cross-References
See Also
Peptide Vaccines for Cancer
Definition
Characteristics
Principles of Antitumor Immunity
Tumor Antigens for T Lymphocytes
Selection of Peptides for Vaccination
Immunization Strategies Using Synthetic Peptides
Remaining Challenges for Developing Effective Antitumor Peptide Vaccines
References
Peptidylarginine Deiminase Enzymes
Synonyms
Definition
Characteristics
Introduction
PAD Isoforms and Targets
PAD 1, 2, and 3
PAD4-Mediated Histone Tail Citrullination: An Emerging Role for PADs in Gene Regulation and Inflammation
Neutrophil Extracellular Traps (NETosis)
p53-PAD4 Pathway: Regulating Chromatin Decondensation and Apoptosis
Role of PADs in Cancer Pathogenesis
PAD4
PAD2
PAD Inhibitors Block Cancer Progression
PAD-Mediated Citrullination: Linking Inflammation with Cancer Progression?
Conclusion
References
Periostin
Synonyms
Definition
Characteristics
References
Peripheral Neurofibromatosis
Peripheral Neuropathy
Synonyms
Definition
Characteristics
Mononeuropathies and Plexopathies
Paraneoplastic Neuropathies
Toxic, Treatment-Related Neuropathies
References
Peripheral Primitive Neuroectodermal Tumor
Peritoneal Carcinomatosis
Peritoneal Dissemination
Peritoneal Malignancy
Peritoneal Mucinous Carcinoma
Peritoneal Tumor
Peroxisome Proliferator-Activated Receptor
Synonyms
Definition
Characteristics
PPARα
PPARbeta/delta (NUC1, FAAR)
PPARgamma
Cross-References
References
Personalized Cancer Medicine
Synonyms
Definition
Characteristics
Why Is Personalized Medicine Important in Cancer?
To What Extent Is Cancer Medicine Already Personalized?
The Future of Personalized Medicine in Cancer
The Challenges for Achieving Personalized Medicine
Conclusions
Cross-References
References
See Also
Peutz-Jeghers Syndrome
Synonyms
Definition
Characteristics
Lentiginosis (Pigment Spots)
Hamartomatous Polyposis
Increased Risk for Malignant Tumors
Pathogenesis
Therapy
Screening Recommendations
References
Peutz-Touraine Syndrome
P-Glycoprotein
Synonyms
Definition
Characteristics
Expression
Cellular and Molecular Regulation
Clinical Relevance
References
pgp-1
Phage Display
Synonyms
Definition
Characteristics
Development of Therapeutic and Imaging Agents for Cancer
Identification of Cancer-Specific Antigen, Interacting Partners, and Immune Response
Cross-References
References
See Also
Phagocytic Glycoprotein-1
Pharma Foods
Pharmacogenetics
Pharmacogenomics
Pharmacogenomics in Multidrug Resistance
Definition
Characteristics
Single Nucleotide Polymorphism (SNP)
ABC Transporters
ABCB1 (P-Glycoprotein/MDR1)
ABCC1 (MRP1/GS-X Pump) and ABCC2 (MRP2/cMOAT)
ABCG2 (BCRP/MXR1/ABCP)
Perspectives
Cross-References
References
See Also
Pharmacokinetics and Pharmacodynamics in Drug Development
Definition
Pharmacokinetics
Pharmacodynamics
Characteristics
Pharmacokinetics
Pharmacodynamics
Demonstrating the PK-PK-Clinical Relationship
Conclusions
References
Pharmacophore
Definition
Characteristics
Cross-References
References
Phase 2 Antioxidant Enzymes
Phase 2 Conjugation Enzymes
Phase 2 Detoxification Enzymes
Phase 2 Drug-Metabolizing Enzymes
Phase 2 Proteins
Phase II Enzymes
Synonyms
Definition
Characteristics
Multiple Isoforms with Some Phase 2 Enzymes
Functions of Phase 2 Enzymes
Regulation of Phase 2 Enzymes by Nrf2
Nrf2 Activation by Inducers of Phase 2 Enzymes
Cross-References
References
See Also
Pheochromocytoma
Definition
Characteristics
Epidemiology
Clinical presentation
Adrenal Incidentaloma
Genetics
Neurofibromatosis 1
Multiple Endocrine Neoplasia Type 2
Von Hippel-Lindau Syndrome
Paraganglioma Syndromes
Biochemical Evaluation
Imaging
Treatment
Preoperative Management
Operative Approach
Postoperative Management
Histopathological and Molecular Markers of Malignant Disease
Management of Malignant Disease
Genetic Testing
References
Phosphatase
Synonyms
Definition
Characteristics
Classes Based on the Optimum pH Condition
Classes Based on the Substrate
Phosphatases and Cancer
PTEN
PP2A
PP5
Protein Tyrosine Phosphatases (PTPs)
Conclusion
Cross-References
References
Phosphatidylinositol 3-Kinase
Phosphatidylinositol 3-Kinase (PI3-kinase)
Phosphatidylinositol-Binding Clathrin Assembly Protein
Phosphoinositide 3-Kinase
Phospholipase A2
Definition
Characteristics
Function
Regulation
Cancer Relevance
Cross-References
References
See Also
Phospholipase D
Definition
Characteristics
Hydrolysis and Transphosphatidylation Reactions
PLD Isoforms
Regulation of PLD Activity
Targets of PLD-Generated PA
Phospholipase D and Cancer
Conclusions
Cross-References
References
See Also
Photocarcinogenesis
Synonyms
Definition
Characteristics
Ultraviolet Radiation and Skin Cancer
Ultraviolet Radiation-Induced Skin Cancer or Photocarcinogenesis
Oxidative Stress and Photocarcinogenesis
DNA Damage and Photocarcinogenesis
p53 and Photocarcinogenesis
Inflammation, Immunosuppression, and Photocarcinogenesis
Defects in Signaling Pathways and Photocarcinogenesis
Conclusions and Recommendations
Cross-References
References
See Also
Photochemoprevention
Definition
Characteristics
References
Photodynamic Diagnostics
Photodynamic Laser Therapy
Photodynamic Therapy
Synonyms
Definition
Characteristics
Historical Perspective
Principle of PDT
Available Photosensitizers
Principles of Clinical Application
Molecular Mechanisms
Cross-References
References
Photodynamic Therapy-Induced Apoptosis
Photodynamic Tumor Therapy
Photo-Induced DNA Damage
Synonyms
Definition
Characteristics
UV-Induced Formation of Bipyrimidine Photoproducts
UVA Mediated Oxidation Reactions
Other Reactions Induced by Exogenous Photosensitizers
Detection of DNA Photodamage in Cells and Skin
Repair of DNA Photodamage
Skin Carcinogenesis
Cross-References
References
Photothermal Ablation
Photothermal Therapy
Synonyms
Definition
Characteristics
Principles of Photothermal Therapy
Heat Generation in a Laser-Tissue Interaction
Cell Death Mechanisms in Photothermal Therapy
Selective Photothermal Therapy with the Aid of Nano-sized Particles
Applications of Photothermal Therapy in Cancer Treatment
Conclusion
Cross-References
References
Further Reading
Photothermal Treatment
Ph-positive Chronic Leukemia
Phyto-cannabinoids
Phytochemicals in Cancer Prevention
Synonyms
Definition
Characteristics
Epidemiology
Cancer Chemopreventive Mechanisms
Cancer Chemopreventive Activity in Rodents
Clinical Trials
Cross-References
References
Phytoestrogens
Definition
Characteristics
Potential Benefits of Phytoestrogens
Potential Drawbacks of Phytoestrogens
Cross-References
References
PI3K Signaling
Synonyms
Definition
Characteristics
PI3K Classification and Signaling Pathways
Class IA PI3K
Class IB PI3K
Class II PI3K
Class III PI3Ks
Class I PI3Ks
Class II PI3Ks
Class III PI3Ks
PI3K Signaling Pathways
Negative Regulation of PI3K Signaling Pathways
Main Phenotypes Induced by PI3K Signaling Pathways
PI3K Signaling and Cell Growth
PI3K Signaling, Tumorigenesis, and Cancers
PI3K, Insulin Signaling, and Diabetes
Future Directions
Cross-References
References
See Also
PI3-Kinase
PI-3-Kinase
PICALM
Synonyms
Definition
Characteristics
PICALM Is Involved in Clathrin-Mediated Endocytosis
PICALM Is Necessary for Clathrin Assembly at the Trans-Golgi Network
The Role of PICALM in Normal Hematopoiesis and Iron Homeostasis In Vivo and In Vitro
PICALM and Malignant Hematopoiesis
A Potential Role for PICALM in Alzheimer Disease
References
PIK3CA
Synonyms
Definition
Characteristics
Genetics
Clinical Relevance
References
Pilocytic Astrocytoma
PIM Protein Kinase Family
Synonyms
Definition
Characteristics
Regulation of Expression and Activity
Role in Carcinogenesis
PIM Kinases and Protein Synthesis
Role in Drug Resistance
PIM Kinases as Therapeutic Targets
Cross-References
References
Pinocytosis
Pituitary Tumor-Transforming Gene 1
PJS
PKC
PL74
PLAB
Placenta Growth Factor
Synonyms
Definition
Characteristics
Clinical Relevance
Cross-References
References
Placental Growth Factor
Placental Site Trophoblastic Tumor
Synonyms
Definition
Characteristics
Clinical Features
Pathological Features
Molecular Etiology
References
Plant-Derived Agents
Plasma Cell Disorder
Plasmacytoma
Definition
Characteristics
Epidemiology
Etiology
Pathogenesis
Clinical Features
Imaging Studies
Treatment
Follow-Up Outpatient Care and Progression to MM
Medical and Legal Pitfalls
Basic Research
Cross-References
References
See Also
Plasminogen-Activating System
Definition
Characteristics
The Plasminogen-Activating System in Cancer Progression
Clinical Significance of PAS Components Expression in Cancer
PAS as Target for Anticancer Therapy
Cross-References
References
See Also
Platelet
Synonyms
Definition
See Also
Platelet-Activating-Factor
Synonyms
Definition
Cross-References
Platelet-Derived Endothelial Cell Growth Factor
Platelet-Derived Growth Factor
Definition
Characteristics
Genes
Bioactivity
Clinical Relevance
Cross-References
References
Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors
Platinating Agents
Platinum Antitumor Agents
Platinum Antitumor Compounds
Platinum Complexes
Synonyms
Definition
Characteristics
Mode of Action
Bioactivation
Formation of Platinum-DNA Adducts
Cellular Response
Mechanisms of Resistance
Reduced Accumulation
Increased Inactivation
Increased Adduct Tolerance and Failure of Apoptotic Pathways
Increased Repair
Approved Platinum Complexes
Cisplatin
Carboplatin
Oxaliplatin
Nedaplatin
Lobaplatin
Heptaplatin
New Platinum Complexes Under Development
Platinum(IV) Complexes
Sterically Hindered Platinum Complexes
Multinuclear Platinum Complexes
Drug Delivery Conjugates with Platinum Complexes
References
Platinum Drugs
Platinum-Refractory Testicular Germ Cell Tumors
Synonyms
Definition
Characteristics
Mechanisms of Platinum Resistance
Treatment of Patients with Cisplatin-Refractory GCTs
Drugs with No or Minor Activity in Refractory GCTs
Active Agents
Salvage Surgery
References
Platyfish-Swordtail Melanoma
Pleiotrophin
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Pleomorphic Xanthoastrocytoma
Pleural Effusion
Synonyms
Definition
Characteristics
Clinical Manifestations and Diagnosis
Thoracentesis
Treatment
Therapeutic Thoracentesis
Tunneled Pleural Catheter
Pleurodesis
Mechanical Pleurodesis
Other Treatment Options
Chemotherapy
Cross-References
References
See Also
Pleurodesis
Plexin Ligands
Plexins
Synonyms
Definition
Characteristics
Plexin Function
Plexins in Tumor Biology
Plexin D1
B Plexins
Cross-References
References
Plexopathy
PLG61
PLP
PLXN
PMP
Podoplanin
Synonyms
Definition
Characteristics
Podoplanin Expression in Human Tumors
Regulation
Podoplanin and Tumor Invasion
Podoplanin and Metastasis
Podoplanin as a Marker for Tumor-Associated Lymphatic Vessels
Conclusions
Cross-References
References
See Also
Podosomes and Invadopodia
POEMS Syndrome
Synonyms
Definition
Cross-References
Pol I Transcription
Poly(ADP-Ribosyl)ation
Definition
Characteristics
Catalytic Function of PARP-1 and Life Cycle of Poly(ADP-Ribose)
Molecular Functions Related with Regulation of DNA Strand Breaks and of DNA Repair
Molecular Functions Related with the Maintenance of Genomic Stability
Molecular Functions Related with DNA Replication
Molecular Functions Related with Gene Expression
Molecular Functions Related with Energy Metabolism and Mitochondrial Changes
Cellular Functions
Cytoprotection and Maintenance of Genomic Stability
Cell Death Induction
Clinical Relevance
Genetic Cancer Risk Assessment
Therapy
Cross-References
References
See Also
Polyamines
Definition
Characteristics
Polyamine Metabolism and Transport
Polyamine Levels and Cancer Cell Growth
Mechanisms of Polyamine Upregulation in Cancer
Development of Polyamine Analogs for Cancer Chemotherapy and Their Mechanism of Action
Polyamines and DNA Promoter Methylation
Polyamine Pathway in Cancer Chemoprevention
Cross-References
References
Polyaromatic Compounds
Polycomb Group
Definition
Characteristics
PcG Protein Complexes
PcG Proteins and Stem Cell Maintenance
PcG Proteins in Human Malignancies and Their Clinical Relevance
References
Polycyclic Aromatic Hydrocarbons
Synonyms
Definition
Characteristics
Anthropogenic Origin and Carcinogenicity
Molecular Mode of Action
Cross-References
References
Polycystic Kidney Disease
Synonyms
Definition
Characteristics
Clinical Features
ADPKD
ARPKD
Molecular Basis
ADPKD
ARPKD
Cilia and PKD
Animal Models
Therapy and Perspective
Cross-References
References
Polycythemia
Synonyms
Definition
Characteristics
References
Polymeric Nanoparticles
Polymorphonuclear Leukocyte Elastase
Polyneuropathy
Polyomavirus Enhancer Binding Protein 2αB
Polyphenols
Definition
Characteristics
Cellular and Molecular Studies
Clinical Studies
Cross-References
References
See Also
Polyserase (TMPRSS9)
Polyunsaturated Fats
Polyunsaturated Fatty Acids
Synonyms
Definition
Characteristics
PUFA Intake and Cancer Risk
Cellular and Molecular Mechanisms Mediating omega-3-PUFA Anticancer Activity
Omega-3 PUFAs Regulate Membrane Lipid Composition and Structure
Omega-3 PUFAs and Bioactive Hydroxylated Polyunsaturated Metabolites
Binding of Omega-3 PUFAs and of Their Bioactive Lipid Mediators to Fatty Acid Receptors
Omega-3 PUFA-Based Therapeutic Strategies and Clinical Trials in Cancer
Cross-References
References
Population Candidate Gene Association Study
Population-Based Cancer Research
Positron Emission Tomography
Synonyms
Definition
Characteristics
Principle of Positron Emission Tomography (PET)
Radiolabeled Biomarkers for PET Imaging Specifically Addressing Metabolic Pathways or Target Molecules
Clinical Applications of PET and PET/CT
Differentiation of Benign from Malignant Tumors and Detection of the Primary Tumor (Cancer of Unknown Primary)
Staging of Cancer, Prognostic Potential of PET
Assessment of Response to Therapy
Restaging of Cancer, Detection of Recurrence
Radiation Treatment Planning
PET for Anticancer Drug Development
Cross-References
References
See Also
Postirradiation Sarcoma
Postnatal Stem Cells
Postreplication Repair
Postsurgical Systemic Therapy
pp60
pp60
PPAR
pRB
Preclinical Drug Safety Evaluation
Preclinical Safety Testing
Preclinical Testing
Synonyms
Definition
Characteristics
Discovery Phase
Phase I Clinical Study
Phase II and III Clinical Studies
References
Pregnane X Receptor (PXR)
Preinvasive Breast Cancer
Preleukemia
Pre-mRNA Splicing
Definition
Characteristics
Spliced Forms in Cancer
Therapeutic Intervention
Cross-References
References
See Also
Preneoplastic Lesions
Definition
Characteristics
Some Experimental Models of Preneoplastic Lesions
Human Preneoplasia
Genetic Predisposition to Neoplasia
Clinical Relevance
References
Preoperative Chemotherapy
Presenilin
Synonyms
Definition
Characteristics
Function
PS/gamma-Secretase-Mediated Notch Signaling and Cancers
gamma-Secretase-Independent Functions of Presenilins
References
See Also
Prevention
Primary Chemotherapy
Primary Liver Cancer
Primary Myelofibrosis
Synonyms
Definition
Characteristics
Background
Historical Perspective
Disease Mechanisms
Clinical and Laboratory Characteristics
Diagnosis
Prognosis and Treatment
References
Primary Systemic Therapy
Probiotics and Cancer
Definition
Characteristics
Use of Probiotic in Cancer Management
Probiotic Formulations
Future Prospects
Limitations Association with Probiotics Application
Conclusion
Cross-References
References
Progestin
Synonyms
Definition
Characteristics
Mechanisms
Clinical Significance
Cross-References
References
Progestogen
Programmed Cell Death
Programmed Cell Death 4
Synonyms
Definition
Characteristics
Tumor Suppressor Activity
Control of Translation
Control of Transcription
Induction of Apoptosis
Control Mechanisms of PDCD4 Expression
PDCD4 in ROS, Inflammation, and Infection
Roles in Cell Differentiation
Clinical Aspects
References
Programmed Form of Necrotic Cell Death
Programmed Necrosis
Progression
Definition
Characteristics
Tumor Stem Cell and Metastasis
The Tumor Stroma and Metastasis
The Metastatic Cascade: Effector and Suppressor Molecules
Clinical Relevance
References
Prohormone Convertase
Prolactin
Synonyms
Definition
Characteristics
Pituitary Prolactin Secretion and Its Regulation
Prolactin Structure and Activation of Cellular Signaling
Biological Effects of Prolactin
Disease States Related to Prolactin
Hyperprolactinemia and Prolactinoma
Prolactin and Breast Cancer
References
Proppins
2-Propylpentanoic Acid
Prosome
Prostaglandins
Definition
Characteristics
Biosynthesis
Regulation of Prostaglandin Synthesis and Degradation
Prostaglandin Signaling Pathway
Prostaglandin Signaling Pathway in Cancer
Cross-References
References
Prostate Cancer
Definition
Characteristics
Risk for Prostate Cancer
Symptoms for Prostate Cancer
Screening
Cross-References
Further Reading
Prostate Cancer Chemoprevention
Prostate Cancer Chemotherapy
Definition
Characteristics
Androgen Deprivation Therapy
Docetaxel (Taxotere) Chemotherapy
Bone Targeting
Tyrosine Kinase Inhibitors
Angiogenesis
Apoptosis
Other Chemotherapy Agents
Cabazitaxel Chemotherapy
Nutraceuticals
Conclusion
References
Prostate Cancer Clinical Oncology
Definition
Characteristics
Clinical Epidemiology and Risk Factors
Etiology
Tumor Biology and Genetics
Clinical Presentation
Diagnosis and Staging
Management
References
Prostate Cancer Designed Treatment
Prostate Cancer Diagnosis
Synonyms
Definition
Characteristics
Slow Development of Prostate Cancer
The Detection Process
Deciding That Diagnosis Is Likely to Be Beneficial
Survival
Non-survival Considerations
Active Surveillance
5-Alpha-Reductase Therapy
At-Risk Patients
Screening
Calculators
Testing for Prostate Cancer
PSA Detection of Coexisting Prostate Cancer
Attempts to Improve PSA Detection of Prostate Cancer
PSA and 5-Alpha-Reductase Therapy
PSA Blood Test in Detection of Future Prostate Cancer
PCA3 Post-Prostatic Massage Urine Test
Multiparametric Magnetic Resonance Imaging (mpMRI)
Biopsy
Initial Biopsy
Repeat Biopsies
Conclusions
Cross-References
References
See Also
Prostate Cancer Epidemiology
Definition
Characteristics
Global Epidemiology of Prostate Cancer
Age-Specific Risk of Prostate Cancer
Pathogenesis of Prostate Cancer
Etiology of Prostate Cancer
Finasteride and Prostate Cancer
Dietary Fat and Prostate Cancer
Nonsteroidal Anti-inflammatory Drugs (NSAIDs) and Prostate Cancer
Genetic Risk for Prostate Cancer
Nutritional Supplements and Prostate Cancer
Prostate Cancer and Sexual Activity
Screening for Prostate Cancer
Zinc and Survival of Prostate Cancer
References
Prostate Cancer Experimental Therapeutics
Synonyms
Definition
Characteristics
Gene Therapy
Vaccine Therapy
Chemotherapeutic Agents
Antiandrogen Therapy
Photodynamic Therapy
Other Agents
Conclusion
Cross-References
References
See Also
Prostate Cancer Genetic Toxicology
Synonyms
Definition
Characteristics
Evidence from Migrant Studies
Candidate Causative Agents
Metabolic Activation in the Prostate
Genetic Damage in the Prostate
Future Directions
Cross-References
References
Prostate Cancer Hormonal Therapy
Definition
Characteristics
Epidemiology
Hormonal Management of Prostate Cancer
Androgen Receptor Biology in Prostate Cancer
Novel Hormonal Compounds for Prostate Cancer Treatment
Conclusions
Cross-References
References
See Also
Prostate Cancer Molecular Carcinogenesis
Prostate Cancer Molecularly Targeted Therapies
Synonyms
Definition
Characteristics
Tyrosine Kinase Inhibitors
Epidermal Receptor Growth Factor Tyrosine Kinase Inhibitors
Platelet-Derived Growth Factor Inhibitors
Vascular Endothelial Growth Factor Inhibitors
PI3K/mTOR/Akt Inhibitors
Endothelin A Receptor Antagonists
Epigenetic Pathway Inhibitors
Antiapoptotic Protein Inhibitors
Insulin-Like Growth Factor Pathway Inhibitors
Conclusion
References
Prostate Cancer Prevention
Synonyms
Definition
Characteristics
Androgens and Prostate Cancer Prevention
Selenium and Vitamin E
Green Tea
Pomegranate
Conclusion
Cross-References
References
Prostate Cancer Radionuclide Imaging
Definition
Characteristics
Bone Scintigraphy
Radiolabeled Monoclonal Antibodies
Positron Emission Tomography
18F-Fluorodeoxyglucose
Novel Positron-Emitter Radiotracers
11C-Acetate
11C-Choline and 18F-Fluorocholine
Androgen Receptor Imaging
Molecular Imaging
Prostate Lymphoscintigraphy
Conclusion
Cross-References
References
See Also
Prostate Cancer Radionuclide Therapy
Definition
Characteristics
Indications and Contraindications for Radionuclide Therapy in Bone Pain Management
Administration, Efficacy, and Toxicity of Radionuclide Therapy
Radiopharmaceuticals
89Strontium Chloride
153Samarium Lexidronam
186Rhenium HEDP
188Rhenium HEDP
Advantages of Radionuclide Therapy
New α-Particle-Emitting Radiopharmaceutical Radium Dichloride (Ra-223)
Conclusions
Cross-References
References
See Also
Prostate Cancer Stem Cells
Definition
Characteristics
Assays to Identify PCSC
Markers of PCSC
Origin of PCSC
Implications for Prostate Cancer Treatment
References
Prostate Cancer Targeted Therapy
Definition
Characteristics
Targeting Prostate Cancer Cells
Targeting Tumor Angiogenesis
Targeting Cell Signal Pathways
Targeting Bone Metastasis
Conclusion
Cross-References
References
See Also
Prostate Carcinoma
Prostate Tumor
Prostate-Specific Antigen
Synonyms
Definition
Characteristics
Clinical Aspects
PSA as Diagnostic Marker
Percent-Free PSA
PSA, %fPSA for Staging and Grading
PSA for Recurrence of PCa
Monitoring After Radical Surgery
Use of PSA to Monitor Radiation Therapy
PSA Monitoring After Androgen Deprivation Therapy
Conclusion and Future
Cross-References
References
Prostate-Specific Membrane Antigen
Synonyms
Definition
Characteristics
PSMA Expression
Enzymatic Functions of PSMA
Does PSMA Have a Signaling Function?
PSMA and Imaging
PSMA as a Therapeutic Target
References
Prostatic Adenocarcinoma
Prostatic Carcinoma
Protease-Activated Receptor Family
Definition
Characteristics
Melanoma
Breast Cancer
Prostate Cancer
Pancreatic Cancer
Lung Cancer
Colon Cancer
References
Protease-Activated Receptors
Synonyms
Definition
Characteristics
References
26S Proteasome
Proteasome
Synonyms
Definition
Characteristics
Structural and Functional Organization of the Proteasome
The 26S Proteasome
The 20S Core Particle
The 19S Regulatory Particle
Assembly of Proteasomes
The Ubiquitin-Proteasome System and Ubiquitin-Independent Functions of the Proteasome
The Ubiquitin-Proteasome System
Ubiquitin-Independent Functions of the Proteasome
Proteasomes in Normal Cell Biology
The Cellular Contexts of Proteasome Function
Cellular Distribution of Proteasomes
Immunoproteasomes
Proteasomes in Cancer Cell Biology
Proteasomes and the Cellular Hallmarks of Cancer
Apoptosis Control and Resistance
Epithelial-Mesenchymal Transition
Immunoproteasomes
Cancer Genes, Hereditary Cancer Syndromes, and the Proteasome
Proteasomal Regulation of Cancer Genes
Proteasomal Regulation by Cancer Genes
Cancer Cachexia
Targeting the Proteasome in Cancer Therapy
Cross-References
References
20S Proteasome Catalytic Core Particle
Proteasome Complex
Proteasome Inhibitors
Definition
Characteristics
Rationale for Therapeutic Utility of Targeting Proteasome
Proteasome and Its Functional Inhibitors
Mechanisms Mediating Bortezomib-Induced Apoptosis in MM Cells
Blockade of Pro-Survival Signaling
Induction of Proapoptotic Signaling
Clinical Relevance
New Proteasome Inhibitor NPI-0052/Marizomib
Cross-References
References
See Also
19S Proteasome Regulatory Particle
Protein Acetyltransferases (PATs)
Protein Arginine Deiminases
Protein Array
Protein Disulfide Reductase
Protein Kinase C Family
Synonyms
Definition
Characteristics
Regulation
PKC Expression in Cancer
PKC Activation in Carcinogenesis
Targets of PKC and Its Cancer-Related Functions
PKC as a Target in Cancer Therapy
Cross-References
References
See Also
Protein Kinases
Definition
Characteristics
Chemical Activity
Classification
Protein Interaction Domains
Regulation
Function
Structure
Deregulation
Pharmacological Strategies
Kinomics and Profiling
Future Direction
References
See Also
Protein Synthesis
Proteinase-Activated Receptor-1
Synonyms
Definition
Characteristics
References
Proteinase-Activated Receptor-2
Synonyms
Definition
Characteristics
Proteinase-Activated Receptor-3
Synonyms
Definition
Characteristics
Proteinase-Activated Receptor-4
Synonyms
Definition
Characteristics
Proteinchip
Synonyms
Definition
Characteristics
Technologies
Analytical Approaches
Applications in Cancer Research
Challenges and Perspectives
References
14-3-3 Proteins
Definition
Cross-References
Protein-Tyrosine Kinase Inhibitors
Proteoglycanase
Proteomics
Definition
Characteristics
Subproteomes and Their Advantages
Common Protein Profiling Methods
Proteome Informatics
Clinical Aspects
Cross-References
References
See Also
Protoadjuvant Therapy
Proton Beam Therapy
Synonyms
Definition
Characteristics
Physical Properties
Biological Properties
Clinical Aspects
Uveal (Choroidal) Melanoma
Sarcomas of the Skull Base and Spine
Head and Neck Cancers
Lung Cancer
Hepatocellular Carcinoma
Prostate Cancer
Childhood Cancers
Miscellaneous
Facilities and Costs for Proton Beam Therapy
Cross-References
References
Proton Radiation Therapy
Proton Therapy
Protracted Low-Dose Chemotherapy
Proviral Integration of Moloney Virus
Prozac
Prune
Definition
Characteristics
Regulation
Clinical Relevance
Inhibition
References
PS
PS 341
PSEN1
PSEN2
Pseudomyxoma Peritonei
Synonyms
Definition
Characteristics
Epidemiology
Historical Perspective
Natural History
Appendiceal Origin
Pathological Classification
Presentation
Staging Systems
Cytoreductive Surgery
Rationale for Intraperitoneal Chemotherapy
Prognosis
Palliative Treatment Options
Basic Science
Cross-References
References
PSMA
PSMD10
PSTT
PtdIns 3-Kinase
PTEN Hamartoma-Tumor Syndrome
Pterophyllus salisburiensis
PTGF-b
PTHLH
PTHrP
PTKIs
PTN
PTTG1
PUFAs
PUMA
Synonyms
Definition
Characteristics
Discovery and Function
Regulation
Clinical Considerations
Cross-References
References
See Also
Purine Nucleoside
Putative NF-Kappa-B-Activating Protein 002N
Pyrexia of Unknown Origin
Pyroptosis
Definition
Characteristics
List of Abbreviations
Inducing Agents for Pyroptosis
Caspase 1-Activated Cellular Responses in Pyroptotic Cells
Contrasts Between Pyroptosis and Apoptosis
Function of Pyroptosis
References
Q
QSAR
Quantitative Structure Activity Relationship
Synonyms
Definition
Characteristics
Cross-References
References
Quantitative Trait Loci
Quinone Methide Friedelane Tripterene (2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,
R
R2
RAC3
RAD001
RAD001 (Everolimus)
Radiation Carcinogenesis
Synonyms
Definition
Characteristics
Radiation
Interaction of Radiation with Matter
Interaction with Living Cells
Clinical Issues
References
Radiation Oncology
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Radiation Sensitivity
Synonyms
Definition
Characteristics
Clinical Radiation Sensitivity or ``Adverse Radiation Effects´´
References
Radiation Therapy
Radiation-Induced Bystander Effect (RIBE)
Radiation-Induced Neoplastic Transformation
Radiation-Induced Sarcomas
Radiation-Induced Sarcomas After Radiotherapy
Synonyms
Definition
Characteristics
Carcinogenesis
Dose-Effect Relation
Megavoltage or Orthovoltage Radiotherapy
Latent Period
Histological Findings
Age and RIS
Incidence
Genetic Findings
Treatment
Conclusion
Cross-References
References
See Also
Radioactive Seed Therapy
Radiochemotherapy
Radioimmunotherapy
Synonyms
Definition
Characteristics
Principle of RIT
Tumor Characteristics
Monoclonal Antibody and Tumor Antigen
Radionuclides
RIT in Malignant Lymphoma
Cross-References
References
See Also
Radioisotope Therapy
Radiological Response Criteria
Definition
Characteristics
Historical Developments
Strengths of Radiological Response Criteria
Limitations of RECIST
Future Developments and Possible Solutions
References
Radioprotective Molecules
Radioprotectors
Synonyms
Definition
Characteristics
Necessity of Radioprotectors in Cancer Radiotherapy
Necessity of Radioprotectors Against Population Exposure
Radiation-Induced Damaging Species
DNA Is a Critical Target of Ionizing Radiations
Mechanisms of DNA Radioprotection
Natural DNA Radioprotectors
Pharmaceutical Radioprotectors
Ethyol (Amifostine) as Radioprotector
Cross-References
References
Radiosensitivity
Radiosensitization
Definition
Characteristics
Evaluation of a Radiosensitization Effect
Mechanisms of Radiosensitization
References
Radiosensitizer
Definition
Characteristics
Background
Target-Based Radiosensitizers
Signaling Molecules as Targets
DNA Damage Response as a Target
Tumor Microenvironment as a Target
Cross-References
References
See Also
Radiotherapy
Radon
Synonyms
Definition
Characteristics
Occurrence
Exposure Situations
Population Under Risk
Health Hazards
Mechanisms
References
Radon Daughter Products
Radon Decay Products
Radon Progeny
1-radyl-sn-glycerol-3-phosphate
RAF
Raf Kinase
Synonyms
Definition
Characteristics
Raf Signaling
Raf Function
Raf in Cancer
Raf Pathway Inhibitors
References
Raf-1
RAFB1
Ral-Interacting Protein 76kDa
RANDAM-2
RANK - Receptor Activator of NF-kappaB, TNFRSF11A, OFE, ODFR, TRANCE-R, ODAR, and CD265
RANKL - Receptor Activator of NF-kappaB Ligand, TNFSF11, OPGL, ODF, TRANCE, and CD254
RANK-RANKL Signaling
Synonyms
Definition
Characteristics
RANKL Function in Bone Remodeling
RANKL Signaling
RANKL and the Vicious Cycle of Bone Metastases
Additional Functions of RANKL in Cancer Cells
References
Rap1 and Sipa-1
Definition
Characteristics
Biological Functions of Rap1
Interference with Ras-Mediated ERK Activation
Activation of MAP Kinases and Other Signaling Pathways
Regulation of Cell Adhesion and Migration
Regulation of Rap1 Signal by Spa-1
Rap1 and Spa-1 in Leukemia
Rap1 and Spa-1 in Cancer Metastasis
Clinical Aspects
References
Rapamune
Rapamycin
Synonyms
Definition
Characteristics
Discovery of Rapamycin
Mechanism of Action
Mechanism(s) of Sensitivity and Resistance
Clinical Activity of Rapamycins
Cross-References
References
See Also
RAS Activation
Definition
Characteristics
Clinical Aspects
Cross-References
References
See Also
RAS-Association Domain Family 1
Synonyms
Definition
Characteristics
RASSF1A as a Tumor Suppressor Gene
RASSF1A Methylation as a Cancer Biomarker
Functional Characterization of RASSF1A
Conclusion
References
RAS Genes
Definition
Characteristics
Structure
Cellular and Molecular Regulation
Clinical Relevance
Cross-References
References
See Also
RAS Homologous Proteins
RAS Pathway-Responsive Transcriptome
RAS Transformation Targets
Synonyms
Definition
Characteristics
Background
RAS Proteins Affect Gene Transcription via Cytoplasmic Effectors
Identification of RAS Transformation Targets
A Selected List of RAS Transformation Targets
Clinical Relevance
References
RASH1
RASK2
RAS-Regulated Genes
RAS-Related Small
RAS-Responsive Genes
RASSF1
RB
RB1
RB/p105
RCC
RCLS
rDNA Transcription
rDNA-Derived Therapeutic Proteins
RE1-Silencing Transcription Factor
Synonyms
Definition
Characteristics
Alternative Splicing of REST
Cancer
Lung Cancer
Breast Cancer
Glioma
Sarcoma
Melanoma
Medulloblastoma
Neuroblastoma
Conclusion
References
See Also
Reactive Oxygen Species
Synonyms
Definition
Characteristics
Chemistry of Major ROS
Generation and Scavenger of ROS
Oxidative Stress
Clinical Aspects
Cross-References
References
See Also
Receptor Cross-Talk
Synonyms
Definition
Characteristics
Survival Signaling/p53 and Suppressor Cross-Talk
Signaling to Angiogenesis
Integrin: Growth Factor Receptor Cross-Talk
Cross-References
References
See Also
Receptor for CXCL12
Receptor for Stromal Cell-Derived Factor-1 Alpha
Receptor Interactions
Receptor Tyrosine Kinase Inhibitors
Synonyms
Definition
Characteristics
Development
Target: Epidermal Growth Factor Receptor (EGFR)
Target: Vascular Endothelial Growth Factor Receptor (VEGFR)
Target: Fibroblast Growth Factor Receptor (FGFR)
Target: Platelet-Derived Growth Factor Receptor (PDGFR)
Molecular Mechanisms
Specificity and Potency
Cross-References
References
Receptor Tyrosine Kinases
Synonyms
Definition
Characteristics
Physiological RTKs Activation
Deregulation of RTKs in Malignant Cells
Clinical Relevance
Cross-References
References
See Also
Receptor-Associated Coactivator 3
Receptor-Mediated Endocytosis
Receptors
Definition
Characteristics
Receptor Classification
Extracellular Receptors
Intracellular Receptors
References
Receptors Regulated by Ligands
Receptors with Empty Ligand-Binding Pockets
Receptors with No Ligand-Binding Pocket
Receptors with Structural Ligands
Recessive Oncogenes
RECK Glycoprotein
Synonyms
Definition
Characteristics
Regulation
Clinical Relevance
Cross-References
References
See Also
Recombinant Therapeutics
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Rectal Cancer
Synonyms
Definition
Characteristics
Presentation
Diagnosis
Staging
Treatment
Surgery
Neoadjuvant Therapy
Adjuvant Therapy
Salvage and Palliative Therapy
Survivorship
Cross-References
References
Rectal Carcinoma
Rectal Tumor
Reductases
Synonyms
Definition
Characteristics
Carbonyl Reduction by SDRs and AKRs
The ``Pro´´
Reductases Protect Against Lung Cancer
Clinical Aspects
The ``Contras´´
Reductases in Chemotherapy Resistance
Cardiotoxicity
Clinical Aspects
References
Regeneration
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Regorafenib
Synonyms
Definition
Characteristics
Preclinical and Phase I Activity of Regorafenib
Activity of Regorafenib in mCRC
Activity of Regorafenib in GIST
Activity of Regorafenib in Other Solid Organ Tumors
References
Regulator of Extracellular Matrix Integrity
Regulatory T Cells
Synonyms
Definition
Characteristics
Molecular Markers of Treg Cells
Treg Cell in Cancer and their Antigen Specificity
Suppressive Mechanisms of Treg Cells
Manipulation of the Number or Suppressive Function of Treg Cells
Cross-References
References
See Also
Rel
Synonyms
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Aspects
Cross-References
See Also
Relaxin
Definition
Characteristics
References
Renal Cancer
Renal Cancer Clinical Oncology
Definition
Characteristics
Clinical Epidemiology
Risk Factors
Renal Cancer Classification
Genetics
Tumor Biology and Implications for Treatment
Clinical Presentation
Incidental Findings
Local Findings
Metastatic Findings
Diagnosis
Staging
Treatment
Localized Disease
Metastatic Disease
Cross-References
References
See Also
Renal Cancer Diagnosis
Synonyms
Definition
Characteristics
Diagnosis by Symptoms
Diagnosis by Blood Test
Diagnosis by Imaging
Diagnosis by Biopsy
Staging
Pathological Diagnosis (Histologic Subtype)
Diagnosis of Metastases
Follow-Up
Cross-References
References
See Also
Renal Cancer Genetic Syndromes
Synonyms
Definition
Characteristics
Early Diagnosis
Treatment
Types of Renal Cancer
Renal Cancer Genes
Types of Inherited Renal Cancer
Clinical Features of Renal Cancer Suggesting Hereditary Renal Cancer
Mechanisms of Development of Inherited Renal Cancers
Cross-References
References
See Also
Renal Cancer Molecular Therapy
Synonyms
Definition
Characteristics
Introduction
Genetics of RCC
Therapy
Molecular Therapy for RCC
Experimental Molecular Therapy
Conclusion
Cross-References
References
See Also
Renal Cancer Pathogenesis
Characteristics
Papillary Type 1 Renal Cell Carcinoma
Papillary Type 2 Renal Cell Carcinoma
Chromophobe Renal Cell Carcinoma
Oncocytoma
Clinical Presentation
Staging
Treatment
Cytokine Therapy
Angiogenesis Inhibition
Tyrosine Kinase Inhibitors
mTOR Inhibitors
Temsirolimus
Everolimus
Concluding Comments
Cross-References
References
See Also
Renal Cancer Pharmacologic Therapy
Renal Cancer Targeted Therapies with Tyrosine Kinase Inhibitors
Definition
Characteristics
Tyrosine Kinase Inhibitors in the Treatment of Renal Cancer
Depletion of Growth Factor as Treatment Modality
Inhibition of a Master Regulator: mTOR
Treatment Challenges
References
Renal Cancer Therapy
Synonyms
Definition
Characteristics
Planning of Treatment
Operative Tumor Removal
The Small Renal Mass: Nephron-Sparing Surgery Versus Experimental Therapy
Active surveillance and watchful waiting
Operative Therapy of Metastatic Disease
Drug Therapy of Metastatic Disease
Other Therapy
Cross-References
References
See Also
Renal Cancer Treatment
Definition
Characteristics
Treatment of Localized Renal Cancer
Surgical Treatment
Nonsurgical Treatment Options, Known as ``Mini-invasive´´
Treatment of Metastatic Renal Cancer
Immunotherapy
Target Therapies
References
Renal Cancer Trends in Molecularly Targeted Therapies
Definition
Characteristics
Research Priorities
Mechanisms of Acquired VEGFR Therapy Resistance
New Targets, Particularly Those Within the Tumor
Predictive and Surrogate Biomarkers (Surrogate Marker)
Combination Versus Sequential Therapy
Mechanisms of Response
References
Renal Cell Carcinoma
Renal Cell Carcinoma Diagnosis
Renal Cell Carcinoma Therapy
Repair of DNA
Definition
Characteristics
Reversal of Base Damage
Removal of Damage
Repair of DNA and Cancer
Repair of DNA Strand Breaks
References
Replication Factories and Replication Foci
Synonyms
Definition
Characteristics
Protein Composition of Replication Factories
Regulation of the Formation of Replication Factories
Clinical Relevance
Cross-References
References
Replication Sites
Replication-Activated Adenovirus
Replication-Competent Adenovirus
Replication-Selective Adenovirus
Replication-Selective Viruses
Replicative DNA Lesion Bypass
Reproductive System Cancers
REPSA
Reserve Cell Carcinoma
Residual Disease
Resistance Modulation
Resistance Reversion
Resistance to Chemotherapy
Resistin
Synonyms
Definition
Characteristics
The Discovery
Molecule Structure
Distribution
Blood Levels
Biological Effects
Effect on Carbohydrate Metabolism or Insulin Sensitivity
Effect on Lipid Metabolism
Effect on Food Uptake
Effect on Body Weight or Obesity
Effect on Inflammation
Effect on Atherosclerosis
Effect on Bone Metabolism
Effect on Growth of Cancerous and Normal Cells
Regulation
Nutritional Regulation
Hormonal Regulation
Cytokine Regulation
Transcription Factor Regulation
Genetic Regulation
Cross-References
References
Resistin-Like Molecule Gamma (RELMgamma)
Restriction Landmark Genomic Scanning
Definition
Characteristics
Application
Identification of Low-Level DNA Amplification in Human Cancers
Global Scanning for DNA Methylation Changes in Human Cancers
Detection of Imprinted Genes
References
Resveratrol
Synonyms
Definition
Characteristics
Mechanism of Action
Clinical Relevance
Cross-References
References
See Also
RET
Definition
Characteristics
Cellular and Molecular Regulation
Knockout Mice
Clinical Relevance
Mutations Found in Medullary Thyroid Carcinoma
Mutations Found in Papillary Thyroid Carcinoma
References
Reticulin
Retinoblastoma
Synonyms
Definition
Characteristics
Clinical Aspects
Diagnosis of Rb
Presentation and Family History
Therapy and Prognosis
Second Tumors
Molecular Genetics
Rb Is Initiated by Two Mutations
Structure of the RB1 Gene
Spectrum of RB1 Gene Mutations
Genotype-Phenotype Associations
Genomic Alterations Associated with the Progression of Rb
Risk Prediction and Genetic Testing
Cross-References
References
See Also
Retinoblastoma1
Retinoblastoma Protein, Biological and Clinical Functions
Synonyms
Definition
Characteristics
The RB1 Gene Encodes the pRB Protein
pRB Is Posttranslationally Modified
Phosphorylation of pRB
Acetylation of pRB
Functions of pRB
Cell Cycle Control
Maintenance of Genome Integrity
Differentiation and Development
Apoptosis
Clinical Relevance
Inactivation of RB1 Initiates Retinoblastoma Development
RB1 Loss in Other Cancers
References
Retinoblastoma Protein, Cellular Biochemistry
Definition
Characteristics
pRb and Its Family Members
Cell-Cycle Regulation of pRb
Role in Terminal Differentiation
Other Targets of pRb
Animal Models
Summarizing Remarks
References
Retinoblastoma Susceptibility Protein
Retinoblastoma Tumor Suppressor Gene
Retinoic Acid
Definition
Characteristics
Mechanism of Action
Regulation
RA Homeostasis
Delivery of RA to Its Nuclear Receptors
Other Regulatory Mechanisms
Clinical Relevance
Cross-References
References
See Also
Retinoid Acid Receptors - RAR
Retinoid Receptor Cross-Talk
Synonyms
Definition
Characteristics
Retinoids
Retinoid Receptors
Retinoid Acid Receptors (RARs)
Rexinoid Receptors (RXRs)
Retinoid Receptors Cross-Talk During Carcinogenesis
Cross-References
References
Retinoid X (Rexinoid) Receptors - RXR
Retinoids
Definition
Characteristics
Retinoid Metabolism
Retinoid Signaling in Normal Cells
Retinoids in Cell Proliferation and Apoptosis
Retinoids and Stem Cells
Retinoids and Cancer
Retinoids in Cancer Therapy
Cross-References
References
Retroviral Insertional Mutagenesis
Synonyms
Definition
Characteristics
History
Provirus Structure
Tumor Types
LTRs Regulate Transcription of Host Genes near the Insertion Site
Promoter Insertion
Enhancer Activation
Protein Fusion
Transcript Stabilization
Cloning of Insertion Sites
Common Integration Sites
Randomness of Insertion
References
Retroviral Insertional Tagging
Retroviral Transduction
Rhabdoid Tumor
Definition
Characteristics
Genetics
References
Rhabdomyosarcoma
Definition
Characteristics
Cross-References
References
See Also
Rho Family Proteins
Synonyms
Definition
Characteristics
Cellular and Molecular Regulation
Functions
Clinical Relevance
References
Rho-GTPase-Activating Protein 7
RhumAb Recombinant Humanized Anti-HER-2 Monoclonal Antibody
rhuMabHER2
Ribonuclease 5
Ribosome-Inactivating Proteins
Definition
Characteristics
Structure and Function
Targeted RIPs for Cancer Therapy
Clinical Trials
Cross-References
References
Rigaud and Schmincke Types of Lymphoepithelioma
RING Finger Protein 196
RIS
Rituxan (US)
Rituximab
Synonyms
Definition
Characteristics
Mechanism of Action
Administration
Indications
Adverse Events
Clinical Trials with Rituximab Monotherapy
Clinical Trials with Rituximab Combination Regimens
Cross-References
References
See Also
RLIP76
RNA Interference
Synonyms
Definition
Characteristics
RNA Interference as a Research Tool
RNA Interference as a Novel Therapeutic Agent Against Cancer
Prospects and Obstacles Regarding RNAi Therapeutics
References
RNA Polymerase I Transcription
Synonyms
Definition
Characteristics
Machinery
Regulation of Activity
Link to Carcinogenesis
Therapeutic Targeting of rRNA Biogenesis
Cross-References
References
RNAi
RNase A Family 5
RNF116
RNF196
Ron Receptor
Synonyms
Definition
Characteristics
Expression and Function
Ligand
Signaling Through Ron
Ron Overexpression in Multiple Tumor Types
Ron Variants
Ron-Dependent Transformation
Transgenic Mouse Models Overexpressing Ron
Ron Loss in Murine Tumor Models
Oncogenic Ron Signaling
Ron as a Therapeutic Target in Human Cancer
References
ROS Reactive Oxygen Species
rRNA Synthesis
RTI40
RTP, cap43, rit42, TDD5 (Mouse), Ndr1 (Mouse), Bdm1 (Rat)
Runx1
Synonyms
Definition
Characteristics
Expression and Regulation
RUNX1 in Leukemia
Cross-References
References
See Also
RUNX1/CBFA2T1
RUNX1/MDS1/EVI1
RUNX1/RUNX1T1
S
S(1-5), T16, EFEMP1, FBLN-3
S-100 Proteins
S100 Proteins
Definition
Characteristics
S100 and Its Role in Cancer
S100B
S100P
S100A1
S100A2
S100A3
S100A4
S100A7
S100A8/9
S100A10
S100A11
S100A13
Conclusions
Cross-References
References
See Also
SAAB
SAC
SAHA
Salicylate
Salisburia adiantifolia
Salisburia macrophylla
Salivary agglutinin (SAG, Human)
Salivary Gland Malignancies
Definition
Characteristics
Anatomy and Physiology
Epidemiology and Risks
Diagnosis
Staging/Classification
Treatment
Conclusions
References
Salt Intake
Synonyms
Definition
Characteristics
Nasopharyngeal Carcinoma
Gastric Cancer
References
Salting
SAPKα
SAPKbeta
SAPKgamma
Saporin
Definition
Characteristics
Structure and Function
Targeted Saporin for Cancer Therapy
Clinical Trials
Cross-References
References
Scaffold Proteins
Synonyms
Definition
Characteristics
Introduction
Roles of Scaffold Proteins
Components of Scaffold Complexes
Structural Characteristics of Scaffold Proteins
Compartmentalization of MAPK Signaling by Scaffold Proteins, Time, Space, and Biological Specificity
Scaffold Proteins in DNA Ddamage Sensing and Repair Responses
Cross-References
References
See Also
Scatter Factor
Synonyms
Definition
Characteristics
The SF Family
SF Receptor (The c-Met Proto-oncogene Product)
Cellular and Molecular Regulation
SF Producer and Responder Cell Types
Regulation of SF Production
Biologic Responses Induced by SF and Their Regulation
SF and c-Met Participate in Various Physiologic and Pathologic Processes
Development
Oncogenesis
Angiogenesis
Clinical Relevance
Cross-References
References
See Also
SCF Receptor
SCF-R
SCGE
SCH52365
Schwannoma-Derived Growth Factor
Schwannomin
Scirrhous
Sclerosing Angiogenic Tumor
Sclerosing Endothelial Tumor
Sclerosing Epithelioid Angiosarcoma
Sclerosing Interstitial Vascular Sarcoma
Sclerosing Stromal Cell Tumor
SDF-1α
SDGF
Sdi1
Second Malignant Neoplasm SMN
Second Primary Cancer SPC
Second Primary Cancers
Second Primary Malignancy SPM
Second Primary Tumors
Synonyms
Definition
Introduction
Causal and Risk Factors
Shared Etiologic Exposure
Genetic Predisposition
Therapy-Related Risk Factors
Diagnosis
Treatment
Prevention
Cross-References
References
See Also
Secondary Metabolites
Secondary Tumor
Secretagogues
gamma-Secretase
Secreted Phosphoprotein 1
Secreted Protein Acidic and Rich in Cysteine
Synonyms
Definition
Characteristics
Gene Organization
Protein Structure
Functional Properties
Animal Models
SPARC in Cancer
Cross-References
References
See Also
Securin
Synonyms
Definition
Characteristics
Securin Function
Securin Function in Tumor Development
Cross-References
References
See Also
``Seed and Soil´´ Theory of Metastasis
Definition
Characteristics
Historical Development of the ``Seed and Soil´´ Theory
Evidence Shows that the ``Seeds´´ Can Even Prepare the ``Soils´´
References
SEL-12
Selective Effect
Selective Serotonin Reuptake Inhibitors
Selenium
Definition
Characteristics
References
SELEX
SEMA
Semanov II Syndrome
Semaphorin
Synonyms
Definition
Characteristics
Subclasses
Structure and Processing
Brief History of the Semaphorin Family
Semaphorins and the Composition of Their Receptors
Brief History of Semaphorins in Cancer Research
Mechanistic Principles Underlying Semaphorin Functions in Cancer Biology
Mechanistic Principle I: Semaphorins Engage in Cross Talk with VEGF Signaling
Mechanistic Principle II: Semaphorins Act to Regulate Integrin Function
Mechanistic Principle III: Semaphorins May Act Through Complexes of Receptor Tyrosine Kinases and Plexins
Mechanistic Principle IV: Semaphorins May Act as Regulators of the Immune Response
Mechanistic Principle V: Evasion of Apoptosis Through Blockade of Dependence Receptor Signaling
Mechanistic Principle VI: Regulation of Cellular Levels of Reactive Oxygen Species
Cross-References
References
See Also
Semaphorin Receptors
Semaxanib (SU5416)
Seminoma
Seminomatous Germ Cell Tumor
Senescence and Immortalization
Definition
Characteristics
Senescence
Immortalization
Relevance of Senescence and Immortalization to Cancer
A Cell Division Counting Mechanism
Telomerase
Alternative Lengthening of Telomeres (ALT)
Tumor Suppressor Genes
Clinical Relevance
Cross-References
References
See Also
Senescence-Associated Chronic Inflammation
Senile Involution
Sentinel Lymph Node
Synonyms
Definition
Characteristics
General Anatomy and Physiology of the Lymphatic System
The Sentinel Node
Lymphoscintigraphy
Sentinel Node Biopsy
Breast Cancer
Melanoma
Concluding Remarks
Cross-References
References
See Also
Sentinel Node
Synonyms
Definition
Characteristics
Sentinel Node Procedure
Clinical Relevance
Immunology of the Sentinel Node
Cross-References
References
SEREX
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
Cross-References
References
Serine Elastase
Serine Protease Inhibitor-Like Domain
Serine Proteases (Type II) Spanning the Plasma Membrane
Synonyms
Definition
Characteristics
Regulation of Activity
Biological Responses
Expression in Cancer
Clinical Relevance
Cross-References
References
See Also
Serine-Threonine Kinase Receptor-Associated Protein
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects
Cross-References
References
See Also
Sertoli-Leydig Cell Tumor
Serum Biomarkers
Definition
Characteristics
Alpha-Fetoprotein (AFP)
Beta Subunit of Human Chorionic Gonadotropin (beta-hCG)
Cancer-Associated Antigen 15-3
Cancer-Associated Antigen 19-9
Cancer-Associated Antigen 27-29
CA125
Carcinoembryonic Antigen (CEA)
Cytokeratin 19 Fragments
Lactate Dehydrogenase (LDH)
Matrix Metalloproteinase-9 (MMP-9)
Placental Alkaline Phosphatase (PLAP)
Prostate-Specific Antigen (PSA)
YKL-40
Cross-References
References
See Also
Seven in Absentia Homologue
Severe Hypoxia
Sex Cord-Stromal Tumors - Granulosa Cell Tumors
Sex Cord-Stromal Tumors with Annular Tubules
Sezary Syndrome
Definition
Characteristics
Staging and Treatment
References
See Also
SH2/SH3 Domains
Synonyms
Definition
Characteristics
Clinical Relevance
References
SH2/Src Homology 3
SH3 Domains
SH3p17
SH3p18
SH3P9
Shark Cartilage
Definition
Characteristics
References
Shedding of Cells
Short Hairpin RNA
shRNA
SIAH
Synonyms
Definition
Characteristics
The Ubiquitin-Proteasome System (UPS) in Health and Disease
Protein Can Be Poly-ubiquitinylated and Targeted for Proteasomal Degradation
SIAH Structure
Regulation of SIAH Expression
Regulation of SIAHs
Role for SIAHs in Tumorigenesis
Conclusions
Acknowledgments
References
Sicca Complex
Sicca Syndrome
Side Population Cells
Synonyms
Definition
Characteristics
Side Population Cells in Cancer
Targeting Cancer SP Cells
References
Signal Transducer and Activator of Transcription
Signal Transducer and Activator of Transcription 3
Signal Transducers and Activators of Transcription in Oncogenesis
Synonyms
Definition
Characteristics
STATs in Cytokine and Growth Factor-Induced Signaling
Bioactivity
STAT Activation During Progression of Human Cancer
Targeting STATs for Cancer Therapy
Targeting the Tumor Cell
Targeting the Tumor Microenvironment
Summary
Cross-References
References
See Also
Signal Transduction
Definition
Characteristics
Receptors
Targets of Signaling Pathways
Signal Transduction in Cancer Pathogenesis
Signal Transduction in Cancer Therapy
Cross-References
References
See Also
Signal Transduction Inhibitor-571
Silent Mating Type Information Regulation 2 Homolog 1
SIMA135 (Subtractive Immunization M(+)HEp3 Associated 135 kDa Protein)
Simian Virus 40
Single-Cell Gel Electrophoresis Assay
Single-Cell Microgel Electrophoresis Assay
Single-Chain Fv Dimer
SIR2L1
SiRNA
Synonyms
Definition
Characteristics
Clinical Aspects
Delivery
Nonspecific Immune Stimulation
Off-Target Interference
Cross-References
References
See Also
siRNA
Sirolimus
SIRT1
Synonyms
Definition
Characteristics
Function of SIRT1 in Cancer
Molecular Substrates of SIRT1
Regulation of SIRT1
Small Molecule SIRT1 Activators
Conclusion
References
Sirtuin 1
Site-Specific Drug Delivery
Sivelestat
Definition
Characteristics
Mechanisms of Action
Clinical Aspects
Clinical Application in Practice
Application to Cancer Treatment as Molecular Targeting Therapy
References
Sjögren Syndrome
Synonyms
Definition
Characteristics
Incidence
Etiology
Clinical Manifestations
Presentation
Involvement of Exocrine Glands
Extraglandular Involvement
Diagnosis
SS Is Diagnosed on the Basis of the European Classification
Laboratory Abnormalities
Anti-Ro/SSA and Anti-La/SSB Antibodies
Anti-CENP-H Antibodies and SS
Pathogenesis
Immunopathology
Humoral Immunity
Cellular Immunity
Immune-Mediated Tissue Destruction
Immunopathogenesis (Summary)
Treatment and Prognosis
SS and HCV
SS Cause Lymphoma?
SS and Lymphoproliferative Disorders
Future Directions
Cross-References
References
See Also
Skeletal Complications (Skeletal-Related Events)
Skeletal Metastasis
Skeletal Secondary Tumors
Skeletrophin
Synonyms
Definition
Characteristics
Clinical Aspects
References
SKI390
Skin Cancer
Synonyms
Definition
Characteristics
Skin Structure and Cancer Frequency
Epidermal Skin Cancers
Dermal Skin Cancers
Skin Cancer of the Subcutis
Multistep Cancer Theory and Ultraviolet Radiation (UV Light)
Genetic Predispositions (Familial Skin Cancers)
Cross-References
References
See Also
Skin Carcinogenesis
Definition
Characteristics
Genetic Basis of Skin Carcinogenesis
Initiation
Promotion
Cellular and Molecular Basis
Relevance to Human Disease
References
Skinny Needle
Skipper-Schabel Model
Skipper-Schabel-Wilcox Model
Skp2
SL-1
Slit
Definition
Characteristics
Slit Function in the Nervous System
Slit-Robo Signaling
Slit Functions Outside the Nervous System
Slit Proteins and Cancer
Slit and Medulloblastoma
Cross-References
References
See Also
Sloughing of Cells
SM-5887
SMA- and MAD-Related Protein 4
Smad Proteins in TGF-Beta Signaling
Definition
Characteristics
Cellular and Molecular Regulation
Clinical Relevance
References
SMAD4
Small Cell and Large Cell Neuroendocrine Carcinomas (NEC) at the Highly Aggressive Pole of the Spectrum of NENs
Small Cell Lung Cancer
Synonyms
Definition
Characteristics
Pathology of SCLC
Clinical Presentation
Staging of SCLC Patients
Therapy
Limited Stage of SCLC
Prophylactic Cranial Radiation
Extensive-Stage SCLC
Recurrent SCLC
Management of Brain Metastases
Conclusion
Cross-References
References
See Also
Small Cell Neuroendocrine Carcinoma
Small Cleaved Cell Lymphoma
Small GTP-Binding Proteins
Small Interfering RNA
Small Molecule Drugs
Definition
Characteristics
Differences to Large Molecule Drugs
High-Throughput Screening
Structure Determination and Modeling
Physicochemical Properties and Drug Uptake
Drug Formulations
Side Effects
Some Examples for the Mechanism of Cancer Drug Action
Systemic Effects of Tumor Growth
Cross-References
References
See Also
Small Molecule Screens
Synonyms
Definition
Characteristics
Types of Screens
``Small´´ Molecules
Screening Technology and Tools
Screening Assays
The Screening Epilog
Conclusions
Cross-References
References
See Also
Small Round Cell Tumor
SMARCB1
SMAX1
SMR
Snail Transcription Factors
Synonyms
Definition
Characteristics
Structural Organization
Transcriptional Repression/Activation
Regulation of Expression and Functional Activity
Additional Snail Functions
Clinical Significance
Cross-References
References
SOCS
Sodium Chloride
Soft Tissue Sarcoma
Solar Light-Induced Cancer
Soluble ATP-Dependent Non-Lysosomal Proteolytic System
Soluble Mesothelin-Related Proteins
Somatic Recombination of V, D, and J Segments
Somatic Stem Cells
Somatocrinin
Somatoliberin
Somites
Definition
Sonochemotherapy
Sonodynamic Therapy
Synonyms
Definition
Characteristics
Physical Mechanisms of Ultrasound-Induced Antineoplastic Activity
Chemical Mechanisms of Ultrasound-Induced Antineoplastic Activity
Classes of Sonosensitizers: Reactive Oxygen Species Agents
Classes of Sonosensitizers: Cytoskeletal-Directed Agents
Classes of Sonosensitizers: Vascular Disrupting Agents
Classes of Sonosensitizers: Echo Contrast Agents
Comparison of Sonodynamic Therapy to Photodynamic Therapy
Conclusion
Cross-References
References
See Also
Sorafenib
Synonyms
Definition
Characteristics
Therapeutic Range
Molecular Targets of Sorafenib
Complex Mechanism of Raf as a Target of Sorafenib
Non-Raf Molecular Targets of Sorafenib
Sorafenib Resistance
Trial of the Sorafenib-Based Combination Therapy
Conclusion
References
Sorafenib (Nexavar)
Sorafenib [INN]
Sorafenibum
Soy Phytoestrogen
SPARC
Spasmolytic Polypeptide SP/TFF2
SPB
SPC1
Specific Immunotherapy for Melanoma
Spectral Karyotyping
Definition
Characteristics
Advantages of SKY
Disadvantages
Clinical Significance
References
Sphacelus
S-Phase Damage-Sensing Checkpoints
Definition
Characteristics
References
S-Phase-Kinase-Associated Protein 2
Sphingolipid Metabolism
Definition
Characteristics
Polydrug Chemotherapy and Multidrug Resistance
Chemotherapy via Ceramide Control
Immunological Attack Against Cancer Cells
Mechanisms of Sphingolipid Antineoplastic Action
Cross-References
References
See Also
Spinal and Bulber Muscular Atrophy (SBMA)
Spindle Assembly Checkpoint
Synonyms
Definition
Characteristics
Discovery
Arrest Mechanism
Known Components
Mechanism of Mitotic Inhibition
SAC Control
Attachment Model
Tension Model
The Spindle Assembly Checkpoint in Cancer
Causes of a Weakened Checkpoint
Therapeutic Targeting of the Spindle Assembly Checkpoint
Pharmacologic Strategies
Biological Strategies
References
Spindle Pole Body
Spinesin (TMPRSS5)
Spleen Tyrosine Kinase
Sprouty
Synonyms
Definition
Characteristics
Mechanisms of Action
Regulation of the Activity of Sproutys
Deregulation of Spry Expression in Cancer
Ability of Sprys to Inhibit Cancer
Cross-References
References
Spry
Squamous Cell Carcinoma
Synonyms
Definition
Characteristics
Etiology
Diagnosis and Clinical Features
Disease Management
Molecular Aspects
Cross-References
References
See Also
Src
Synonyms
Definition
Characteristics
Domain Structure
Subcellular Localization
Interacting Proteins
Cellular and Molecular Aspects
Clinical Relevance
Cross-References
References
See Also
Src Homology 2
SRC-1: NCOA1
SRC-2: NCOA2
SRC-3
SRC-3: NCOA3
Src8
SSRIs
Stage IA-C Ovarian Cancer
Staging of Tumors
Synonyms
Definition
Characteristics
Purpose
Methods
Perspectives
References
See Also
STARD12
START Domain-Containing Protein
STAT
STAT3
Synonyms
Definition
Characteristics
STAT3-Targeted Genes
STAT3 in Oncogenesis
STAT3 Overexpression and Constitutive Activation in Human Cancers
STAT3 as a Therapeutic Target
Conclusion
References
Staurosporine
Definition
Characteristics
Kinase Inhibition
Staurosporine in Apoptosis
Antitumor Activity
References
Steel Ligand Receptor
Stefins
Synonyms
Definition
Characteristics
Physiological and Pathological Roles
Stefins in Cancer
References
Stem Cell Factor
Stem Cell Imaging
Definition
Characteristics
Markers of Adult SCs in Candidate Tissues
Conventional Imaging of SCs
Possibility of a Cancer SC
A Candidate Example: Prostate
New Methodologies: Toward a Biophysical Imaging Approach
Cross-References
References
Stem Cell Markers
Definition
Characteristics
Methods Used to Detect Stem Cell Markers in Cancer Biology
Clinical Applications
Cross-References
References
See Also
Stem Cell Mobilization
Definition
Characteristics
Cross-References
References
See Also
Stem Cell Plasticity
Synonyms
Definition
Characteristics
Metaplasia
The Bone Marrow and Tumor Stroma: Clinical Relevance
References
Stem Cell Telomeres
Synonyms
Definition
Characteristics
References
Stem-Like Cancer Cells
Synonyms
Definition
Characteristics
Identification
Clinical Implications
Steroid Receptor Coactivator-3
Steroid Receptor Coactivators
Synonyms
Definition
Characteristics
Functional Domains
Molecular Mechanisms
Dysregulations of SRCs in Cancers
Roles of SRCs in Tumor Growth and Progression
SRCs as Prognostic Biomarkers and Potential Therapeutic Drug Targets
Conclusions
Cross-References
References
Steroid Receptor Coactivators (SRCs): Nuclear Receptor Coactivators (NCOAs)
Steroid Sulfatase
Synonyms
Definition
Characteristics
Regulation of STS
STS in Hormone-Dependent Breast Cancer (HDBC)
STS Inhibitors
Cross-References
References
See Also
Steroid Sulfohydrolase
Steroid Tumor
Steroid X Receptor (SXR)
Synonyms
Definition
Characteristics
SXR in Cancer
Breast Cancer
Prostate Cancer
Ovarian Cancer
Endometrial Cancer
Colon Cancer
Other Cancers
Antitumorigenic Properties of SXR
SXR as a Therapeutic Target
Cross-References
References
Steryl Sulfohydrolase
STI571
STI-571
Synonyms
Definition
Characteristics
Physicochemical Properties
Pharmacology and Pharmacokinetics
Clinical Applications
Chronic Myeloid Leukemia (CML)
Gastrointestinal Stromal Tumors (GISTs)
Other Diseases
Side Effects
Resistance to STI-571
Low Concentrations in the Central Nervous System (CNS)
Cross-References
References
Sticker Sarcoma
Synonyms
Definition
Cross-References
See Also
Stivarga
STK12
STK13
STK15
STK5
STK6
STK7
STMY1
Stomach Cancer
STR1
STRAP
Stress
Definition
Characteristics
Possible Pathways of an Association of Stress and Cancer Risk
Methodological Problems
Findings
Conclusion
References
Stress Response
Synonyms
Definition
Characteristics
Cellular Regulation
Life or death decisions under stress
Relevance for cancer progression and therapy
Cross-References
References
Stress-Activated Protein Kinase
Stress-Induced Bystander Effect
Stroma
Stromagenesis
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Stromal Cell Response
Stromal Progression
Stromatogenesis
Stromelysin-1
Synonyms
Definition
Characteristics
Enzymatic Properties of Stromelysin-1
Regulation of Stromelysin-1
Stromelysin-1 and Cancer
References
Structural Biology
Definition
Characteristics
Cross-References
References
See Also
Structural Vascular Stabilization
STS
Subarachnoidal Spread
Subependymal Giant Cell Astrocytoma
Suberoylanilide Hydroxamic Acid
Substrate Channeling
Synonyms
Definition
Characteristics
The Key Features
The Prototypical Paradigm
Details
Mechanisms
References
Further Reading
Subtype AML-M7
Suicide Gene Therapy
Definition
Cross-References
Sulfated Glycoprotein-2 (SGP-2)
Sulfokinases
Sulforaphane
Synonyms
Definition
Characteristics
Actions and Pharmacology
Mechanisms of Action
Metabolism and Pharmacokinetics
Summary
References
Sulfotransferases
Synonyms
Definition
Characteristics
Sulfotransferases and Cancer
Cross-References
References
See Also
Sunitinib (Sutent)
Sunlight-Induced Cancer
Supervised Classification
Synonyms
Definition
Characteristics
Clinical Relevance
Caveats and Recommendations
Cross-References
References
See Also
Supervised Learning
Supportive Care
Synonyms
Definition
Characteristics
Supportive Care Needs Require an Individualized Approach
Patients and Carers Are Key Members of the Supportive Care Team
A Multidisciplinary and Coordinated Approach
The Development of an Evidence Base to Interventions to Improve Supportive Care Outcomes
Conclusion
References
Suppressive T Cells
Suppressor of Invasion, Metastasis, and Angiogenesis
Suppressors of Cytokine Signaling
Synonyms
Definition
Characteristics
Role of SOCS-1 in Cancer
SOCS-3 in Human Cancer
SOCS-2 in Malignant Diseases
Cross-References
References
See Also
Surface Molecules
Surface Plasmon Resonance
Definition
Characteristics
Detection Principle of SPR Biosensor
Properties of the SPR Biosensor
Biomedical Applications
Biomolecular Interaction Analysis (BIA)
High-Throughput Screening (HTS)
Proteomics Research
Cross-References
References
See Also
Surgical Pathology
Surgical Trauma and Cancer Recurrence
Definition
Characteristics
Trauma and Inflammation
Laboratory Investigations
Clinical Applications
References
Surrogate Endpoint
Synonyms
Definition
Characteristics
Role of Surrogate Endpoints in Cancer Research
Methods of Validation
Caveats in the Use of Validated Surrogate Endpoints
Cross-References
References
See Also
Surrogate Endpoint Biomarker
Surrogate Outcome
Survivin
Synonyms
Definition
Characteristics
Functional and Cellular Characteristics
Clinical Relevance
Cross-References
References
See Also
Susceptibility Loci
SV40
Synonyms
Definition
Characteristics
Replication
Clinical Relevance
References
SWI/SNF-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin Subfamily b, Member 1
Syk
Syk Tyrosine Kinase
Synonyms
Definition
Characteristics
Distribution and Function
Experimental Evidence for a Tumor/Metastasis Suppressor Role of Syk
Mechanism of Syk Activation and Signaling
Clinical Studies of Syk Expression and Activity
Cross-References
References
SYLD (syld709613 Protein)
Synovial Sarcoma
Definition
Characteristics
References
Synthetic Cannabinoids
Synuclein
Definition
Characteristics
Synuclein Expression in Cancer
Role in Tumor Progression
References
Systemic Antibody-Directed Radionuclide Therapy
Systemic Spread of Cancer
Systemic Therapy
T
T-Cell Receptor Gene Rearrangement
T-Cell Response
Definition
Characteristics
References
T-Cells Recognizing Autoantigens
t(8;21)
t(8;21)(q22;q22)
T1α2
TACE
Tachykinins
Synonyms
Definition
Characteristics
Tachykinin Genes
Tachykinin Receptors
Implications of Tachykinins in Malignancies
Breast Cancer
Lung Cancer
Brain Cancer
Colon Cancer
Pancreatic Cancer
Melanoma
Hematopoietic Malignancies
Aprepitant for the Treatment CINV
Cross-References
References
See Also
Takatsuki Disease or PEP Syndrome
Tamoxifen
Definition
Characteristics
Background
Adjuvant Therapy
Chemoprevention
Mechanism of Action
Side Effects
References
Tankyrases
Synonyms
Definition
Characteristics
Structure
Intracellular Distribution
Binding Partners
Functions
Telomere Elongation
Cell Division Control
Insulin-Stimulated Glucose Uptake
Clinical Aspects
Cross-References
References
Tanshinone C
Tarceva
Targeted Drug Delivery
Synonyms
Definition
Characteristics
Intratumoral Drug Administration
Liposomal Drug Delivery
Tumor-Activated Prodrug (TAP) Therapy
Antibody-Directed Enzyme Prodrug Therapy
Gene-Directed Enzyme Prodrug Therapy
Folate-Targeted Drug Delivery
Transferrin-Targeted Drug Delivery
Albumin-Drug Conjugate for Targeted Delivery
Cross-References
References
See Also
Targeted Drug Design
Targeted Radioimmunotherapy
Targeted Toxins
Targeted Viruses
Targeting Cancer Stem Cells
Synonyms
Definition
Characteristics
Cancer Stem Cells Are Resistant to Radiation Therapy
Cancer Stem Cells Are Resistant to Chemotherapy
Cancer Stem Cells Have Deregulated Embryonic Signaling Pathways
Targeting Cancer Stem Cells
Cross-References
References
Targeting Tumor-Initiating Cells
Tasigna
TAT Protein of HIV
Definition
Characteristics
Transcriptional Regulation: Control of HIV Replication
AIDS-Associated Pathologies: A Direct Contribution by Tat
Conclusion
References
Tax Binding Protein-181
Tax Helper Protein
Taxol
Definition
Characteristics
Mode of Action
Clinical Pharmacology
Antitumor Activity
Absorption and Excretion
Toxicity
Structurally and Functionally Related Compounds
Structurally Related Compounds
Nontaxane, Microtubule-Stabilizing Agents
References
Taxotere
Synonyms
Definition
Characteristics
Chemistry
Mechanisms of Action
Clinical Use
Cross-References
References
See Also
T-body
TCC
2,3,7,8-TCDD
TCDD
TCL1
Definition
Characteristics
Clinical Characteristics of the T-PLL
Cytogenetic and Molecular Characteristic of the TCL1 Locus
Animal Models
TCL1 Protein
TCL1 Expression in Normal and Pathological Tissues
Conclusions
References
TDA
TDGF-1
Technical Knockout
Definition
Characteristics
Isolation of Genes in Apoptosis
Clinical Aspects
References
Telomerase
Definition
Characteristics
What Are Telomeres and What Do They Do?
Why Do Telomeres Shorten?
What Is Cellular Senescence?
If You Can Stop the Shortening of Telomeres, Will This Prevent Cellular Aging?
Can Telomerase Be Used as a Product to Extend Cell Lifespan?
Cell and Molecular Regulation
Clinical Relevance
Could Telomerase Be the ``Achilles Heel´´ of Cancer?
Will Inhibiting Telomerase Restore the Senescence Program in Cancer Cells and If So Will This Therapy Cure Cancer?
Will Telomerase Activity Be Useful in Cancer Diagnostics?
Have Any Telomerase Therapeutic Agents Been Identified and What Are the Potential Complications of Such Strategies?
Cross-References
References
See Also
Telomeric Repeats of Stem Cells
Temodal
Temodar
Temoget
Temozolomide
Synonyms
Definition
Characteristics
Temozolomide and MGMT
Mismatch DNA Repair and Temozolomide Response
Repair of N7-Methylguanine and N3-Methyladenine DNA Lesions
References
Temsirolimus
Synonyms
Definition
Characteristics
Mechanism of Action
Activity in Cancer Models
Activity in Clinical Trials
Cross-References
References
See Also
Temsirolimus (Torisel)
Tenascin-C
Synonyms
Definition
Characteristics
Molecular Organization
Induction and Processing
Interaction Partners
Cell Rounding and Tumor Cell Proliferation
Metastasis
Angiogenesis
Phenotype of Tenascin-C Knockout Mice and Cancer
Clinical Aspects
References
Tensional Homeostasis
Definition
Characteristics
References
Teratocarcinoma-Derived Growth Factor-1
Teratoma
Testicular Cancer
Synonyms
Definition
Characteristics
Diagnosis
Therapy
Non-seminomatous Germ Cell Tumors (NSGCT)
Low-Stage (IIA/B) NSGCT
Clinical Stages IIC and III
Seminomatous Germ Cell Tumors
Clinical Stage I Seminoma
Low-Stage (Clinical Stage IIA/B) Seminoma
Clinical Stages IIC and III
Salvage Chemotherapy, High-Dose Chemotherapy
Genetics
Future Directions in TC
Cross-References
References
See Also
Testicular Feminization (TFM)
Testicular Germ Cell Tumor
Testicular Germ Cell Tumors
Synonyms
Definition
Characteristics
Incidence
Risk Factors
Histological Classification
Clinical Presentation and Diagnosis
Staging and Risk Stratification
Management
Seminomas
Nonseminomatous Germ Cell Tumors (NSGCT)
Conclusions
Cross-References
References
See Also
Testicular Tumors
Testosterone-Repressed Prostate Message 2 (TRPM-2)
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Tetraspanin
TG2
TGc
TGF-beta-Stimulated Clone 36, Tsc-36
Theragnostics
Theranostics
Synonyms
Definition
Characteristics
Diagnostic Component
Therapeutic Component
Other Components
Impact
Cross-References
References
Therapeutic Prevention
Therapeutic Viruses
Thiazolidin
Synonyms
Characteristics
Cross-References
References
See Also
Thiazolidinone
Thin Needle
Thiol-Protease Inhibitors
Thioredoxin System
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Thiostatins
Third-Generation Bisphosphonate
Third-Generation Nitrogen-Containing Bisphosphonate
Thoracentesis
Three-Dimensional Culture
Synonyms
Definition
Characteristics
Comparison of 3D and 2D Cultures
Advantages of 3D Cultures in Cancer Biology
3D Culture Models for Drug Sensitivity Screening
3D Models of Mammary Gland and Breast Cancer
Conclusion
References
Three-Dimensional Tissue Cultures
Definition
Characteristics
Multicellular Spheroids
Culture
Diffusion Limited Growth, ECM, and Differentiation
Dissociation, Histology, and Other Study Methods
Cell Proliferation
pH and Metabolite Gradients
Cancer Biology
Drug Effects and Multicellular Resistance
Drug Transport
Individualized Therapy
Multicellular Layers (MCL) or Multilayered Cell Cultures (MCC)
Multilayered Post-Confluent Cultures (MPCC)
Cocultures of Tumor and Normal Cells
Histocultures
Organotypic Cultures
Organ Cultures and Tissue Engineering
Conclusions and Future Directions
References
Thrombin Receptor
Thrombin Receptor-Like 1
Thrombin Receptor-Like 2
Thrombin Receptor-Like 3
Thrombocyte
Thrombospondin
Definition
Characteristics
Classification
TSP-1 and TSP-2 as Inhibitors of Tumor Angiogenesis
Mechanism
Cross-References
References
See Also
Thymidine Phosphorylase
Synonyms
Definition
Characteristics
References
Thymoma
Definition
Characteristics
Diagnosis
Classification
Treatment and Prognosis
References
Thyroid Cancer Treatment
Synonyms
Definition
Characteristics
Differentiated Thyroid Cancer
Radioactive Iodine: Targeted Therapy
Thyroglobulin: A Tumor Marker for Surveillance
Thyroid Hormone Replacement and TSH Suppression
Medullary Thyroid Cancer
Anaplastic Thyroid Cancer
Tyrosine Kinase Inhibitors
Vandetanib
Cabozantinib
Sorafenib
Lenvatinib
Conclusion
Cross-References
Thyroid Carcinogenesis
Definition
Characteristics
General Features
Role of Ionizing Radiation
Genetic Aberrations
RET Gene Changes
Cross-References
References
See Also
Thyroid Carcinoma Treatment
Thyroid Hormone Receptor Activator Molecule 1
Tiam1
Synonyms
Definition
Characteristics
Cross-References
References
See Also
TIF2
Tight Junction
Definition
Characteristics
References
Tight Junction Protein-1
TIL
Time of Flight
Definition
Cross-References
See Also
Time-Resolved Fluorescence
Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery
Synonyms
Definition
Characteristics
Cross-References
References
See Also
Time-to-Event Analysis
TIMPs
Tissue Inhibitors of Metalloproteinases
Synonyms
Definition
Characteristics
References
Tissue Kallikreins
Tissue Proliferation
Tissue Spectroscopy
Tissue Stem Cells
Synonyms
Definition
Characteristics
Defining the ``Stem Cell´´ Concept
Distinctive Features of Tissue Stem Cells
Tissue Stem Cells and Cancer
Tissue Stem Cells and ``Cancer Stem Cells´´
Cross-References
References
Further Reading
Tissue Transglutaminase
Tissue-Specific Stem Cells
TJP-1
TKIs
TLR4
Definition
Characteristics
TLR4 and Cancer
TLR4 Expression and Cancer
Endogenous TLR4 Ligands and Cancer
TLR4 as a Target for Cancer Therapy
TLR Agonists for Cancer
TLR Antagonists
References
T-Lymphoma Invasion and Metastasis
TMPRSS11-13 (Human Genes)
TMPRSS2/ERG Fusions
Definition
Characteristics
References
TMPRSS4
TMZ
TNBC
TNF-Related Apoptosis-Inducing Ligand
Synonyms
Definition
Characteristics
Introduction
TRAIL and Its Cell Surface Receptors
Organization of TRAIL-Induced Apoptotic Signaling
Mechanisms to Attenuate TRAIL-Induced Apoptosis
Augmentation of TRAIL-Induced Apoptosis
Clinical Potential for TRAIL Therapy
Perspectives
Cross-References
References
See Also
TNF-α in HIV Infection
Definition
Characteristics
Physiopathological Role of TNF-α in HIV Infection
Relationships Between HIV Replication and TNF-α
Role of TNF-α in NeuroAIDS
Role of TNF-α in Cachexia, Opportunistic Infections, and Tumors
Clinical Relevance of Molecules Inhibiting TNF-α Synthesis
References
TNK
Tobacco Addiction
Tobacco Carcinogenesis
Definition
Characteristics
Tobacco Products and Human Cancer
Tumor Induction in Laboratory Animals
Chemistry of Tobacco Smoke
Mechanisms of Tumor Induction
References
Tobacco Dependence
Tobacco-Related Cancers
Definition
Characteristics
Tobacco as a Carcinogen
Cancers Related with Tobacco
Evidence for Other Cancers
Passive Smoking
Conclusions
References
Toll-Like Receptors
Definition
Characteristics
TLRs and Cancer Treatment
Cross-References
References
Tongue Cancer
Synonyms
Definition
Characteristics
Epidemiology
Pathology and Genetic Aberration
Clinical Features
Subclinical Nodal Metastasis
TNM Staging and Prognostic Factors
Pretreatment Assessment
Treatment Options
Controversy of Elective Neck Dissection Versus Observation of N0 Neck
Outcome of Treatment
Cross-Reference
References
See Also
Topoisomerases
Definition
Characteristics
Topoisomerase Families
Molecular Mechanism
Cellular Functions
Topoisomerase Inhibition and Cancer Therapy
References
Torisel
Toxicity Testing
Toxicogenomics
Definition
Characteristics
Genomics
Approaches, Technology, and Data Analysis
Data Interpretation
Applications in Toxicology
Epigenomics
Approaches, Technology, and Data Analysis
Data Interpretation
Applications in Toxicology
Transcriptomics
Approaches, Technology, and Data Analysis
Data Interpretation
Applications in Toxicology
Proteomics
Approaches, Technology, and Data Analysis
Data Interpretation
Applications in Toxicology
Metabolomics
Approaches, Technology, and Data Analysis
Data Interpretation
Application to Toxicology
References
Toxicological Carcinogenesis
Synonyms
Definition
Characteristics
History
Cross-References
References
See Also
TP53
Synonyms
Definition
Characteristics
Upstream of p53: Signaling of DNA Damage
Downstream of p53: Cell Cycle Control, Apoptosis, and DNA Repair
Clinical Relevance
Cross-References
References
TP63
TP73
TP73L
TPI
TRA-8
Trabectedin
Synonyms
Definition
Characteristics
Structural and Biophysical Characterization of Trabectedin-DNA Adducts
Biological Activity
Clinical Studies
References
Trace Elements
Traditional Chinese Medicine
Traffic ATPases
TRAIL
TRAIL Receptor Antibodies
Synonyms
Definition
Characteristics
Clinical Aspects
Mapatumumab (Anti-DR5)
Lexatumumab (Anti-DR5)
TRA-8 (CS1008)
Perspectives
TRAM1
TRAM-1
Transabdominal Dissemination
Transabdominal Metastasis
Transcoelomic Metastasis
Synonyms
Definition
Characteristics
Mechanisms of Transcoelomic Metastasis
Models of Metastasis
Cell Detachment
Peritoneal Fluid and Anatomy
Ascites: A Metastatic Milieu
Immune Evasion
Tumor Implantation
Clinical Aspects
Cross-References
References
See Also
Transdifferentiation
Transduction of Oncogenes
Synonyms
Definition
Characteristics
Identification of Cellular Oncogenes
Mechanism of Oncogene Transduction
Lessons from Retroviral Transduction of Oncogenes
Cross-References
References
See Also
Transfer of T Cells
Transforming Gene
Transforming Growth Factor-Beta
Definition
Characteristics
TGF-beta Receptors and Signaling Mechanisms
Latency TGF-beta
Matrix Association and Release of TGF-beta
LTBPs: Expression and Functions
Activation of Soluble and Extracellular Matrix Forms of Latent TGF-beta
TGF-beta and LTBP Knockout Mice
Perspective
Cross-References
References
See Also
Transfusion of T Cells
Transgenic Mice
Transgenic Mouse
Definition
Characteristics
Methodology
Random Versus Targeted Gene Insertion
Knockout Mice
Tissue-Specific Knockout Mice (The Cre/loxP System)
References
Transglutaminase Type-2
Transglutaminase-2
Synonyms
Definition
Characteristics
TG2 and Apoptosis
TG2 in Drug Resistance and Metastasis
Clinical Relevance
References
Transin-1
Transitional Cell Carcinoma
Synonyms
Definition
Characteristics
Epidemiology
Etiology
Signs and Symptoms
Evaluations
Staging (1997 AJCC-UICC, TNM Staging)
Treatment of Superficial Bladder Cancer
Treatment of Invasive Bladder Cancer
Treatment of Metastatic Bladder Cancer
Cross-References
References
Transitional Cell Carcinoma of Bladder
Transitional Cell Carcinoma of Renal Pelvis
Transitional Cell Carcinoma of the Urinary Bladder
Transitional Cell Carcinoma of Ureter
Translesion DNA Polymerases
Definition
Characteristics
References
Transmembrane 4 Superfamily Protein
Transmembrane Protease Serine 1-13: Hepsin (TMPRSS 1)
Transplacental Carcinogenesis
Synonyms
Definition
Characteristics
Types of Transplacental Carcinogens
Mechanisms of Cancer Causation
References
Trask (Transmembrane and Associated with Src Kinases)
Trastuzumab
Synonyms
Definition
Characteristics
Antibody
Molecular Target of Trastuzumab
Murine Anti-HER2 Monoclonal Antibody (mAb) 4D5
Clinical Indications
HER2 Status
Mechanisms of Action of Trastuzumab
Mechanisms of Trastuzumab Resistance
Trastuzumab-Induced Cardiotoxicity
Cross-References
References
Treatment-Refractory Germ Cell Tumors
Trefoil Factors
Synonyms
Keywords
Definition
Characteristics
TFF Discovery and Expression
TFF in Mucosal Repair and Protection
TFF in Chronic Inflammation and Cancer Progression
Aberrant Expression of TFF as Clinical Markers of the Neoplasia
Conclusions
References
Trefoil Peptides pS2/TFF1
Treg
TRF1-Interacting, Ankyrin-Related ADP-Ribose Polymerases
TR-FRET
Trident
4,5,7-Trihydroxyisoflavone
3,4,5-Trihydroxystilbene
Triple-Negative Breast Cancer
Synonyms
Definition
Characteristics
Breast Cancer Treatment by Radiotherapy
Radiation and Metastases Development
Biomarkers of TNBC Poor Responders
Cross-References
References
Tripterine
TROP-1
Trophectoderm
Definition
Cross-References
Trp63
Trp73
Trx
TSC
TSC1
tTGase
Tuberous Sclerosis Complex
Synonyms
Definition
Characteristics
Clinical Aspects
The Genetic Basis of TSC
The Molecular Pathology of TSC
Targeted Therapy for TSC
References
Tuberous Sclerosis Complex 1
Tubulin-Interacting Proteins
Tumor Antigens
Definition
Characteristics
Tumor Antigens Classification, Expression, and Immune Response
Cross-References
References
Tumor Cell Invasion
Tumor Cell-Induced Platelet Aggregation
Synonyms
Definition
Characteristics
Mechanisms
Clinical Aspects and Therapy
Cross-References
References
See Also
Tumor Cell-Platelet Aggregate
Tumor Cell-Platelet Interaction
Tumor Glucose Metabolism
Tumor Grading
Tumor Markers
Tumor Metabolism
Tumor Microenvironment
Synonyms
Definition
Characteristics
Complex Regulatory Circuits
The Yin-yang (Double Edged Sword) Interplay in the Tumor Microenvironment
Vicious Cycles in the Tumor microenvironment
The Nontumor Cells in the Tumor Microenvironment may Express a Different Phenotype than Their Counterparts at Distant Sites
Site Specific Metastasis and the Metastatic Microenvironment
Dormant Micrometastasis and Microenvironmental Control
The Tumor Microenvironment as Target in Cancer Therapy
Cross-References
References
See Also
Tumor Necrosis Factor
Synonyms
Definition
Characteristics
Discovery
Sources
Genes and Proteins
Biological Functions
Anti-TNF Therapy
Molecular Mechanisms
Activation of the IKK/NF-kappaB and the MAPK/AP-1 Pathways
Induction of Apoptosis
Conclusion
References
Tumor Necrosis Factor-Alpha Converting Enzyme
Tumor Pathology
Tumor Progenitors
Tumor Rejection of Non-Irradiated Tumor Areas
Tumor Spread
Tumor Staging
Tumor Suppression
Definition
Characteristics
Clinical Aspects
References
Tumor Suppressor Genes
Synonyms
Definition
Characteristics
What Was the Evidence for Tumor Suppressors?
Retinoblastoma: The First Suppressor
Are There Other Tumor Suppressors and What Cellular Role Do They Normally Play?
Clinical Relevance
Cross-References
References
See Also
Tumor Typing
Tumor-Associated Macrophages
Definition
Characteristics
Macrophages
Tumor-Associated Macrophages
TAM Recruitment
TAM Express Selected M2 Protumoral Functions
Modulation of Adaptive Immunity by TAM
Conclusion
Cross-References
References
See Also
Tumor-Associated Stromal Progression
Tumor-Endothelial Communication
Tumor-Endothelial Cross-Talk
Synonyms
Definition
Characteristics
Adhesion Molecules in Tumor-Endothelial Cross-Talk Mediating Extravasation
Tumor-Endothelial Cross-Talk Leading to Prothrombotic Endothelial Activation
Conclusion
References
Tumor-Induced Platelet Aggregation
Tumor-Initiating Cells
Tumor-Reinitiating Cells
Tumor-Repopulating Cells
Tumor-Transforming 1
Tunicamycin
Definition
Characteristics
Structure and Function
Antiviral Effect of Tunicamycin
Apoptosis Induction by Tunicamycin
Tunicamycin Increases Effects of Anticancer Agents
Tunicamycin Enhances Death Ligand TRAIL-Induced Apoptosis
Cross-References
References
Turban Tumor Syndrome
Turcot Syndrome
Definition
Characteristics
Clinical Criteria
Genetics
PMS2 Gene
Microsatellite Instability
Clinical Aspects
References
Turmeric Yellow
TUTR1
TXBP181
Type I Cystatins
Type I Interferon
Type-1 Hypersensitivity
Typical Neurocytoma
Tyrosine Kinase Inhibitors
Synonyms
Definition
Characteristics
Cross-References
References
U
Ubiquitin Ligase SCF-Skp2
Synonyms
Definition
Characteristics
Regulation
Clinical Significance
References
Ubiquitination
Definition
Characteristics
Cellular Regulation
Clinical Relevance
p53 and HPV-Associated Cervical Carcinoma
von Hippel-Lindau (VHL) Disease
beta-Catenin Turnover in Colorectal Tumors
Degradation of p27, a CDK Inhibitor
Cbl and Receptor Tyrosine Kinase Degradation
Fanconi Anemia
Induced Degradation of PML-RARα in Acute Promyelocytic Leukemia
Summary
Cross-References
References
See Also
Ubiquitous TPR Motif Protein UTY
Ubiquitously Transcribed Tetratricopeptide Repeat, X Chromosome
Ubiquitously Transcribed TPR Protein on the X Chromosome
Ubiquitously Transcribed TPR Protein on the Y Chromosome
Ubiquitously Transcribed X Chromosome Tetratricopeptide Repeat Protein
Ubiquitously Transcribed Y Chromosome Tetratricopeptide Repeat Protein
UCN-01 Anticancer Drug
Synonyms
Definition
Characteristics
Inhibition of Chk1 Kinase and G2 Checkpoint Abrogation by UCN-01
Mechanisms of UCN-01-Induced Tumor Cell Death
Cross-References
References
See Also
Ultrasound Biomicroscopy
Ultrasound Microimaging
Synonyms
Definition
Characteristics
Two- and Three-Dimensional Anatomical Imaging
Doppler Blood Flow Imaging
Contrast-Enhanced Imaging
Cross-References
References
See Also
Ultrasound Therapy
Ultraviolet Light-Induced DNA Damage
Ultraviolet Radiation-Induced Cancer
Uncertain or Unknown Histogenesis Tumors
Definition
Characteristics
Distinctive Tumors of Uncertain Histogenesis
Synovial Sarcoma
Epithelioid Sarcoma
Alveolar Soft Part Sarcoma
Clear Cell Sarcoma of Soft Tissue
Extraskeletal Myxoid Chondrosarcoma
Desmoplastic Small Round Cell Tumor
Extrarenal Rhabdoid Tumor
Neoplasms with Perivascular Epithelioid Cell Differentiation (PEComas)
Hemangioblastoma
Angiomatoid Fibrous Histiocytoma
Myxoid Tumors
Other (Benign) Tumors
Cross-References
References
See Also
Unfolded Domain
Unfolded Protein Response and Cancer
Definition
Characteristics
The Key Players of the UPR Pathway
Activation and Function of the IRE1α-XBP1 Branch
Activation and Function of the PERK-eIF2α Branch
Activation and Function of the ATF6 Branch
The Role of UPR in Cancer Development
UPR in the Survival and Death of Cancer Cells
UPR and Inflammation
UPR and Cancer Angiogenesis
UPR and Cancer Dormancy
Targeting UPR in Cancer Therapy
Promote Apoptosis by Increasing ER Stress and UPR Activation in Cancer Cells
Promote Apoptosis by Inhibiting the Adaptive Function of UPR
Cross-References
References
UNII-9ZOQ3TZI87
Unrip: Unr-interacting Protein
Unstructured Domain
UPA
Uranium Miners
Definition
Characteristics
Uranium Mining
Miners´ Cohorts
Exposures
The Risk of Lung Cancer
Histopathological Findings
The Risk of Cancers Other than Lung Cancer
Conclusion
Cross-References
References
See Also
Urokinase-Type Plasminogen Activator
Synonyms
Definition
Cross-References
See Also
Urothelial Cancer Molecular Therapy
Urothelial Carcinoma
Synonyms
Definition
Characteristics
Distribution, Histology, and Clinical Course
Molecular Subtypes and Tumor Stem Cells
Causes
Therapy
Diagnosis and Monitoring
Molecular Genetics
Epigenetics
Cross-References
References
Urothelial Carcinoma, Clinical Oncology
Synonyms
Definition
Characteristics
Diagnosis
Therapy
Genetics
Cross-References
References
See Also
Urothelial Tumor
Uterine Cancer
Uterine Fibroid
Uterine Fibroma
Uterine Leiomyoma
Synonyms
Definition
Characteristics
Clinical, Epidemiological, and Histological Characteristics
Cellular Characteristics
Molecular Genetic and Cytogenetic Characteristics
Heritability and Genetic Epidemiological Characteristics
Cross-References
References
See Also
Uterine Leiomyoma, Clinical Oncology
Synonyms
Definition
Characteristics
Incidence
Gross Features
Microscopic Features
Symptoms
Diagnosis
Treatment
Cytogenetic Changes
References
Uterus Cancer
UTX
Synonyms
Definition
Characteristics
Introduction: Chromatin Structure and Epigenetic Modifications
Polycomb Repressive Complex 2
UTX/KDM6A is a H3K27 Demethylase
UTX Demethylase Target Specificity
UTX Associates with Histone Methyltransferases
UTX is a Specific Epigenetic Regulator
UTX Targets the Retinoblastoma Pathway
UTX Has JmjC Domain-Independent Functions
UTX Controls Life Span Through the Insulin/IGF-1 Pathway
UTX is a Tumor Suppressor Gene
A Two-Hit Model of UTX/UTY Inactivation
UTX Mutations were Identified in Various Tumor Types
Inhibition of UTX Demethylase Activity in IDH Mutated Cells
UTX and Tumorigenesis Through Notch and Rb
Cross-References
References
UTY, Ubiquitously Transcribed Tetratricopeptide Repeat Gene on the Y Chromosome
UV Radiation
Definition
Solar and UV Radiation
Characteristics
Cancers of the Skin
Epidemiological Evidence of the Carcinogenic Potential of UV Radiation
Host Factors
Solar UV Radiation as an Environmental Risk Factor
Biological Effects of UV Radiation Relevant to Carcinogenesis
DNA Damage
Cellular Damage
Differential Effects of UVA and UVB
Changes in the Immune Response
Drug-Induced Photosensitivity
Cancer Preventive Effects of UV Radiation
UV-Emitting Tanning Devices
Characteristics of UV-Emitting Tanning Devices
Evidence of the Carcinogenic Potential
References
Uveal Melanoma
Synonyms
Definition
Characteristics
Epidemiology
Risk Factors
Clinical Diagnosis
Signs and Symptoms
Diagnostic Modalities
Treatment Options
Pathology
Systemic Evaluation and Metastasis
Prognostic Factors
Systemic Therapy and Future Directions
References
Uvomorulin
V
V(D)J Recombination
Synonyms
Definition
Characteristics
Mechanism
Clinical Aspects
References
Valproic Acid
Synonyms
Definition
Characteristics
Valproic Acid Affects Cell Behavior by Multiple Mechanisms
Valproic Acid Inhibits Histone Deacetylase Activity
Valproic Acid Interferes with MAPK Signaling
Valproic Acid Affects the Beta-Catenin Pathway
Valproic Acid May Influence Additional Pathways
Cross-References
References
See Also
Vanadium
Definition
Characteristics
Vanadium in Cancer Treatment
Chemopreventive/Anticancer Mechanisms
Future Direction
References
Vascular Disrupting Agents
Synonyms
Definition
Characteristics
Vascular Disrupting Agents
The Biologic Approach
Small Molecule VDAs
Flavonoid Agents
Tubulin-Binding Agents
VDAs as Adjuvants to Conventional Therapy
Cross-References
References
Vascular Endothelial Growth Factor
Synonyms
Characteristics
VEGF Family
VEGF Receptor Family
VEGF in Vasculogenesis and Physiological Angiogenesis
Clinical Relevance
VEGF in Pathophysiological Angiogenesis
VEGF/VEGF Receptor System: Therapeutic Opportunities
Cross-References
References
See Also
Vascular Endothelial Growth Factor Receptor (VEGFR) Inhibitors
Vascular Maturation
Vascular Permeability Factor
Vascular Remodeling
Vascular Stabilization
Synonyms
Definition
Characteristics
Normal Vascular Hierarchy
Vascular Destabilization and Activation of Angiogenesis
Vascular Stabilization
Clinical Implications
References
Vascular Targeted Therapies
Vascular Targeting Agents
Synonyms
Definition
Characteristics
Background
Mechanisms
Clinical Aspects
Cross-References
References
See Also
Vascular-Targeted Therapies
Vasculogenic Mimicry
Definition
Characteristics
Introduction
Current Status of Studies on VM in Tumors
Molecular Mechanisms Underlying VM
Dedifferentiation of Tumor Cells Is the Key to Formation of VM Channels
Linearly Patterned Programmed Cell Necrosis and Three-Stage Phenomenon
The Effect of Local Tumor Microenvironment on the Formation of VM Channels
Clinical Significance of VM
Advances and Challenges
VM and Lymphogenesis in Tumors
VM-Targeted Therapy
References
Vasoactive Intestinal Contractor
Vasopressin
Definition
See Also
VDAs
VEGF
VEGF Trap
Velcade
v-erb-B2
Vesical Cancer
VHL
VHL Tumor Suppressor Gene
VIC
Villi
Definition
Villin 2
Viral Oncology Epigenetics
Definition
Characteristics
Host Epigenetic Changes Due to Viruses and Virus-Associated Cancers
Kaposi Sarcoma-Associated Herpesvirus
Epstein-Barr Virus
Hepatitis B Virus
Human Papillomavirus
Polyomaviruses (SV40, BK Virus, and JC Virus)
Adenovirus
Human T-Cell Lymphotropic Viruses
Epigenetic Alterations in the Virus Due to the Host
Conclusions
Cross-References
See Also
Viral Vector-Mediated Gene Transfer
Synonyms
Definition
Characteristics
References
Virology
Definition
Characteristics
Tumor Virus Epidemiology
Immunosurveillance and Viral Oncogenesis
Tumor Viruses
Herpesviridae
Epstein-Barr Virus
EBV-Encoded microRNAs
Burkitt Lymphoma
Nasopharyngeal Carcinoma
Hodgkin Lymphoma
Lymphoproliferative Disease in Immunosuppressed Patients
Other Tumors
Kaposi Sarcoma Herpesvirus
Polyomaviridae
Papillomaviridae
Hepadnaviridae
Hepatitis B Virus
Flaviviridae
Hepatitis C Virus
Human T-Lymphotropic Virus 1
Cross-References
References
See Also
Virotherapy
Synonyms
Definition
Characteristics
Oncolytic Adenoviruses and Cancer Gene Therapy
CRAds in Human Clinical Trials and the Limitations
CAR Deficiency on Tumor Cells Results in the Poor Infectivity of Ad Agents
Infectivity-Enhanced CRAd Agents Retargeting CRAds to Tumor Cells
Tumor-Specific CRAd Agents: Using a Tumor-Specific Promoter to Drive Specific Viral Replication
A Double-Targeted CRAd for Ovarian Cancer
Future Directions for Improvements
Cross-References
References
See Also
Virus Vector
Virus Vector-Mediated Gene Transfer
Vitamin D
Definition
Characteristics
Mechanisms
Conclusions
Cross-References
References
See Also
v-Ki-ras2
Vomeroglandin (Mouse)
Von Hippel-Lindau Disease
Synonyms
Definition
Cross-References
See Also
Von Hippel-Lindau Tumor Suppressor Gene
Synonyms
Definition
Characteristics
Molecular Features
Role in Diseases: Clinical, Molecular, and Cellular Characteristics
Clinical and Molecular Features
Classical Clinical Definition
Germline Mutations
Genotype/Phenotype Correlation
Sporadic Tumors
Somatic Mutations
Cellular/Functional Features
Applications in Diagnosis and Clinical Management
Remaining Questions
Cross-References
References
See Also
Von Recklinghausen Disease
Vorinostat
Synonyms
Definition
Characteristics
Background
Mechanism of Antitumor Action
Clinical Studies
Activity of Vorinostat in Non-oncology Indications
Cross-References
References
VP-16
VPF
v-raf Murine Sarcoma Viral Oncogene Homolog B1
v-Src
VTAs
W
WA
Waf1
Warburg Effect
Synonyms
Definition
Characteristics
Mitochondrial DNA Mutations and the Warburg Effect
Tumor Suppressors and the Warburg Effect
Oncogenic Activation of the Warburg Effect
Does the Warburg Effect Contribute to Tumorigenicity?
Clinical Aspects and Summary
Cross-References
References
Warts (wts)-Drosophila Lats
WARTS (WTS)-Mammalian LATS1
WD-Repeat Proteins Interacting with Phosphoinositides
Well-Differentiated Neurocytoma
Wermer Syndrome
Wilms Tumor
Wilms´ Tumor
Definition
Characteristics
How Common Is Wilms´ Tumor?
What Causes Wilms´ Tumor?
Clinical Characteristics
Is Wilms´ Tumor One Disease?
Molecular Characteristics of Wilms´ Tumor
Treatment of Relapsed Wilms´ Tumor and Future Therapeutic Possibilities
Heritability and Screening
Cross-References
References
WIN
WIPI
Synonyms
Definition
Characteristics
Autophagy
The Mechanism of Autophagy
Autophagy, WIPI, and Cancer
Human WIPI Proteins
WIPI-1 Puncta-Formation Assessment to Monitor Autophagy
Outlook: WIPI as New Targets for Anticancer Therapy?
References
Withaferin A
Synonyms
Definition
Characteristics
References
Wnt Signaling
Definition
Characteristics
Wnt Proteins
Wnt Signaling Pathways
Other Ligands and Receptors that Directly Regulate Wnt Signaling
Cross-References
References
See Also
WWOX
Definition
Characteristics
The Gene
New Genetic Findings
The Protein
Biological Role
In Normal Cells
In Cancer Cells
Cross-References
References
See Also
X
X Box Repressor (XBR)
Xenobiotic Biotransformation
Xenobiotic Metabolism
Xenobiotic Receptor
Xenobiotics
Synonyms
Definition
Characteristics
Origin of Xenobiotics
Metabolism of Xenobiotics
Effects of Xenobiotics
Interactions
Individual Variability
Xenobiotics and Cancer
Exploitation of Knowledge About Xenobiotics and XMEs
References
Xeroderma Pigmentosum
Definition
Characteristics
Xeroderma Pigmentosum (XP) Subgroups
Clinical Aspects
Cellular Parameters
Molecular Parameters
Animal Models
Other NER-Related Syndromes
Cross-References
References
See Also
Xerophthalmia
Xerostomia
XIAP
Synonyms
Definition
Characteristics
Structure
Functions
Gene and Regulation
XIAP and Cancer
Conclusions
Cross-References
References
See Also
Xiphophorus
Synonyms
Definition
Characteristics
Genetics of Melanoma Formation
The Xmrk Oncogene
UV-Induced Tumors
Carcinogen-Induced Tumors
Further Information
Cross-References
References
See Also
X-Linked Inhibitor of Apoptosis
XLP2
X-Ray-Induced Cancer
Y
Yinxing
YM-529
Yolk Sac Tumor
Yondelis
Z
Z-Factor
Definition
Characteristics
Connections to Other Assay Data Quality Indicators
Z-Factor and Lower Limit of Detection of an Assay
References
ZO-1
Zoledronic Acid
Definition
Characteristics
Structural Characteristics
In Vitro Activities of Zoledronic Acid and Mechanisms of Action
Antitumor and Bone Resorption Activity of Zoledronic Acid in Preclinical Models
Clinical Aspects
Cross-References
References
See Also
Zolinza
Zollinger-Ellison Syndrome
Zonula Adherens
Zonula Occludens Protein-1
Synonyms
Definition
Characteristics
Molecular Biology of ZO-1
Regulation and Function of ZO-1
ZO-1 in Cancer
Cross-References
References
See Also
ZZANK1

Citation preview

Manfred Schwab Editor

Encyclopedia of Cancer Fourth Edition

Manfred Schwab Editor

Encyclopedia of Cancer Fourth Edition

With 1230 Figures and 260 Tables

Editor Manfred Schwab German Cancer Research Center (DKFZ) Tumorgenetik, Heidelberg, Germany

ISBN 978-3-662-46874-6 ISBN 978-3-662-46875-3 (eBook) ISBN 978-3-662-47424-2 (print and electronic bundle) DOI 10.1007/978-3-662-46875-3 Library of Congress Control Number: 2017933328 # Springer-Verlag Berlin Heidelberg 2001, 2008, 2011, 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer-Verlag GmbH Germany The registered company address is: Heidelberger Platz 3, 14197 Berlin, Germany

Preface to the Fourth Edition

Welcome to the fourth edition of the Encyclopedia of Cancer. The third edition had appeared in 2011, and the tremendous response by the scientific community has encouraged us to prepare a subsequent edition that is now available. The past 5 years have seen an enormous progress in cancer research, with particular emphasis on the bench-to-bed paradigm and the application of personalized cancer medicine. For this new edition, the multidisciplinary approach bridging basic science and clinical application was further developed. Numerous new entries by authorities from the international scientific community were added to meet the substantial progress in molecular cancer etiology, diagnostics, and therapy. Entries from the third edition were updated, and new entries were added addressing central areas of basic and clinical cancer research, such as personalized cancer medicine, immunotherapy, pediatric and adult oncology, and epigenetics. The Encyclopedia of Cancer, fourth edition, will be available both in print and online version. The online version is designed as an interactive and dynamic database where authors at any time will be able to modify and update presentations in order to keep the content up to date. Additionally, new entries can be entered at any time, and contributors are encouraged to suggest new topics that they feel are insufficiently covered. The technical preparation of the Encyclopedia of Cancer would not have been possible without the competent and dedicated input by Daniela Graf and Melanie Thanner. Their excellent and pleasant cooperation is highly appreciated. Thanks also to the publisher who has taken every effort to develop this prestigious Encyclopedia of Cancer into a useful instrument from which both basic scientists and clinicians may benefit. Heidelberg, March 1, 2016 Manfred Schwab

v

Preface to the Third Edition

Recent developments in the rapidly developing field of cancer research are seeing a dynamic progress in basic and clinical cancer science, with translational research increasingly becoming a new paradigm. In particular, the identification of a large number of prognostic and predictive clinically validated biomarkers now allows exciting and promising new approaches in both personalized cancer medicine and targeted therapies to be pursued. The third edition of the Encyclopedia of Cancer is now available 10 years after the first edition had come out in 2001. Numerous new entries addressing topics of basic cancer research have been added. As a major new feature, upto-date and authoritative essays present a comprehensive picture of topics ranging from pathology, to clinical oncology and targeted therapies for personalized cancer medicine for major cancers types, such as breast cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cancer, lung cancer, and hematological malignancies, leukemias, and lymphomas. This information source should be of great value to both the clinical and basic science community. The Encyclopedia of Cancer, Third Edition, is available both in print and online versions. Contributors to the Encyclopedia of Cancer are encouraged to keep their presentations up-to-date by online editing. Clinical and basic scientists are encouraged to suggest new essays to the editor-in-chief. The technical preparation of the Encyclopedia of Cancer would not have been possible without the competent input of Jutta Jaeger-Hamers, Melanie Thanner, and Saskia Ellis; their excellent and pleasant cooperation is highly appreciated. Heidelberg, Germany Manfred Schwab

vii

Preface to the Second Edition

Given the overwhelming success of the first edition of the Cancer Encyclopedia, which appeared in 2001, and the amazing development in the different fields of cancer research, it has been decided to publish a second fully revised and expanded edition, following the principal concept of the first edition that has proven so successful. Recent developments are seeing a dynamic merging of basic and clinical science, with translational research increasingly becoming a new paradigm in cancer research. The merging of different basic and clinical science disciplines toward the common goal of fighting against cancer has long ago called for the establishment of a comprehensive reference source both as a tool to close the language gap between clinical and basic science investigators and as a platform of information for advanced students and informed laymen alike. It is intended to be a resource for all interested in information beyond their own specific expertise. While the first edition had featured contributions from approximately 300 scientists/clinicians in one volume, the second edition includes more than 1,000 contributors in four volumes with an A–Z format of more than 7,000 entries. It provides definitions of common acronyms and short definitions of both related terms and processes in the form of keyword entries. A major information source are detailed essays that provide comprehensive information on syndromes, genes and molecules, and processes and methods. Each essay is well structured, with extensive cross-referencing between entries. Essays represent original contributions by the corresponding authors, all distinguished scientists in their own field, editorial input has been carefully restricted to formal aspects. A panel of field editors, each an eminent international expert for the corresponding field, has served to ensure the presentation of timely and authoritative Encyclopedia entries. These new traits are likely to meet the expectance that a wide community has toward a cancer reference work. An important element in the preparation of the Encyclopedia has been the competent support by the Springer crew, Dr. Michaela Bilic, Saskia Ellis, and lately, Jana Simniok. I am extremely grateful for their excellent and pleasant cooperation.

ix

x

Preface to the Second Edition

The Cancer Encyclopedia, Second Edition, will be available both in print and online versions. Clinicians, research scientists, and advanced students will find this an amazing resource and a highly informative reference for cancer. Heidelberg, Germany Manfred Schwab

Preface to the First Edition

Cancer, although a dreadful disease, is at the same time a fascinating biological phenotype. Around 1980, cancer was first attributed to malfunctioning genes and, subsequently, cancer research has become a major area of scientific research supporting the foundations of modern biology to a great extent. To unravel the human genome sequence was one of those extraordinary tasks, which has largely been fueled by cancer research, and many of the fascinating insights into the genetic circuits that regulate developmental processes have also emerged from research on cancer. Diverse biological disciplines such as cytogenetics, virology, cell biology, classical and molecular genetics, epidemiology, biochemistry, together with the clinical sciences, have closed ranks in their search of how cancer develops and to find remedies to stop the abnormal growth that is characteristic of cancerous cells. In the attempt to establish how, why, and when cancer occurs, a plethora of genetic pathways and regulatory circuits have been discovered that are necessary to maintain general cellular functions such as proliferation, differentiation, and migration. Alterations of this fine-tuned network of cascades and interactions, due to endogenous failure or to exogenous challenges by environmental factors, may disable any member of such regulatory pathways. This could, for example, induce the death of the affected cell, may mark it for cancerous development or may immediately provide it with a growth advantage within a particular tissue. Recent developments have seen the merger of basic and clinical science. Of the former, particularly genetics has provided instrumental and analytical tools with which to assess the role of environmental factors in cancer, to refine and enable diagnosis prior to the development of symptoms, and to evaluate the prognosis of patients. Hopefully, even better strategies for causal therapy will become available in the future. Merging the basic and clinical science disciplines toward the common goal of fighting cancer calls for a comprehensive reference source to serve both as a tool to close the language gap between clinical and basic science investigators and as an information platform for the student and the informed layperson alike. Obviously this was an extremely ambitious goal, and the immense progress in the field cannot always be portrayed in line with the latest developments. The aim of the Encyclopedia is to provide the reader with an entrance point to a particular topic. It should be of value to both basic and clinical scientists working in the field of cancer research. Additionally, both students and lecturers in the life sciences should xi

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Preface to the First Edition

benefit highly from this database. I therefore hope that this Encyclopedia will become an essential complement to existing science resources. The attempts to identify the mechanisms underlying cancer development and progression have produced a wealth of facts, and no single individual is capable of addressing the immense breadth of the field with undisputed authority. Hence, the “Encyclopedic Reference of Cancer” is the work of many authors, all of whom are experts in their fields and reputable members of the international scientific community. Each author contributed a large number of keyword definitions and in-depth essays and in so doing it was possible to cover the broad field of cancer-related topics within a single publication. Obviously this approach entails a form of presentation, in which the author has the freedom to set priorities and to promote an individual point of view. This is most obvious when it comes to nomenclature, particularly that of genes and proteins. Although the editorial intention was to apply the nomenclature of the Human Genome Organisation (HUGO), the more vigorous execution of this attempt has been left to future endeavors. In the early phase of planning the Encyclopedia, exploratory contacts to potential authors produced an overwhelmingly positive response. The subsequent contact with almost 300 contributory authors was a marvelous experience, and I am extremely grateful for their excellent and constructive cooperation. An important element in the preparation of the Encyclopedia has been the competent secretarial assistance of Hiltrud Wilbertz of the Springer-Verlag and of Ingrid Cederlund and Cornelia Kirchner of the DKFZ. With great attention to detail they helped to keep track of the technical aspects in the preparation of the manuscript. It was a pleasure to work with the Springer crew, including Dr. Rolf Lange as the Editorial Director (Medicine) and Dr. Thomas Mager, Senior Editor for Encyclopedias and Dictionaries. In particular I wish to thank Dr. Walter Reuss, who untiringly has mastered all aspects and problems associated with the management of the numerous manuscripts that were received from authors of the international scientific community. It has been satisfying and at times comforting to see how he made illustration files come alive. Thanks also to Dr. Claudia Lange who, being herself a knowledgeable cell biologist, has worked as the scientific editor. Her commitment and interest have substantially improved this Encyclopedia. As a final word, I would like to stress that although substantial efforts have been made to compose factually correct and well-understandable presentations, there may be places where a definition is incomplete or a phrase in an essay is flawed. All contributors to this Encyclopedia will be extremely happy to receive possible corrections, or revisions, in order for them to be included in any future editions of the “Encyclopedic Reference of Cancer.” Heidelberg, Germany Manfred Schwab

Editor-in-Chief

Manfred Schwab, Dr. rer. nat. University-Professor of Genetics Neuroblastoma Genomics B087 German Cancer Research Center (DKFZ)

xiii

Contributors

Apart from few editorial input, the respective authors are responsible for the content of their own texts. Vesa Aaltonen Department of Ophthalmology, University of Turku, Turku, Finland Trond Aasen Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Cory Abate-Shen Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA Phillip H. Abbosh Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Kotb Abdelmohsen RNA Regulation Section, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, MD, USA Fritz Aberger Department of Molecular Biology, University of Salzburg, Salzburg, Austria Hinrich Abken Tumor Genetics, Clinic I Internal Medicine, University Hospital Cologne, and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Amal M. Abu-Ghosh Department of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Rosita Accardi Infections and Cancer Biology Group, International Agency for Research on Cancer, Lyon, France Filippo Acconcia Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Christina L. Addison Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Vaqar M. Adhami School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA Farrukh Afaq Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA Chapla Agarwal SOP-Administration, University of Colorado Denver – Anschutz Medical Campus, Aurora, CO, USA xv

xvi

Garima Agarwal College of Pharmacy, The Ohio State University, Columbus, OH, USA Rajesh Agarwal Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO, USA Patrizia Agostinis Department of Cellular and Molecular Medicine, Cell Death Research and Therapy Lab, KU Leuven Campus Gasthuisberg, Leuven, Belgium Terje C. Ahlquist Roche Norway, Oslo, Norway Kazi Mokim Ahmed Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA Khalil Ahmed Minneapolis VA Health Care System and University of Minnesota, Minneapolis, MN, USA Shahid Ahmed Department of Oncology, University of Saskatchewan, Saskatoon, SK, Canada Joohong Ahnn Department of Life Science, Hanyang University, Seoul, South Korea Cem Akin University of Michigan, Ann Arbor, MI, USA Gada Al-Ani Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA Ami Albihn Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden Adriana Albini IRCCS Multimedica, Milano, Italy Jérôme Alexandre Faculté de Médecine Paris – Descartes, UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul, Paris, France Amal Yahya Alhefdhi Department of Surgery – MBC 40, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia Shadan Ali Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA Malcolm R. Alison Centre for Diabetes and Metabolic Medicine, Queen Barts and the London School of Medicine and Dentistry, Institute of Cell and Molecular Science, London, UK Catherine Alix-Panabieres University Medical Center, Lapeyronie Hospital, Montpellier, France Alison L. Allan Cancer Research Laboratories, London Regional Cancer Program and Departments of Oncology and Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

Contributors

Contributors

xvii

Paola Allavena Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy Damian A. Almiron Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Angel Alonso Deutsches Krebsforschungszentrum, Heidelberg, Germany Gianfranco Alpini Departments of Medicine and Medical Physiology, Texas A&M Health Science Center, College of Medicine, Central Texas Veterans Health Care System, Baylor Scott & White Health, Temple, TX, USA Marie-Clotilde Alves-Guerra Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Pierre Åman LLCR, Department of Pathology, Institute of Biomedicine, Sahlgrenska Academy, Goteborg University, Gothenburg, Sweden Kurosh Ameri Department of Medicine, Division of Cardiology, Translational Cardiac Stem Cell Program, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Cardiovascular Research Institute, University of California San Francisco (UCSF), San Francisco, CA, USA Mounira Amor-Guéret Institut Curie – UMR 3348 CNRS, Orsay Cedex, France Grace Amponsah Department of Pathology, Comprehensive Cancer Centre, The Ohio State Medical Centre, Columbus, OH, USA John W. Anderson Dream Master Laboratory, Chandler, AZ, USA Kenneth C. Anderson Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Boston, MA, USA Nicolas André Centre for Research in Oncobiology and Oncopharmacology, INSERM U911, Marseille, France Metronomics Global Health Initiative, Marseille, France Department of Pediatric Hematology and Oncology, La Timone Children’s Hospital, Marseille, France Peter Angel Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Heidelberg, Germany Andrea Anichini Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy Talha Anwar Medical Scientist Training Program and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA Peter D. Aplan Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

xviii

Natalia Aptsiauri UGC Laboratorio Clínico Hospital Universitario Virgen de las Nieves Facultad de Medicina, Universidad de Granada, Granada, Spain Rami I. Aqeilan He Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research-Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel Tsutomu Araki Departments of Obstetrics and Gynecology, Nippon Medical School, Kawasaki and Tokyo, Japan Sanchia Aranda School of Nursing, The University of Melbourne, Carlton, VIC, Australia Diego Arango CIBBIM - Nanomedicina Oncologia Molecular, Vall d’Hebron Hospital Research Institute, Barcelona, Spain David J. Araten NYU School of Medicine, Laura and Isaac Perlmutter Cancer Center and the New York VA Medical Center, New York, NY, USA Laura Arbona Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain Valentina Arcangeli Department of Oncology, Instituto Scientifico Romagnolo per lo s, Infermi Hospital, Rimini, Italy Gemma Armengol Faculty Biosciences, U. Biological Anthropology, Universitat Autonoma de Barcelona, Barcelona, Spain Elias S. J. Arnér Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Marie Arsenian-Henriksson Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden Stefano Aterini Department of Experimental Pathology and Oncology, University of Firenze, Florence, Italy Scott Auerbach Biomolecular Screening, National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA Katarzyna Augoff Department of Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland Marc Aumercier CNRS, INRA, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, Villeneuve d’Ascq, France Riccardo Autorino Clinica Urologica, Seconda Università degli Studi, Naples, Italy Matias A. Avila Division of Hepatology, CIMA, University of Navarra, Pamplona, Spain Hava Karsenty Avraham Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA, USA

Contributors

Contributors

xix

Shalom Avraham Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA, USA Sanjay Awasthi United States Longview Cancer Center, Longview, TX, USA Yogesh C. Awasthi City of Hope, Duarte, CA, USA Debasis Bagchi Department of Pharmacy Sciences, Creighton University Medical Center, Omaha, NE, USA Xue-Tao Bai State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Michael J. Baine Department of Radiation Oncology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Jürgen Bajorath Department of Life Science Informatics, B-IT, University of Bonn, Bonn, Germany Stuart G. Baker Biometry Research Group, National Cancer Institute, Bethesda, MD, USA Elizabeth K. Balcer-Kubiczek Department of Radiation Oncology, Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA Enke Baldini Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy Graham S. Baldwin Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia Sherri Bale GeneDx, Rockville, MD, USA Laurent Balenci INSERM Unité Mixte 873, Grenoble, France Sushanta K. Banerjee Cancer Research Unit, Research Division, VA Medical Center, Kansas City, MO, USA Michal Baniyash The Lautenberg Center for Immunology and Cancer Research, Israel-Canada Medical, Research Institute Faculty of Medicine, The Hebrew University, Jerusalem, Israel Shyam S. Bansal Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, AL, USA Nektarios Barabutis Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA Aditya Bardia Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA Rafijul Bari Departments of Medicine and Molecular Sciences, Vascular Biology Center, Cancer Institute, University of Tennessee Health Science Center, Memphis, TN, USA

xx

Contributors

Nicola L. P. Barnes Department of Academic Surgery, South Manchester University Hospital, Manchester, UK Robert Barouki Inserm UMR-S 1124, Université Paris Descartes, Paris, France Juan Miguel Barros-Dios Department of Preventive Medicine and Public Health, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Harry Bartelink Department of Radiotherapy, The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Stefan Barth Institute of Infectious Disease and Molecular Medicine and Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa Helmut Bartsch Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Thomas Barz Max-Panck-Institut für Psychiatrie, Munich, Germany Holger Bastians Abt. Molekulare Göttingen, Göttingen, Germany

Onkologie,

Universitätsmedizin

Anna Batistatou Ioannina University Medical School, Ioannina, Greece Surinder K. Batra Eppley Institute for Research in Cancer and Allied Diseases and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Frederic Batteux Faculté de Médecine Paris – Descartes, UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul, Paris, France Jacques Baudier INSERM Unité Mixte 873, Grenoble, France Paul Bauer Pfizer Research Technology Center, Cambridge, MA, USA Tobias Bäuerle Institute of Radiology, University Medical Center Erlangen, Erlangen, Germany Asne R. Bauskin Department of Medicine, Centre for Immunology, St. Vincent’s Hospital, University of New South Wales, Sydney, NSW, Australia Boon-Huat Bay Department of Anatomy, National University of Singapore, Singapore, Singapore Jean-Claude Béani Clinique Universitaire de Dermato-Vénéréologie, Photobiologie et Allergologie, Pôle Pluridisciplinaire de Médecine, CHU de Grenoble, Grenoble, France Nicole Beauchemin Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada John F. Bechberger Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, BC, Canada

Contributors

xxi

Gerhild Becker Department of Palliative Care, University Hospital Freiburg, Freiburg, Germany Katrin Anne Becker Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany Marie E. Beckner Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Roberto Bei Department of Clinical Sciences and Translational Medicine, Faculty of Medicine, University of Rome “Tor Vergata”, Rome, Italy Claus Belka Department of Radiation Oncology, University of Tübingen, Tübingen, Germany Anita C. Bellail Department of Pathology and Laboratory Medicine, Henry Ford Health System, Detroit, MI, USA Larissa Belov School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW, Australia P. Annécie Benatrehina College of Pharmacy, The Ohio State University, Columbus, OH, USA Maurizio Bendandi Department of Clinical Medicine, School of Medicine, Ross University, Roseau, Commonwealth of Dominica Yaacov Ben-David Division of Molecular and Cellular Biology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada Martin Benesch Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescence Medicine, Medical University of Graz, Graz, Austria Suzanne M. Benjes Cancer Genetics Research, University of Otago, Christchurch, New Zealand Carmen Berasain Division of Hepatology, CIMA, University of Navarra, Pamplona, Spain Alan Berezov Department of Pathology, Laboratory Medicine and Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Rob J. W. Berg University Medical Center Utrecht, Utrecht, The Netherlands Corinna Bergelt Institute of Medical Psychology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Rene Bernards The Netherlands Cancer Institute, Amsterdam, The Netherlands Zwi Berneman Vaccine and Infections Disease Institute (VAXINFECTIO) Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium

xxii

Jérôme Bertherat Endocrinology, Metabolism and Cancer Department, INSERM U567, Institut Cochin, Paris, France Saverio Bettuzzi Department of Biomedicine, Biotechnology and Translational Research, University of Parma, Parma, Italy Arun Bhardwaj Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA Kumar M. R. Bhat Department of Anatomy, Kasturba Medical College, Manipal University, Manipal, Karnataka, India Malaya Bhattacharya-Chatterjee University of Cincinnati and The Barrett Cancer Center, Cincinnati, OH, USA Caterina Bianco Division of Extramural Activities, National Institutes of Health, Rockville, MD, USA Tina Bianco-Miotto Robinson Research Institute and School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA, Australia Jean-Michel Bidart Department of Clinical Biology, Institut GustaveRoussy, Villejuif, France Jaclyn A. Biegel Department of Pathology and Laboratory Medicine, Children’s Hospital of Los Angeles, Los Angeles, CA, USA Margherita Bignami Istituto Superiore di Sanita’, Rome, Italy Irene V. Bijnsdorp Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Chen Bing Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK Angelique Blanckenberg Department of Chemistry and Polymer Science, Stellenbosch University, Matieland, South Africa Giovanni Blandino Translational Oncogenomic Laboratory, Regina Elena Cancer Institute, Rome, Italy David E. Blask Laboratory of Chrono-Neuroendocrine Oncology, Department of Structural and Cellular Biology, Tulane University School of Medicine, New Orleans, LA, USA Jonathan Blay Department of Pharmacology, Dalhousie University, Halifax, NS, Canada Peter Blume-Jensen Xtuit Pharmaceuticals, Boston, MA, USA Sarah Bocchini Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy Ann M. Bode The Hormel Institute, University of Minnesota, Austin, MN, USA Paolo Boffetta Icahn School of Medicine at Mount Sinai, New York, NY, USA

Contributors

Contributors

xxiii

Stefan K. Bohlander Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Valentina Bollati EPIGET - Epidemiology, Epigenetics and Toxicology Lab - Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Subbarao Bondada Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY, USA Maria Grazia Borrello Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Giuseppe Borzacchiello Department of Veterinary Medicine and Animal Productions, University of Naples “Federico II”, Naples, Italy Valerie Bosch Forschungsschwerpunkt Infektion und Krebs, F020, German Cancer Research Center (DKFZ), Heidelberg, Germany Chris Boshoff Cancer Research Campaign Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, London, UK Irina Bosman Institute of Pharmacy, University of Bonn, Bonn, Germany Galina I. Botchkina Department of Pathology, Stony Brook University, Stony Brook, NY, USA Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY, USA Franck Bourdeaut Département de pédiatrie, INSERM 830, Biologie et génétique des tumeurs, Institut Curie, Paris, France Jean-Pierre Bourquin Pediatric Oncology, University Children’s Hospital Zurich, Zurich, Switzerland Hassan Bousbaa Instituto Investigação Formação Avançada Ciências Tecnologias Saúde, CESPU – Cooperativa de Ensino Superior Politecnico e Universitario, Gandra PRD, Portugal Norman Boyd Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, Toronto, ON, Canada Sven Brandau Department of Otorhinolaryngology, University DuisburgEssen, Essen, Germany Burkhard H. Brandt Institute of Clinical Chemistry, University Medical Centre Schleswig-Holstein, Kiel, Germany Hiltrud Brauch Breast Cancer Susceptibility and Pharmacogenomics, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tübingen, Stuttgart, Germany Massimo Breccia Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy

xxiv

Samuel N. Breit Cytokine Biology and Inflammation Research Program, St Vincent’s Centre for Applied Medical Research (AMR), St Vincent’s Hospital, Sydney, NSW, Australia Edwin Bremer Department of Pathology and Laboratory Medicine, Section Medical Biology, Laboratory for Tumor Immunology, University Medical Center Groningen, Groningen, The Netherlands Catherine Brenner INSERM UMR-S 769, Labex LERMIT, ChâtenayMalabry, University of Paris Sud, Paris, France David J. Brenner Department of Radiation Oncology, Columbia University, New York, NY, USA Amanda E. Brinker Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA Nikko Brix Clinic for Radiotherapy and Radiation Oncology, LMU Munich, Munich, Germany Katja Brocke-Heidrich Praxis für Naturheilkunde und ganzheitliche Therapie, Leipzig, Germany Angela Brodie University of Maryland School of Medicine, Baltimore, MD, USA Jonathan Brody Department of Surgery, Thomas Jefferson University, Philadelphia, PA, USA Christopher L. Brooks Institute for Cancer Genetics, and Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Mai N. Brooks Surgical Oncology, School of Medicine, University of California, Los Angeles, CA, USA David A. Brown St. Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital, University of New South Wales, Sydney, NSW, Australia Karen Brown Department of Cancer Studies, University of Leicester, Leicester, UK Kevin Brown University of Florida, College of Medicine, Gainesville, FL, USA Tilman Brummer Institut für Molekulare Medizin und Zellforschung, Zentrum für Biochemie und Molekulare Zellforschung (ZBMZ), AlbertLudwigs-Universität Freiburg, Freiburg, Germany Antonio Brunetti Department of Health Sciences, University of Catanzaro “Magna Græcia”, Catanzaro, Italy Andreas K. Buck Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany

Contributors

Contributors

xxv

Laszlo Buday Department of Medical Chemistry, Semmelweis University Medical School, Budapest, Hungary Marie Annick Buendia Hopital Paul Brousse, Inserm U785, Centre Hépatobiliaire, Villejuif, France Ralf Buettner City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Nigel J. Bundred Department of Academic Surgery, South Manchester University Hospital, Manchester, UK Alexander Bürkle Department of Biology, University of Konstanz, Konstanz, Germany Barbara Burwinkel Division Molecular Biology of Breast Cancer, University of Heidelberg, Department of Gynecology and Obstetrics, Heidelberg, Germany Xavier Busquets Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain Jagdish Butany Laboratory Medicine and Pathobiology, University Health Network/Toronto, Toronto, ON, Canada Neville J. Butcher School of Biomedical Sciences, University of Queensland, St Lucia, QLD, Australia Timon P. H. Buys Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, BC, Canada Miguel A. Cabrita Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Jean Cadet Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada Yi Cai Department of Pathology, Baylor College of Medicine, Houston, TX, USA Yiqiang Cai Section of Nephrology, Yale University School of Medicine, New Haven, CT, USA Bruno Calabretta Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA, USA Daniele Calistri Molecular Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Meldola, Italy Javier Camacho Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del I.P.N., Mexico City, D.F., Mexico William G. Cance Departments of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA

xxvi

Amparo Cano Departamento de Bioquímica, Facultad de Medicina, UAM, Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM IdiPAZ, Madrid, Spain Anthony J. Capobianco Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Emilia Caputo Institute of Genetics and Biophysics – ABT, Napoli, Italy Salvatore J. Caradonna Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, NJ, USA Michele Carbone University of Hawaii Cancer Center, Honolulu, HI, USA Vinicio Carloni University of Florence, Florence, Italy Neil O. Carragher Drug Discovery Group, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh, UK Michela Casanova Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy Wolfgang H. Caselmann Medizinische Klinik und Poliklinik I, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany Giuliana Cassinelli Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Webster K. Cavenee Ludwig Institute for Cancer Research, UCSD, La Jolla, CA, USA Esteban Celis Georgia Cancer Center, Augusta University, Augusta, GA, USA Chiswili Chabu Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Wook-Jin Chae Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Ho Man Chan Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, UK Shing Leng Chan Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Dawn S. Chandler Department of Pediatrics, Columbus Children’s Research Institute, Center for Childhood Cancer, The Ohio State University School of Medicine, Columbus, OH, USA Guru Chandramouly Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Mau-Sun Chang Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

Contributors

Contributors

xxvii

Mei-Chi Chang Biomedical Science Team, Chang Gung Institute of Technology, Taoyuan, Taiwan Lung-Ji Chang Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA Jane C. J. Chao School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan Christine Chaponnier Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Konstantinos Charalabopoulos Ioannina University Medical School, Ioannina, Greece Malay Chatterjee Department of Pharmaceutical Technology, Jadavpur University, Calcutta, West Bengal, India Sunil K. Chatterjee University of Cincinnati and The Barrett Cancer Center, Cincinnati, OH, USA Gautam Chaudhuri Department of Molecular and Medical Pharmacology and Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA M. Asif Chaudry University Department of Surgery, Royal Free and University College London Medical School, London, UK Dharminder Chauhan Department of Medical Oncology, The Jerome Lipper Multiple Myeloma Center, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Jeremy P. Cheadle Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK Ai-Ping Chen Department of Gynecology, Affiliated Hospital of Qingdao University, Qingdao, China Chienling Chen Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA Herbert Chen Department of Surgery, University of Alabama - Birmingham (UAB) School of Medicine, UAB Hospital and Health System, University of Alabama Comprehensive Cancer Center, Birmingham, AL, UK Jie Chen Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China Sai-Juan Chen State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Taosheng Chen Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, USA

xxviii

Wenxing Chen Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China Yingchi Chen Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA Zhu Chen State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China George Z. Cheng Harvard Medical School, Boston, MA, USA Jin Q. Cheng Molecular Oncology Program and Research Institute, H. Lee Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, FL, USA Liang Cheng Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Chun Hei Antonio Cheung Department of Pharmacology and Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China Ya-Hui Chi Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan Martyn A. Chidgey School of Cancer Sciences, University of Birmingham, Birmingham, UK Sudhakar Chintharlapalli Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA Alexandre Chlenski Department of Pediatrics, Section of Hematology/ Oncology, University of Chicago, Chicago, IL, USA Daniel C. Cho Beth Israel Deaconess Medical Center, Boston, MA, USA William Chi-Shing Cho Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong Michael Chopp Neurology Research, Henry Ford Health System, Detroit, MI, USA Pei-Lun Chou Division of Allergy-Immunology-Rheumatology, Department of Internal Medicine, Lin Shin Hospital, Taichung, Taiwan Claus Christensen Department of Cancer Genetics, Danish Cancer Society, Copenhagen, Denmark Rikke Christensen Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark Gerhard Christofori Department of Biomedicine, University of Basel, Basel, Switzerland Richard I. Christopherson School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia

Contributors

Contributors

xxix

Fong-Fong Chu Department of Cancer Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA Wen-Ming Chu Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI, USA Bong-Hyun Chung BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Republic of Korea Fung-Lung Chung Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA Jacky K. H. Chung Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada Sue Clark Imperial College London, London, UK Pier Paolo Claudio The University of Mississippi, Medical Center Cancer Institute, Jackson, MS, USA Elizabeth B. Claus Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT, USA Pascal Clayette SPI-BIO, Service de Neurovirologie, CEA, CRSSA, EPHE, Fontenay aux Roses Cedex, France Dahn L. Clemens Research Service, Veterans Administration Medical Center, Omaha, NE, USA Steven C. Clifford Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK Kevin A. Cockell Nutrition Research Division, Health Canada, Ottawa, ON, Canada Susan L. Cohn Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, IL, USA Graham A. Colditz Washington University in St. Louis, St. Louis, MO, USA Paola Collini Anatomic Pathology Department, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy Andrew R. Collins Department of Nutrition, University of Oslo, Oslo, Norway Nicoletta Colombo Istituto Europeo di Oncologia, Milan, Italy Joan W. Conaway Stowers Institute for Medical Research, Kansas, MO, USA Ronald C. Conaway Stowers Institute for Medical Research, Kansas, MO, USA Bong-Hyun Chung: deceased.

xxx

Lellys Mariella Contreras Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA Amanda E. Conway Molecular Cancer Biology, Duke University Medical Center, Durham, NC, USA Nathalie Cools Vaccine and Infections Disease Institute (VAXINFECTIO) Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium Helen C. Cooney UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland Scott Coonrod Baker Institute for Animal Health, Department of Biomedical Sciences, School of Veterinary Medicine, Cornell University, Ithaca, NY, USA Kumarasen Cooper Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA Laurence J. N. Cooper Division of Pediatrics, Department of Immunology, MD Anderson Cancer Center, Houston, TX, USA Michael K. Cooper Department of Neurology, Vanderbilt Medical Center, Nashville, TN, USA Peter J. Coopman IRCM, INSERM U1194, Montpellier Cancer Research Institute, Montpellier, France Lanfranco Corazzi Department of Experimental Medicine, University of Perugia, Perugia, Italy Maria Paola Costi Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, Modena, Italy Richard J. Cote Department of Pathology, Miller School of Medicine, University of Miami, Miami, FL, USA Massimo Cristofanilli Division of Hematology and Oncology, Robert H Lurie Comprehensive Cancer Center, Chicago, IL, USA Marcus V. Cronauer Department of Urology, University Hospital Schleswig-Holstein – Campus Lübeck, Lübeck, Germany Sidney Croul Department of Pathology, UHN, University of Toronto, Toronto, ON, Canada Ronald G. Crystal Division of Pulmonary and Critical Care Medicine, Weill Cornell Medical College, New York, NY, USA Bruce D. Cuevas Department of Molecular Pharmacology and Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA Jiuwei Cui Jilin University, Changchun, Jilin, China Edna Cukierman Basic Science/Tumor Cell Biology, Fox Chase Cancer Center, Philadelphia, PA, USA

Contributors

Contributors

xxxi

Zoran Culig Department of Urology, Innsbruck Medical University, Innsbruck, Austria David Cunningham Department of Medicine, The Royal Marsden NHS Foundation Trust, London, UK David T. Curiel Division of Cancer Biology, Washington University, St. Louis, MO, USA Franck Cuttitta NCI Angiogenesis Core Facility, National Cancer Institute, National Institutes of Health, Advanced Technology Center, Gaithersburg, MD, USA Andrea Cziffer-Paul Department of Pathology, The Mount Sinai School of Medicine, New York, NY, USA Massimino D’Armiento Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy Yun Dai Department of Gastroenterology, Peking University First Hospital, Beijing, China Yataro Daigo Institute of Medical Science, The University of Tokyo, Tokyo, Japan Lokesh Dalasanur Nagaprashantha City of Hope National Medical Center, Duarte, CA, USA Ashraf Dallol Centre of Innovation in Personalised Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Tamas Dalmay School of Biological Sciences, University of East Anglia, Norwich, UK Ivan Damjanov Department of Pathology, University of Kansas School of Medicine, Kansas City, KS, USA Vincent Dammai Dammai-Morgan Scientific Consultants LLC, Mount Pleasant, SC, USA Chendil Damodaran University of Louisville, Louisville, KY, USA Janet E. Dancey Canadian Cancer Trials Group, Queen’s University, Kingston, ON, Canada Nadia Dandachi Department of Internal Medicine, Division of Oncology, Medical University Graz, Graz, Austria Chi V. Dang Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA Alla Danilkovitch-Miagkova National Cancer Institute-FCRDC, Frederick, MD, USA Kakoli Das Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, Singapore

xxxii

Kaustubh Datta Department of Urology Research, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Pran K. Datta Departments of Surgery and Cancer Biology, VanderbiltIngram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Leonor David IPATIMUP (Institute of Molecular Pathology and Immunology of the University of Porto) and Medical Faculty of the University of Porto, Porto, Portugal David Mark Davies Department of Oncology, South West Wales Cancer Centre, Swansea, UK Juhayna Kassem Davis Carolinas HealthCare System, Charlotte, NC, USA Alexey Davydov Fox Chase Cancer Center, Philadelphia, PA, USA Shaheenah Dawood Department of Medical Oncology, Dubai Hospital, Dubai, United Arab Emirates Robert Day Department of Surgery/Division of Urology, Institut de Pharmacologie, Faculté de Médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada Terry Day Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Suzane Ramos da Silva Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Enrique de Alava Institute of Biomedicine of Sevilla (IBiS), Virgen del Rocio University Hospital /CSIC/University de Sevilla, Seville, Spain Diederik de Bruijn Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Floris Aart de Jong Amgen BV, Breda, The Netherlands Vincenzo de Laurenzi Department of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, Rome, Italy Ben O. de Lumen Department of Nutritional Sciences and Toxicology, University of California at Berkeley, Berkeley, CA, USA Elvira de Mejia Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL, USA Ana Ramirez de Molina Nutritional Genomics and Cancer Unit, IMDEA Food Institute, Madrid, Spain

Contributors

Contributors

xxxiii

Christiane de Wolf-Peeters Department of Pathology, University Hospitals of K.U. Leuven, Leuven, Belgium Jochen Decker Hematology Oncology Medical School Clinic III, University of Mainz, Mainz, Germany P. Markus Deckert Zentrum für Innere Medizin II – Abteilung für Onkologie und Palliativmedizin, Klinikum Brandenburg, Brandenburg an der Havel, Germany Francesca Degrassi Institute of Molecular Biology and Pathology IBMN c/o “Sapienza” University, Italian National Research Council CNR, Rome, Italy Amir R. Dehdashti Division of Neurosurgery, University of Toronto, Toronto, ON, Canada Maryse Delehedde R&D Lunginnov, Campus de l’Institut Pasteur de Lille, Lille, France Olivier Dellis Signalisation Calcique et Interactions Cellulaires dans le Foie, INSERM UMR-S 1174, Université Paris-Sud 11, Orsay, France Renée M. Demarest Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Berna Demircan University of Florida, College of Medicine, Gainesville, FL, USA Miriam Deniz Department of Obstetrics and Gynaecology, University of Ulm, Ulm, Germany Samuel Denmeade The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins, Baltimore, MD, USA David A. Denning Department of Surgery, Marshall University, Huntington, WV, USA Sylviane Dennler Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands Channing J. Der University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Barbara Deschler Comprehensive Cancer Center Mainfranken, Clinical Trials Office, University of Würzburg, Würzburg, Germany Chantal Desdouets Institut Cochin, Université Paris Descartes, CNRS, Paris, France Peter Devilee Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Mark W. Dewhirst Department of Radiation Oncology, Duke University, Durham, NC, USA Girish Dhall Division of Hematology-Oncology, Department of Pediatrics, Children’s Hospital Los Angeles and the Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

xxxiv

Danny N. Dhanasekaran Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Pier Paolo Di Fiore IFOM, the FIRC Institute of Molecular Oncology, Milan, Italy Giuseppe Di Lorenzo Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia molecolare e clinica, Università degli Studi “Federico II”, Naples, Italy Dario Di Luca Department of Medical Sciences, University of Ferrara, Ferrara, Italy Marc Diederich College of Pharmacy, Seoul National University, Seoul, South Korea Joseph DiFranza Department of Family Medicine and Community Health, University of Massachusetts Medical Center, Worcester, MA, USA Martin Digweed Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Berlin, Germany Peter ten Dijke Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands Gerard Dijkstra University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Nathalie Dijsselbloem Lab of Eukaryotic Gene Expression, LEGEST-University Gent, Ghent, Belgium Helen Dimaras The Hospital for Sick Children, Department of Ophthalmology and Vision Science, The University of Toronto, Toronto, ON, Canada Jian Ding State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China Zhaoxia Ding Department of Gynecology, Affiliated Hospital of Qingdao University, Qingdao, China Jürgen Dittmer Klinik für Gynäkologie, Universität Halle-Wittenberg, Halle (Saale), Germany Henrik J. Ditzel Department of Cancer and Inflammation Reserch, Institute fo Molecular Medicine, University of Southern Denmark, Odense C, Denmark Dan Dixon Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA Cholpon S. Djuzenova Klinik für Strahlentherapie der Universität Würzburg, Würzburg, Germany Christian Doehn Urologikum Lübeck, Lübeck, Germany

Contributors

Contributors

xxxv

Yasufumi Doi Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Milos Dokmanovic Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA Qihan Dong The University of Western Sydney, Sydney, NSW, Australia Department of Endocrinology, Central Clinical School, Royal Prince Alfred Hospital, The University of Sydney, Sydney, NSW, Australia Zigang Dong The Hormel Institute, University of Minnesota, Austin, MN, USA Ben Doron Oregon Health and Science University, Portland, OR, USA Qing Ping Dou The Prevention Program, Barbara Ann Karmanos Cancer Institute and Department of Pathology, School of Medicine, Wayne State University, Detroit, MI, USA Thierry Douki Laboratoire “Lésions des Acides Nucléiques”, Institute Nanosciences et Cryogénie, Grenoble, France Harry A. Drabkin Division of Hematology-Oncology, Medical University of South Carolina and the Hollings Cancer Center, Charleston, SC, USA Tommaso A. Dragani Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy Kenneth Drake Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, USA Martin Dreyling Department of Internal Medicine III, University of Munich, Großhadern, Munich, Germany Nathalie Druesne-Pecollo UMR U1153 INSERM, U1125 INRA, CNAM, Université Paris 13, Centre de Recherche Epidémiologie et Statistique Sorbonne Paris Cité, Bobigny, France Brian J. Druker Oregon Health and Science University Cancer Institute, Portland, OR, USA Denis Drygin Pimera, Inc., San Diego, CA, USA Raymond N. DuBois ASU Biodesign Institute, Tempe, AZ, USA Dan G. Duda Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Jaquelin P. Dudley Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA Roy J. Duhé Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA

xxxvi

Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS, USA Ignacio Duran Department of Medical Oncology and Hematology, Robert and Maggie Bras and Family New Drug Development Program, Princess Margaret Hospital, Toronto, ON, Canada Stephen T. Durant R&D, Oncology, Innovative Medicines, AstraZeneca, Little Chesterford, UK Meenakshi Dwivedi Department of Life Science, Hanyang University, Seoul, South Korea Madalene A. Earp Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN, USA Behfar Ehdaie Department of Surgery, Urology Service, Memorial SloanKettering Cancer Center, New York, NY, USA Justis P. Ehlers Cole Eye Institute, Cleveland Clinic, Cleveland, OH, USA Gerhard Eisenbrand Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany Mohamad Elbaz Department of Pathology, Comprehensive Cancer Centre, The Ohio State Medical Centre, Columbus, OH, USA Patricia V. Elizalde Laboratory of Molecular Mechanisms of Carcinogenesis, Institute of Biology and Experimental Medicine (IBYME), CONICET, Buenos Aires, Argentina Bassel El-Rayes Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA Winship Cancer Institute of Emory University, Atlanta, GA, USA Mitsuru Emi Departments of Obstetrics and Gynecology, Nippon Medical School, Kawasaki and Tokyo, Japan Steffen Emmert Clinic for Dermatology and Venereology, University Medical Center Rostock, Rostock, Germany Caroline End Division of Molecular Genome Analysis, DKFZ, Heidelberg, Germany Daniela Endt Department of Human Genetics, Biozentrum University of Würzburg, Würzburg, Germany Rainer Engers Institute of Pathology, University Hospital Düsseldorf, Düsseldorf, Germany Marica Eoli Unit of Clinical Neuro-Oncology, Istituto Neurologico Besta, Milan, Italy Anat Erdreich-Epstein Division of Hematology-Oncology, Department of Pediatrics, Children’s Hospital Los Angeles and the Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Contributors

Contributors

xxxvii

Süleyman Ergün Institut für Anatomie und Zellbiologie, Maximilians-Universität Würzburg, Würzburg, Germany

Julius-

Pablo V. Escribá Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain Nuria Están-Capell Service of Clinical Analysis, Dr. Peset University Hospital, Valencia, Spain Konstantinos Evangelou Molecular Carcinogenesis Group, Laboratory of Histology-Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece Mark F. Evans Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT, USA B. Mark Evers Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA Vera Evtimov Monash University, Melbourne, VIC, Australia Jörg Fahrer Department of Toxicology, University Medical Center Mainz, Mainz, Germany Cristina Maria Failla Experimental Immunology Laboratory, IDI-IRCCS, Rome, Italy Marco Falasca Faculty of Health Sciences, School of Biomedical Sciences, Curtin University, Perth, WA, Australia Fang Fan Department of Pathology, University of Kansas School of Medicine, Kansas City, KS, USA Saijun Fan Long Island Jewish Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Bingliang Fang Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Jinxu Fang Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA Lei Fang Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Valeria R. Fantin Merck Research Laboratories, Boston, MA, USA Z. Shadi Farhangrazi Biotrends International, Denver, CO, USA Omid C. Farokhzad Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology, Brigham and Women’s Hospital, Boston, MA, USA William L. Farrar National Cancer Institute – Frederick, Frederick, MD, USA

xxxviii

Alessandro Fatatis Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA, USA Andrew P. Feinberg Department of Medicine and Center for Epigenetics, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Mark A. Feitelson Department of Biology, Temple University, Philadelphia, PA, USA Francesco Feo Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy Félix Fernández Madrid Department of Internal Medicine, Division of Rheumatology, Wayne State University, Detroit, MI, USA Paula Fernández-García Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain Marie Fernet INSERM U612, Institut Curie-Recherche, Orsay, France Audrey Ferrand INSERM U.858, Institut de Médecine Moléculaire de Rangueil, IFR150, Université Paul Sabatier, Toulouse, France Andrea Ferrari Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy Stefania Ferrari Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, Modena, Italy Robert A. Figlin Division of Hematology Oncology, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Lorena L. Figueiredo-Pontes Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Cristina Fillat Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain Daniel Finley Department of Cell Biology, Harvard Medical School, Boston, MA, USA Gaetano Finocchiaro Unit of Experimental Neuro-Oncology, Istituto Nazionale Neurologico Besta, Milan, Italy Paul B. Fisher Departments of Urology, Pathology and Neurosurgery, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY, USA James Flanagan Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Michael Fleischhacker Universitätsklinikum Halle (Saale), Klinik für Innere Medizin I, Schwerpunkt Pneumologie, Halle (Saale), Germany

Contributors

Contributors

xxxix

Eliezer Flescher Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Jonathan A. Fletcher Albany Medical College, Albany, NY, USA Barbara D. Florentine Department of Pathology, Henry Mayo Newhall Memorial Hospital, Valencia, CA, USA CA and Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Tamara Floyd Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Riccardo Fodde Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands Judah Folkman Children’s Hospital and Harvard Medical School, Boston, MA, USA Hamidreza Fonouni Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Kenneth A. Foon The Pittsburgh Cancer Institute, Pittsburgh, PA, USA Alessandra Forni Department of Occupational and Environmental Health “Clinica del Lavoro L. Devoto”, University of Milan, Milan, Italy David A. Foster Department of Biological Sciences, Hunter College of the City University of New York, New York, NY, USA Paul Foster Department of Endocrinology and Metabolic Medicine, Imperial College Faculty of Medicine, St. Mary’s Hospital, London, UK Paul Fréneaux Département de Pathologie, Institut Curie, Paris, France Rodrigo Franco Redox Biology Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA David A. Frank Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Stuart J. Frank Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, UAB, Endocrinology Section, Birmingham VAMC Medical Service, Birmingham VA Medical Center, University of Alabama, Birmingham, AL, USA Stanley R. Frankel Merck Research Laboratories, Boston, MA, USA Michael J. Franklin Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, USA Aleksandra Franovic Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada Ralph S. Freedman UT MD Anderson Cancer Center, Houston, TX, USA

xl

Michael R. Freeman Urological Diseases Research Center, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA Emil Frei Dana-Farber Cancer Institute, Boston, MA, USA Jean-Noël Freund INSERM U1113 and Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Faculté de Médecine, Strasbourg, France Errol C. Friedberg University of Texas Southwestern Medical Center, Dallas, TX, USA Steven M. Frisch Mary Babb Randolph Cancer Center and Department of Biochemistry, West Virginia University, Morgantown, WV, USA Andrew M. Fry University of Leicester, Leicester, UK Mark Frydenberg Department of Surgery, Monash University, Melbourne, VIC, Australia Hendrik Fuchs Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Atsuko Fujihara Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan Hirota Fujiki Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Saga, Japan Jiro Fujimoto Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan Jun Fujita Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Hiroshi Fukamachi Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Kenji Fukasawa Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Tomoya Fukui Department of Respiratory Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan Simone Fulda Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Frankfurt, Germany Claudia Fumarola Department of Experimental Medicine, Unit of Experimental Oncology, University of Parma, Parma, Italy Kyle Furge Van Andel Research Institute, Grand Rapids, MI, USA

Contributors

Contributors

xli

Mutsuo Furihata Department of Pathology, Kochi Medical School, Kochi, Japan Rhoikos Furtwängler Universitätsklinikum des Saarlandes, Klinik für Pädiatrische Onkologie und Hämatologie, Homburg/Saar, Germany Bernard W. Futscher Department of Pharmacology and Toxicology, Arizona Cancer Center and College of Pharmacy, University of Arizona, Tucson, AZ, USA Ulrich Göbel Clinic of Pediatric Oncology, Hematology and Immunology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany Tobias Görge Department of Dermatology, University of Münster, Münster, Germany Ursula Günthert Institute of Pathology, University Hospital, Basel, Switzerland Shirish Gadgeel Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA Jochen Gaedche Department of General, Visceral and Pediatric Surgery, University Medical Center, Göttingen, Germany Federico Gago Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain William M. Gallagher UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland Bernard Gallez Biomedical Magnetic Resonance, Université Catholique de Louvain, Brussels, Belgium Brenda L. Gallie The Hospital for Sick Children, Department of Ophthalmology and Vision Science, The University of Toronto, Toronto, ON, Canada Antoine Galmiche EA4666, Université de Picardie Jules Verne (UPJV), Amiens, France Service de Biochimie, Centre de Biologie Humaine (CBH), University Hospital of Amiens (CHU Sud), Amiens, France Ramesh K. Ganju Department of Pathology, Comprehensive Cancer Centre, The Ohio State Medical Centre, Columbus, OH, USA Ping Gao Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA Dolores C. García-Olmo Unidad de Investigación, Complejo Hospitalario Universitario de Albacete, Albacete, Spain Roy Garcia City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA

xlii

Robert A. Gardiner School of Medicine, University of Queensland, Brisbane, QLD, Australia Centre for Clinical Research, University of Queensland, Herston, QLD, Australia Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia Edith Cowan University Western Australia, Joondalup, WA, Australia Lawrence B. Gardner The NYU Cancer Institute, New York University School of Medicine, New York, NY, USA Patricio Gariglio Genetic and Molecular Biology, CINVESTAV-IPN, Mexico City, México Cathie Garnis MIT Center for Cancer Research, Cambridge, MA, USA Andrei L. Gartel Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Ronald B. Gartenhaus The University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD, USA Thomas A. Gasiewicz University of Rochester Medical Center, Rocheser, NY, USA Patrizia Gasparini Tumor Genomic Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy Zoran Gatalica Department of Pathology, Creighton University School of Medicine, Omaha, NE, USA Grégory Gatouillat Laboratory of Biochemistry, IFR53, Faculty of Pharmacy, Reims, France Adi F. Gazdar Hamon Center for Therapeutic Oncology Research and Departments of Pathology, Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA Christian Geisler Department of Hematology, The Finsen Centre, Rigshospitalet, Copenhagen, Denmark Klaramari Gellci Department of Biomedical Engineering, Wayne State University, Detroit, MI, USA Eleni A. Georgakopoulou Department of Histology and Embryology, Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece Spyros D. Georgatos Department of Basic Sciences, The University of Crete, School of Medicine, Heraklion, Crete, Greece Julia M. George Queen Mary University of London, London, UK Kimberly S. George Parsons Department of Chemistry, Marietta College, Marietta, OH, USA

Contributors

Contributors

xliii

Armin Gerger Department of Internal Medicine, Division of Oncology, Medical University Graz, Graz, Austria Ulrich Germing Klinik für Hämatologie, Onkologie und Klinische Immunologie, Heinrich-Heine-Universität, Düsseldorf, Germany Jeffrey E. Gershenwald Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Andreas J. Gescher Department of Cancer Studies, Cancer Biomarkers and Prevention Group, University of Leicester, Leicester, Leicester, UK Christian Gespach Laboratory of Molecular and Clinical Oncology of Solid tumors, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, France INSERM U. 673, Paris, France B. Michael Ghadimi Department of General, Visceral and Pediatric Surgery, University Medical Center, Göttingen, Germany Michelle Ghert Department of Surgery, Hamilton Health Sciences, Juravinski Cancer Centre, McMaster University, Hamilton, ON, Canada Riccardo Ghidoni Laboratory of Biochemistry and Molecular Biology, San Paolo Medical School, University of Milan, Milan, Italy Saurabh Ghosh Roy Department of Cell and Developmental Biology, University of California, Irvine, Irvine, CA, USA Ronald A. Ghossein Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Lorenzo Gianni Department of Oncology, Instituto Scientifico Romagnolo per lo s, Infermi Hospital, Rimini, Italy Michael K. Gibson Case Western Reserve University, Cleveland, OH, USA Michael Z. Gilcrease Department of Pathology, Breast Section, MD Anderson Cancer Center, Houston, TX, USA M. Boyd Gillespie Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA François Noël Gilly Department of Digestive Oncologic Surgery, Hospices Civils de Lyon–Université Lyon 1, Lyon, France Thomas Gilmore Biology Department, Boston University, Boston, MA, USA Oliver Gimm Department of Surgery, University Hospital, Linköping, Sweden Alessio Giubellino Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Morten F. Gjerstorff Department of Oncology, Odense University Hospital, Odense C, Denmark

xliv

Shannon S. Glaser Department of Internal Medicine, Texas A&M Health Science Center, Central Texas Veterans Health Care System, Temple, TX, USA Hansruedi Glatt Federal Institute for Risk Assessment (BfR), Berlin, Germany Olivier Glehen Department of Digestive Oncologic Surgery, Hospices Civils de Lyon–Université Lyon 1, Lyon, France Aleksandra Glogowska Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada Thomas W. Glover Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA John C. Goddard Jacksonville Hearing and Balance Institute, Jacksonville, FL, USA Andrew K. Godwin The University of Kansas Medical Center, Kansas City, KS, USA Elspeth Gold Department of Anatomy, Otago School of Medical Sciences, Dunedin, New Zealand Gary S. Goldberg Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA Itzhak D. Goldberg Long Island Jewish Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Susanne M. Gollin Department of Human Genetics, University of Pittsburgh Graduate School of Public Health and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Roy M. Golsteyn Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada Rohini Gomathinayagam Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Ellen L. Goode Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN, USA Gregory J. Gores Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA Vassilis Gorgoulis Department of Histology and Embryology, Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece Noriko Gotoh Division of Cancer Cell Biology, Cancer Research Institute, Kanazawa University, Kanazawa city, Ishikawa, Japan Lynn F. Gottfried LeClairRyan, Rochester, NY, USA

Contributors

Contributors

xlv

Stéphanie Gout Le Centre de recherche du CHU de Québec-Université Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Ammi Grahn Department of Clinical Chemistry and Transfusion Medicin, Institute of Biomedicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Galit Granot Felsenstein Medical Research Center, Beilinson Hospital, Sackler School of Medicine, Tel Aviv University, Petah Tikva, Israel Denis M. Grant Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada Heidi J. Gray Gynecologic Oncology, University of Washington, Seattle, WA, USA Peter Greaves Department of Cancer Studies, University of Leicester, Leicester, UK John A. Green Department of Cancer Medicine, University of Liverpool, Liverpool, UK Mark I. Greene Department of Pathology, Laboratory Medicine and Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Michael Greene Auburn University, Auburn, AL, USA Arjan W. Griffioen Angiogenesis Laboratory, Department of Pathology, Maastricht University, Maastricht, The Netherlands Dirk Grimm BIOQUANT, Cluster of Excellence Cell Networks, University of Heidelberg, Heidelberg, Germany Matthew J. Grimshaw Breast Cancer Biology Group, King’s College London School of Medicine, Guy’s Hospital, London, UK Stephen R. Grobmyer Department of Surgery, Division of Surgical Oncology, University of Florida, Gainesville, FL, USA Bernd Grosche Department of Radiation Protection and Health, Bundesamt für Strahlenschutz (Federal Office for Radiation Protection), Oberschleissheim, Germany Isabelle Gross INSERM U1113, Université de Strasbourg, Strasbourg, France Michael Grusch Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria Wei Gu Institute for Cancer Genetics, and Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Francisca Guardiola-Serrano University of the Balearic Islands, Palma de Mallorca, Spain

xlvi

Juliana Guarize Department of Thoracic Surgery, European Institute of Oncology, Milan, Italy Valentina Guarneri Istituto Oncologico Veneto IRCCS, Division of Medical Oncology 2, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy Tiziana Guarnieri Department of Biology, Geology and Environmental Sciences, Alma Mater Studiorum University of Bologna, Bologna, Italy Liliana Guedez Immunopathology Section, National Eye Institute, Bethesda, MD, USA Frederick Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA Abhijit Guha Division of Neurosurgery, University of Toronto, Toronto, ON, Canada Katherine A. Guindon Department of Pharmacology and Toxicology, Queen’s University, Kingston, ON, Canada Erich Gulbins Department of Molecular Biology, University of DuisburgEssen, Essen, Germany Charles A. Gullo Microbiology NUS (Research), Duke/NUS GMS, Singapore, Singapore Aparna Gupta Life Science Research Associate, Department of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA Sonal Gupta Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Murali Gururajan Department of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA Bristol-Myers Squibb & Co, Princeton, NJ, USA James F. Gusella Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA Graeme R. Guy Signal Transduction Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore Manuel Guzmán Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain Geum-Youn Gwak Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Gangnam-gu, Seoul, South Korea

Contributors

Contributors

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Guy Haegeman Lab of Eukaryotic Gene Expression, LEGEST-University Gent, Ghent, Belgium Stephan A. Hahn University of Bochum, Bochum, Germany Jörg Haier Comprehensive Cancer Center Münster, University Hospital Münster, Münster, Germany Numsen Hail Department of Pharmaceutical Sciences, The University of Colorado at Denver and Health Sciences Center, Denver, CO, USA Pierre Hainaut International Prevention Research Institute, Lyon, France Brett M. Hall Department of Pediatrics, Columbus Children’s Research Institute, The Ohio State University, Columbus, OH, USA Janet Hall Centre de Recherche en Cancérologie de Lyon (CRCL), UMR Inserm 1052 - CNRS 5286, Lyon, France Joyce L. Hamlin Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA Rasha S. Hamouda GeneDx, Rockville, MD, USA Kelsey R. Hampton Department of Cancer Biology, Kansas University Cancer Center, Kansas City, KS, USA The University of Kansas Medical Center, Kansas City, KS, USA Lina Han Department of Leukemia, Section of Molecular Hematology and Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Ross Hannan Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, ANU College of Medicine, Biology and the Environment, Canberra, ACT, Australia Chunhai Hao Department of Pathology and Laboratory Medicine, Henry Ford Health System, Detroit, MI, USA J. William Harbour Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA Mark Harland Section of Epidemiology and Biostatistics, Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK Adrian L. Harris Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Cancer Research UK, Headington, Oxford, UK Randall E. Harris Director Center of Molecular Epidemiology, The Ohio State University, Columbus, OH, USA Marion Hartley Ruesch Center for the Cure of Gastrointestinal Cancers, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA

xlviii

Uzma Hasan CIRI, Oncoviruses and Innate Immunity, INSERM U1111, Ecole Normale Supérieure, Université de Lyon, CNRS-UMR5308, Hospices Civils de Lyon, Lyon, France Mia Hashibe University of Utah, Salt Lake City, UT, USA Masaharu Hata Division of Radiation Oncology, Department of Oncology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan Yosef S. Haviv Division of Nephrology, Hadassah-Hebrew University Medical Center, Department of Medicine, Jerusalem, Israel John D. Hayes Medical Research Institute, Jacqui Wood Cancer Centre, University of Dundee, Dundee, UK Nicole M. Haynes Cancer Therapeutics Program, Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia Hong He Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia Lili He Molecular Oncology Program and Research Institute, H. Lee Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, FL, USA Li-Zhen He Memorial Sloan-Kettering Cancer Center, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA Ruth He Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Yu-Ying He Medicine/Dermatology, University of Chicago, Chicago, IL, USA Stephen S. Hecht The Cancer Center, University of Minnesota, Minneapolis, MN, USA Ingrid A. Hedenfalk Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden Petra Heffeter Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Ahmed E. Hegab Department of Geriatric and Respiratory Medicine, Tohoku University Hospital, Sendai, Japan Axel Heidenreich Division of Oncological Urology, Department of Urology, University of Köln, Köln, Germany Olaf Heidenreich Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK Werner Held Ludwig Center for Cancer Research, Department of Oncology, University of Lausanne, Epalinges, Switzerland

Contributors

Contributors

xlix

Carl-Henrik Heldin Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden Wijnand Helfrich Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen, Department of Pathology and Laboratory Medicine, Section Medical Biology, Laboratory for Tumor Immunology, University Medical Center Groningen, Groningen, The Netherlands Debby Hellebrekers Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Hospital, Maastricht, The Netherlands Ingegerd Hellstrom Department of Pathology, University of Washington, Seattle, WA, USA Karl Erik Hellstrom Department of Pathology, University of Washington, Seattle, WA, USA Paul W. S. Heng Department of Pharmacy, National University of Singapore, Singapore, Singapore Kai-Oliver Henrich DKFZ, German Cancer Research Center, Heidelberg, Germany Rui Henrique Department of Pathology, Portuguese Oncology InstitutePorto, Porto, Portugal Ellen C. Henry University of Rochester Medical Center, Rocheser, NY, USA Elizabeth P. Henske Center for LAM Research and Clinical Care, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Donald E. Henson Uniformed Services University of the Health Sciences, Bethesda, MD, USA Serge Hercberg UMR U1153 INSERM, U1125 INRA, CNAM, Université Paris 13, Centre de Recherche Epidémiologie et Statistique Sorbonne Paris Cité, Bobigny, France Meenhard Herlyn The Wistar Institute, Philadelphia, PA, USA Heike M. Hermanns Med. Klinik II, Hepatologie, Universitätsklinikum Würzburg, Würzburg, Germany Blanca Hernandez-Ledesma Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM, CEI UAM+CSIC), Madrid, Spain Wolfgang Herr Universitätsklinikum Regensburg, Regensburg, Germany Erika Herrero Garcia Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Helen E. Heslop Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, and The Methodist Hospital, Houston, TX, USA

l

Jochen Hess Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Heidelberg, Germany Dominique Heymann Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, University of Nantes, Nantes, France Martha Hickey Obstetrics and Gynaecology, The University of Melbourne, Parkville, VIC, Australia James Hicks Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA Kevin O. Hicks Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand Colin K. Hill Department of Radiation Oncology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Shawn Hingtgen Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, USA Isabelle Hinkel INSERM U1113, Université de Strasbourg, Strasbourg, France Boaz Hirshberg Cardiovascular and Metabolic Diseases, Pfizer Inc, Groton, CT, USA Ari Hirvonen Finnish Institute of Occupational Health, Helsinki, Finland Ricardo Hitt Hospital Universitario Severo Ochoa, Madrid, Spain Eiso Hiyama Natural Science Center for Basic Research and Development, Department of Pediatric Surgery, Hiroshima University Hospital, Hiroshima University, Hiroshima, Japan Falk Hlubek Department of Pathology, Ludwig-Maximilians-University of Munich, Munich, Germany Steven N. Hochwald Departments of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA Mir Alireza Hoda Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria Michael Hodsdon Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA Kasper Hoebe Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Markus Hoffmann Hals-, Nasen- und Ohrenheilkunde, Kopf- und Halschirurgie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Germany

Contributors

Contributors

li

Michèle J. Hoffmann Department of Urology, Heinrich Heine University, Düsseldorf, Germany Lorne J. Hofseth Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA Susanne Holck Department of Pathology, Copenhagen University Hospital, Hvidovre, Denmark Stefan Holdenrieder Institute of Clinical Chemistry and Clinical Pharmacology, Universitatsklinikum Bonn, Bonn, Germany James F. Holland Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Petra Den Hollander Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Caroline L. Holloway BC Cancer Agency, Vancouver Island Centre, Victoria, BC, Canada Arne Holmgren Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Astrid Holzinger Tumor Genetics, Clinic I Internal Medicine, University Hospital Cologne, and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Jun Hyuk Hong Division of Urologic Oncology, The Cancer Institute of NJ, Robert Wood Johnson Medical School, New Brunswick, NJ, USA Adília Hormigo Department of Neurology, Medicine (Division Hematology Oncology) and Neurosurgery, Icahn School of Medicine at Mount Sinai and The Tisch Cancer Institute, New York, NY, USA Joshua Hornig Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Michael R. Horsman Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark Andrea Kristina Horst Inst. Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany David W. Hoskin Departments of Pathology, and Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada Andreas F. Hottinger Departments of Clinical Neuroscience and Oncology, CHUV, Lausanne University Hospital, Lausanne, VD, Switzerland Peter J. Houghton Greehey Children’s Cancer Research Institute, UT Health Science Center, San Antonio, TX, USA Anthony Howell CRUK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Trust, Manchester, UK

lii

Lynne M. Howells Department of Cancer Studies, University of Leicester, Leicester, UK Chia-Chien Hsieh Department of Human Development and Family Studies (Nutritional Science and Education), National Taiwan Normal University, Taipei, Taiwan Shie-Liang Hsieh Department of Microbiology and Immunology, National Yang-Ming University, Immunology Research Center, Taipei Veterans General Hospital; Genomics Research Center, Academia Sinica, Taipei, Taiwan Wei Hu Departments of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Cheng-Long Huang Department of Second Surgery, Kagawa University, Kagawa, Japan Gonghua Huang Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA Shile Huang Department of Biochemistry and Molecular Biology and FeistWeiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, USA Kay Huebner Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA Pere Huguet Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Maureen B. Huhmann Department of Nutrition Sciences, School of Health Related Professions, Rutgers The State University, Newark, NJ, USA Wen-Chun Hung National Institute of Cancer Research, National Health Research Institutes, Tainan Taiwan, Republic of China Tony Hunter Salk Institute, Molecular and Cell Biology Laboratory, La Jolla, CA, USA Teh-Ia Huo Institute of Pharmacology, School of Medicine, National YangMing University, Taipei, Taiwan Jacques Huot Le Centre de recherche du CHU de Québec-Université Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Karen L. Huyck Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA Sam T. Hwang Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Brandy D. Hyndman Department of Pathology and Molecular Medicine, Queen’s University Cancer Research Institute, Queen’s University, Kingston, ON, Canada

Contributors

Contributors

liii

Maitane Ibarguren Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain Takafumi Ichida Department of Hepatology and Gastroenterology, Juntendo University School of Medicine, Shizuoka Hospital, Shizuoka, Japan Yoshito Ihara Department of Biochemistry, School of Medicine, Wakayama Medical University, Wakayama, Japan Hitoshi Ikeda Department of Pediatric Surgery, Dokkyo Medical University Koshigaya Hospital, Koshigaya, Saitama, Japan Landon Inge Norton Thoracic Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA Kazuhiko Ino Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan Juan Iovanna INSERM, Stress Cellulaire, Parc Technologique de Luminy, Marseille Cedex, France

Scientifique

et

Irmgard Irminger-Finger Molecular Gynecology and Obstetrics Laboratory, Department of Gynecology and Obstetrics, Geneva University Hospitals, Geneva, Switzerland Meredith S. Irwin Cell Biology Program and Division of HematologyOncology Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Toshihisa Ishikawa Biochemistry, Molecular Biology, and Pharmacogenomics, NGO Personalized Medicine and Healthcare, Yokohama, Japan Toshiyuki Ishiwata Department of Integrated Diagnostic Pathology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan Mark A. Israel Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Antoine Italiano Early Phase Trials and Sarcoma Units, Institut Bergonie, Bordeaux, France Norimasa Ito Departments of Surgery and Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA Michael Ittmann Department of Pathology, Baylor College of Medicine, Houston, TX, USA Richard Ivell School of Biosciences and School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK Antoni Ivorra Department of Information and Communication Technologies, Universitat Pompeu Fabra (UPF), Barcelona, Spain Nobutaka Iwakuma Department of Surgery, Division of Surgical Oncology, University of Florida, Gainesville, FL, USA

liv

Shai Izraeli Pediatric Hemato-Oncology, Sheba Medical Center and Tel Aviv University, Ramat Gan, Israel Paola Izzo Department of Molecular Medicine and Medical Biotechnology, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy Mark Jackman Wellcome/CRC Institute, Cambridge, UK Alan Jackson Centre for Imaging Sciences, University of Manchester, Manchester, UK Deborah Jackson-Bernitsas Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Stephan C. Jahn Department of Pharmacology and Therapeutics and the UF and Shands Cancer Center, University of Florida, Gainesville, FL, USA David Jamieson School of Clinical and Laboratory Sciences, Newcastle University, Newcastle upon Tyne, UK Siegfried Janz Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA Daniel G. Jay Tufts University School of Medicine, Boston, MA, USA Gordon C. Jayson Cancer Research UK Department of Medical Oncology, Christie Hospital, Manchester, UK Kuan-Teh Jeang National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD, USA Diane F. Jelinek Department of Immunology, Mayo Clinic, College of Medicine, Rochester, MN, USA Jiiang-Huei Jeng Laboratory of Pharmacology and Toxicology, School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan Elwood V. Jensen National Institute of Health, Bethesda, MD, USA Erika Jensen-Jarolim Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria The Interuniversity Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria Carmen Jeronimo Research Center, Portuguese Oncology Institute-Porto, Porto, Portugal Lin Ji Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Shuai Jiang Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

Contributors

Contributors

lv

Yufei Jiang Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Charlotte Jin Departments of Clinical Genetics, University Hospital, Lund, Sweden Chengcheng Jin The David H. Koch Institute of Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Andrew K. Joe Department of Medicine, Herbert Irving Comprehensive Cancer Center, New York, NY, USA Manfred Johannsen Facharztpraxis Urologie Johannsen and Laux, Berlin, Germany Kaarthik John Division of Microbiology, Tulane University, Covington, LA, USA Alan L. Johnson Pennsylvania State University, State College, PA, USA Sara M. Johnson Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA Won-A Joo The Wistar Institute, Philadelphia, PA, USA V. Craig Jordan Breast Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA Serene Josiah Cambridge, MA, USA Richard Jove Vaccine and Gene Therapy Institute of Florida, Port Saint Lucie, FL, USA Jaroslaw Jozwiak Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland Jesper Jurlander Department of Hematology, Rigshospitalet, Copenhagen, Denmark Donat Kögel Experimental Neurosurgery, Center for Neurology and Neurosurgery, Goethe-University Hospital, Frankfurt am Main, Germany Ralf Küppers Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Essen, Germany Chaim Kahana Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Bernd Kaina Department of Toxicology, University Medical Center Mainz, Mainz, Germany Kiran Kakarala Departments of Otolaryngology-Head and Neck Surgery, University of Kansas Medical Center, Kansas City, KS, USA Tadao Kakizoe National Cancer Center, Tokyo, Japan Ganna V. Kalayda Institute of Pharmacy, University of Bonn, Bonn, Germany

lvi

Tuula Kallunki Unit of Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark Takehiko Kamijo Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama, Japan Yasufumi Kaneda Department of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Kazuhiro Kaneko Department of Gastroenterology, Endoscopy Division, National Cancer Center Hospital East, Chiba, Japan Inkyung Kang Department of Surgery, University of California, San Francisco, San Francisco, CA, USA Jayakanth Kankanala Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN, USA Yung-Hsi Kao Department of Life Sciences, College of Science, National Central University, Jhongli City, Taiwan David E. Kaplan Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA, USA Niki Karachaliou Instituto Oncológico Dr. Rosell, Quiron-Dexeus University Hospital, Barcelona, Spain Sophia N. Karagiannis St. John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences and Medicine, King’s College London, London, UK NIHR Biomedical Research Centre at Guy’s and St. Thomas’ Hospitals, Guy’s Hospital, King’s College London, London, UK Michalis V. Karamouzis Department of Biological Chemistry, Medical School, University of Athens, Goudi, Athens, Greece Adam R. Karpf Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA Nilesh D. Kashikar Departments of Surgery and Cancer Biology, VanderbiltIngram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA Matilda Katan CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, London, UK William K. Kaufmann Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Manjinder Kaur Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO, USA Sukhwinder Kaur Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA

Contributors

Contributors

lvii

Ingo Kausch Department of Urology, Ammerlandklinik Westerstede, Westerstede, Germany Koji Kawakami Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Kyoto, Japan Frederic J. Kaye National Cancer Institute, NIH and National Naval Medical Center, Bethesda, MD, USA Stanley B. Kaye Drug Development Unit, Institute of Cancer Research, The Royal Marsden Hospital, Sutton, UK Evan T. Keller Departments of Urology and Pathology, University of Michigan, Ann Arbor, MI, USA Daniel Keppler Department of Biological Science, College of Pharmacy, Touro University-CA, Vallyo, CA, USA Santhosh Kesari Department of Translational Neuro-Oncology and Neurotherapeutics, John Wayne Cancer Institute, Providence St. John’s Health Center, Santa Monica, CA, USA Jorma Keski-Oja Departments of Pathology and of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland Khandan Keyomarsi Department of Experimental Radiation Oncology, Unit 1052, University of Texas MD Anderson Cancer Center, Houston, TX, USA Abdul Arif Khan Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Shahanavaj Khan Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Chand Khanna Comparative Oncology Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Samir N. Khleif GRU Cancer Center, Augusta, GA, USA Roya Khosravi-Far Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA Tobias Kiesslich Department of Internal Medicine I, Paracelsus Medical University, Institute of Physiology and Pathophysiology, Paracelsus Medical University, Salzburg, Austria Fumitaka Kikkawa Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan Nerbil Kilic Kantonspital St. Gallen, St. Gallen, Switzerland Isaac Yi Kim Division of Urologic Oncology, The Cancer Institute of NJ, Robert Wood Johnson Medical School, New Brunswick, NJ, USA Jung-whan Kim Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX, USA

lviii

Miran Kim Division of Gastroenterology, Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI, USA Moonil Kim BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Republic of Korea Seong Jin Kim Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, USA Su Young Kim Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Adi Kimchi Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel A. Douglas Kinghorn College of Pharmacy, The Ohio State University, Columbus, OH, USA David Kirn Jennerex Biotherapeutics Inc., San Francisco, CA, USA Youlia M. Kirova Department of Radiation Oncology, Institut Curie, Paris, France Shinichi Kitada Burnham Institute for Medical Research, La Jolla, CA, USA Karel Kithier Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA Chikako Kiyohara Department of Preventive Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Celina G. Kleer Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA George Klein Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Michael J. Klein Department of Pathology and Laboratory Medicine, Hospital for Special Surgery, New York, NY, USA Elena Klenova Department of Biological Sciences, University of Essex, Colchester, Essex, UK Thomas Klonisch Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada Elizabeth Knobler Department of Dermatology, Columbia College of Physicians and Surgeons, New York, NY, USA Robert Knobler Department of Dermatology, Medical University of Vienna, Vienna, Austria Beatrice Knudsen Cedars-Sinai, Los Angeles, CA, USA

Contributors

Contributors

lix

Stefan Kochanek Division of Gene Therapy, University of Ulm, Ulm, Germany Manish Kohli Medical Oncology, Mayo Clinic, Rochester, MN, USA Katri Koli Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland Christian Kollmannsberger Division of Medical Oncology, British Columbia Cancer Agency, Vancouver Cancer Centre, University of British Columbia, Vancouver, BC, Canada Yutaka Kondo Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Lin Kong Department of Radiation Oncology, Fudan Universtiy Shanghai Cancer Center, Shanghai, China Marina Konopleva Department of Leukemia and Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Roland E. Kontermann Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany Janko Kos Faculty to Pharmacy, University of Ljubljana, Ljubljana, Slovenia Marta Kostrouchova Institute of Cellular Biology and Pathology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Athanassios Kotsinas Molecular Carcinogenesis Group, Laboratory of Histology-Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece Evangelia A. Koutsogiannouli Department of Urology, Heinrich Heine University, Düsseldorf, Germany Heinrich Kovar Children’s Cancer Research Institute, Vienna, Austria Craig Kovitz Department of Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Christian Kowol Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria Barnett S. Kramer Office of Disease Prevention, National Institutes of Health, Bethesda, MD, USA Oliver H. Krämer Department of Toxicology, University Medical Center Mainz, Mainz, Germany Barbara Krammer Department of Molecular Biology, University of Salzburg, Salzburg, Austria Henk J. van Kranen National Institute of Public Health and Environment, Bilthoven, The Netherlands

lx

Robert Kratzke Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, USA Thomas Krausz Department of Pathology, University of Chicago, Chicago, IL, USA Jürgen Krauter Medizinische Klinik III – Hämatologie und Onkologie, Klinikum Braunschweig, Braunschweig, Germany Bernhard Kremens Department of Pediatric Hematology, Oncology and Respiratory Medicine, University Hospitals of Essen, Essen, Germany Betsy T. Kren Minneapolis VA Health Care System and University of Minnesota, Minneapolis, MN, USA Yasusei Kudo Department of Oral Molecular Pathology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan Deepak Kumar Department of Biological and Environmental Sciences, University of the District of Columbia, Washington, DC, USA Parvesh Kumar Department of Radiation Oncology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Rakesh Kumar Department of Biochemistry and Molecular Medicine, George Washington University, Washington, DC, USA Hiroki Kuniyasu Department of Molecular Pathology, Nara Medical University School of Medicine, Kashihara, Nara, Japan Siavash K. Kurdistani Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Elena Kurenova Departments of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA Keisuke Kurose Departments of Obstetrics and Gynecology, Nippon Medical School, Kawasaki and Tokyo, Japan Peter Kurre Department of Pediatrics, Oregon Health and Science University, Portland, OR, USA Robert M. Kypta Cell Biology and Stem Cells Unit, CIC bioGUNE, Derio, Spain Imperial College London, London, UK Juan Carlos Lacal Instituto de Investigaciones Biomedicas, CSIC, Madrid, Spain James C. Lacefield Departments of Electrical and Computer Engineering and Medical Biophysics, University of Western Ontario, London, ON, Canada Stephan Ladisch Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center and The George Washington University School of Medicine, Washington, DC, USA

Contributors

Contributors

lxi

Hermann Lage Institute of Pathology, Charité Campus Mitte, Berlin, Germany Charles P. K. Lai Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, BC, Canada Henry Lai Departments of Bioengineering, University of Washington, Seattle, WA, USA Dale W. Laird Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada Hilaire C. Lam Center for LAM Research and Clinical Care, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Janice B. B. Lam Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China Wan L. Lam Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, BC, Canada Hui Y. Lan The Chinese University of Hong Kong, Hong Kong, China Joseph R. Landolph, Jr. Department of Molecular Microbiology and Immunology, and Department of Pathology; Laboratory of Chemical Carcinogenesis and Molecular Oncology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Health Sciences Campus, University of Southern California, Los Angeles, CA, USA Ari L. Landon The University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD, USA Robert Langer Department of Chemical Engineering and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Sigrid A. Langhans Nemours Center for Childhood Cancer Research, Alfred I duPont Hospital for Children, Wilmington, DE, USA Cinzia Lanzi Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Rosamaria Lappano Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, Rende, Italy Paola Larghi Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy James M. Larner Department of Therapeutic Radiology and Oncology, University of Virginia School of Medicine, Charlottesville, VA, USA Göran Larson Department of Clinical Chemistry and Transfusion Medicin, Institute of Biomedicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

lxii

Lars-Inge Larsson Department of Pathology, Copenhagen University Hospital, Hvidovre, Denmark Susanna C. Larsson Division of Nutritional Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Philippe Lassalle INSERM U774, Institut Pasteur de Lille, Lille, France Antony M. Latham Endothelial Cell Biology Unit, Leeds Institute of Genetics Health and Therapeutics (LIGHT), University of Leeds, Leeds, UK Farida Latif Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham, UK Paule Latino-Martel UMR U1153 INSERM, U1125 INRA, CNAM, Université Paris 13, Centre de Recherche Epidémiologie et Statistique Sorbonne Paris Cité, Bobigny, France Kirsten Lauber Clinic for Radiotherapy and Radiation Oncology, LMU Munich, Munich, Germany Béatrice Lauby-Secretan Section of the IARC Monographs, IARC/WHO, Lyon, France Virpi Launonen Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Martin F. Lavin University of Queensland Centre for Clinical Research at Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD, Australia Brian Law Department of Pharmacology and Therapeutics and the UF and Shands Cancer Center, University of Florida, Gainesville, FL, USA Gwendal Lazennec INSERM, Montpellier, France Pedro A. Lazo CSIC-Universidad de Salamanca, Instituto de Biología Molecular y Celular del Cáncer, Salamanca, Spain Gail S. Lebovic Director of Women’s Services, The Cooper Clinic, Dallas, TX, USA David P. LeBrun Department of Pathology and Molecular Medicine, Queen’s University Cancer Research Institute, Queen’s University, Kingston, ON, Canada Protein Function Discovery Group, Queen’s University, Kingston, ON, Canada Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, USA Sean Bong Lee Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA, USA Seong-Ho Lee Department of Nutrition and Food Science, University of Maryland, College Park, MD, USA

Contributors

Contributors

lxiii

Stephen Lee Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada William P. J. Leenders Department of Pathology, Radboud University Medical Center Nijmegen, Nijmegen, The Netherlands Andreas Leibbrandt Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Manuel C. Lemos CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal Eric Lentsch Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Derek LeRoith Division of Endocrinology, Diabetes and Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA Yun-Chung Leung Lo Ka Chung Centre for Natural Anti-cancer Drug Development and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China Francis Lévi Warwick Medical School, University of Warwick, Coventry, UK Jay A. Levy University of California, School of Medicine, San Francisco, CA, USA Benyi Li Department of Urology, The University of Kansas Medical Center, Kansas City, KS, USA Guideng Li Institute for Immunology, School of Medicine, University of California, Irvine, CA, USA Kaiyi Li Department of Surgery, Baylor College of Medicine, Houston, TX, USA Yan Li Department of Immunology, Cleveland Clinic, Cleveland, OH, USA Daiqing Liao Department of Anatomy and Cell Biology, UF Health Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA Yung-Feng Liao Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Emmanuelle Liaudet-Coopman IRCM, INSERM, UMI, CRLC Val d’Aurelle, Montpellier, France Rossella Libè Endocrinology, Metabolism and Cancer Department, INSERM U567, Institut Cochin, Paris, France Danielle Liddle Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford, UK Jane Liesveld James P. Wilmot Cancer Center, University of Rochester, Rochester, NY, USA

lxiv

Stephanie Lim Medical Oncology, Ingham Research Institute, Liverpool, NSW, Australia Ke Lin Department of Haematology, Royal Liverpool University Hospital, Liverpool, UK Sheng-Cai Lin Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian, China Shiaw-Yih Lin Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Wan-Wan Lin Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan Yong Lin Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, NM, USA Janet C. Lindsey Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK Christopher A. Lipinski Melior Discovery, Waterford, CT, USA Joseph Lipsick Stanford University, Stanford, CA, USA Fei-Fei Liu Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada Department of Radiation Oncology, Princess Margaret Hospital, Toronto, ON, Canada Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Tao Liu Department of Medicine, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA Wen Liu Division of Life Science, Hong Kong University of Science and Technology, Kowloon, Hong Kong Xiangguo Liu School of Life Science, Shandong University, Jinan, Shandong, China Yiyan Liu Department of Radiology, New Jersey Medical School, Rutgers University, New Brunswick, NJ, USA Hui-Wen Lo Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA Ting Ling Lo Signal Transduction Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore Victor Lobanenkov Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, National Institutes of Health, Bethesda, MD, USA

Contributors

Contributors

lxv

Holger N. Lode Klinik und Poliklinik für Kinder und Jugendmedizin, Universitätsmedizin Greifswald, Greifswald, Germany Lawrence A. Loeb University of Washington, Seattle, WA, USA Robert Loewe Department of Dermatology, Division of General Dermatology, Medical University of Vienna, Vienna, Austria Steffen Loft Department of Environmental Health, University of Copenhagen, Copenhagen, Denmark Dietmar Lohmann Institut für Humangenetik, Universitätsklinikum Essen, Essen, Germany Matthias Löhr Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden Vinata B. Lokeshwar Department of Biochemistry and Molecular Biology, Medical College of Georgia; Augusta University, Augusta, GA, USA Alexandre Loktionov DiagNodus Ltd, Babraham Research Campus, Cambridge, UK Elias Lolis Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA Pier-Luigi Lollini Laboratory of Immunology and Biology of Metastasis, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Weiwen Long Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH, USA David J. López University of the Balearic Islands, Palma de Mallorca, Spain Miguel Lopez-Lazaro Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain Ana Lopez-Martin Hospital Universitario Severo Ochoa, Madrid, Spain Charles L. Loprinzi Department of Oncology, Mayo Clinic, Rochester, MN, USA Jochen Lorch Dana Farlur Cancer Institute, Boston, MA, USA Edith M. Lord Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Reuben Lotan Department of Thoracic Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Ragnhild A. Lothe Department of Cancer Prevention, RikshospitaletRadiumhospitalet Medical Centre, Oslo, Norway Michael T. Lotze Department of Surgery and Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA

lxvi

Christophe Louandre EA4666, Université de Picardie Jules Verne (UPJV), Amiens, France Service de Biochimie, Centre de Biologie Humaine (CBH), University Hospital of Amiens (CHU Sud), Amiens, France Chrystal U. Louis Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, and The Methodist Hospital, Houston, TX, USA Dmitri Loukinov Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, National Institutes of Health, Bethesda, MD, USA David B. Lovejoy Department of Pathology, University of Sydney, Sydney, NSW, Australia José Lozano Department of Molecular Biology and Biochemistry, University of Málaga, Málaga, Spain Guanning N. Lu Departments of Otolaryngology-Head and Neck Surgery, University of Kansas Medical Center, Kansas City, KS, USA Jiade J. Lu Department of Radiation Oncology, Fudan Universtiy Shanghai Cancer Center, Shanghai, China Jing Lu Departments of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Tzong-Shi Lu Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA, USA Yuanan Lu Department of Public Health Science, University of Hawaii, Honolulu, HI, USA Irina A. Lubensky National Cancer Institute, Division of Cancer Treatment and Diagnosis, National Institutes of Health, Rockville, MD, USA Jared M. Lucas Divisions of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Andreas Luch German Federal Institute for Risk Assessment (BfR), Berlin, Germany Maria Li Lung Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Jian-Hua Luo Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Gary H. Lyman Public Health Sciences and Clinical Research Divisions, Hutchinson Institute for Cancer Outcomes Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Henry Lynch Department of Preventive Medicine and Public Health, Creighton University, Omaha, NE, USA

Contributors

Contributors

lxvii

Elsebeth Lynge Institute of Public Health, University of Copenhagen, Copenhagen, Denmark Scott K. Lyons Molecular Imaging Group, CRUK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK Wenjian Ma National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA Michael MacManus Department of Radiation Oncology, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia Britta Mädge DKFZ, Heidelberg, Germany Claudie Madoulet Laboratory of Biochemistry, IFR53, Faculty of Pharmacy, Reims, France Rolando F. Del Maestro Montreal, QC, Canada Marcello Maggiolini Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, Rende, Italy Brinda Mahadevan Abbott Nutrition, Regulatory Affairs, Abbott Laboratories, Columbus, OH, USA Joseph F. Maher Cancer Institute, University of Mississippi Medical Center, Jackson, MS, USA Csaba Mahotka Institute of Pathology, Heinrich Heine Universität, Düsseldorf, Germany Sourindra N. Maiti Division of Pediatrics, Department of Immunology, MD Anderson Cancer Center, Houston, TX, USA Isabella W. Y. Mak Department of Surgery, Hamilton Health Sciences, Juravinski Cancer Centre, McMaster University, Hamilton, ON, Canada N. K. Mak Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China Jennifer Makalowski Tumor Genetics, Clinic I Internal Medicine, University Hospital Cologne, and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Cédric Malicet INSERM, Stress Cellulaire, Parc Technologique de Luminy, Marseille Cedex, France

Scientifique

et

Alessandra Mancino Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy Evelyne Manet CIRI-International Center for Infectiology Research, INSERM U1111, Université Lyon 1, ENS de Lyon, Lyon, France Sridhar Mani Department of Medicine, Oncology and Molecular Genetics, Albert Einstein College of Medicine, New York, NY, USA

lxviii

Marcel Mannens Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Alberto Mantovani Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy Ashley A. Manzoor Department of Radiation Oncology, Duke University, Durham, NC, USA Selwyn Mapolie Department of Chemistry and Polymer Science, Stellenbosch University, Matieland, South Africa Lucia Marcocci Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy Maurie Markman Department of Medical Oncology, Eastern Regional Medical Center, Philadelphia, PA, USA Dieter Marmé Tumor Biology Center, Institute of Molecular Oncology, Freiburg, Germany Marie-Claire Maroun Department of Internal Medicine, Division of Rheumatology, Wayne State University, Detroit, MI, USA Deborah J. Marsh Kolling Institute of Medical Research and Royal North Shore Hospital, University of Sydney, Sydney, NSW, Australia John L. Marshall Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Angela Märten National Centre for Tumour Diseases; Department of Surgery, University Hospital Heidelberg, Heidelberg, Germany Francis L. Martin Centre for Biophotonics, Lancaster University, Lancaster, Lancashire, UK Olga A. Martin Division of Radiation Oncology and Cancer Imaging, Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia The Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia Victor D. Martinez British Columbia Cancer Research Centre, Vancouver, BC, Canada Gaetano Marverti Department of Biomedical Sciences, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy Edmund Maser Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Kiel, Germany Thomas E. Massey Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada Noriyuki Masuda Department of Respiratory Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan

Contributors

Contributors

lxix

Atsuko Masumi Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan Yasunobu Matsuda Department of Medical Technology, Niigata University Graduate of Health Sciences, Niigata, Japan Sachiko Matsuhashi Department of Internal Medicine, Saga Medical School, Saga University, Saga, Japan Takaya Matsuzuka Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA Malgorzata Matusiewicz Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland Warren L. May Department of Health Administration, School of Health Related Professions, University of Mississippi Medical Center, Jackson, MS, USA Arnulf Mayer Department of Radiooncology and Radiotherapy, University Medical Center Mainz, Mainz, Germany Matthew A. McBrian Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Joseph H. McCarty MD Anderson Cancer Center, Houston, TX, USA Molliane Mcgahren-Murray Department of Systems Biology, Unit 1058, University of Texas MD Anderson Cancer Center, Houston, TX, USA Katherine A. McGlynn Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA W. Glenn McGregor University of Louisville, Louisville, KY, USA Iain H. McKillop Department of General Surgery, Carolinas Medical Center, Charlotte, NC, USA Margaret McLaughlin-Drubin Brigham and Women’s Hospital, Boston, MA, USA Roger E. McLendon Department of Pathology, Duke University Medical Center, Durham, NC, USA Donald C. McMillan University Department of Surgery, Royal Infirmary, Glasgow, UK David W. Meek Division of Cancer Research, Jacqui Wood Cancer Centre/ CRC, University of Dundee, Dundee, UK Annette Meeson Institute of Genetic Medicine and North East Stem Cell Institute, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK Kamiya Mehla The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA

lxx

Arianeb Mehrabi Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Mohammad Mehrmohammadi Department of Biomedical Engineering, Wayne State University, Detroit, MI, USA Anil Mehta Division of Cardiovascular Medicine, University of Dundee, Dundee, UK Kapil Mehta The University of Texas MD Anderson Cancer Center, Houston, TX, USA Rekha Mehta Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, Ottawa, ON, Canada Yaron Meirow The Lautenberg Center for Immunology and Cancer Research, Israel-Canada Medical, Research Institute Faculty of Medicine, The Hebrew University, Jerusalem, Israel Bar-Eli Menashe Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Wenbo Meng Special Minimally Invasive Surgery, Hepatopancreatobiliary Surgery Institute of Gansu Province, Clinical Medical College Cancer Center, First Hospital of Lanzhou University, Lanzhou University, Lanzhou, Gansu, China Deepak Menon Department of Biological Sciences, Hunter College of the City University of New York, New York, NY, USA Heather Mernitz Alverno College, Milwaukee, WI, USA Karl-Heinz Merz Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany Enrique Mesri Viral Oncology Program, Sylvester Comprehensive Cancer Center and Development Center for AIDS Research, Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, USA Roman Mezencev Georgia Institute of Technology, School of Biology, Atlanta, GA, USA Jun Mi Department of Therapeutic Radiology and Oncology, University of Virginia School of Medicine, Charlottesville, VA, USA Dennis F. Michiel Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA Josef Michl Departments of Pathology, Molecular and Cell Biology, State University of New York, Downstate Medical Center, New York, NY, USA Stephan Mielke Abteilung Hämatologie und Onkologie, Medizinische Klinik und Poliklinik II, Zentrum Innere Medizin (ZIM), Universitätsklinikum Würzburg, Würzburg, Germany

Contributors

Contributors

lxxi

Oleg Militsakh Head and Neck Surgery, Nebraska Medical Center, Nebraska Methodist Hospital, Omaha, NE, USA Mark Steven Miller Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC, USA Takeo Minaguchi Department of Obstetrics and Gynecology, University of Tsukuba, Tokyo, Japan Nagahiro Minato Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Rodney F. Minchin School of Biomedical Sciences, University of Queensland, St Lucia, QLD, Australia Lucas Minig Gynecologic Department, Valencian Institute of Oncology (IVO), Valencia, Spain John D. Minna Hamon Center for Therapeutic Oncology Research and Departments of Pathology, Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA Claudia Mitchell Institut Cochin, Université Paris Descartes, CNRS, Paris, France Kazuo Miyashita Faculty of Fisheries Sciences, Department of Bioresources Chemistry, Hokkaido University, Hakodate, Hokkaido, Japan Eiji Miyoshi Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Japan Jun Miyoshi Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Toshihiko Mizuta Department of Internal Medicine, Imari Arita Kyoritsu Hospital, Saga, Japan Omeed Moaven Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA K. Thomas Moesta Klinik für Chirurgie und Chirurgische Onkologie, Charité Universitätsmedizin Berlin, Berlin, Germany Seyed Moein Moghimi Nanomedicine Research Group, Centre for Pharmaceutical Nanotechnology and Nanotoxicology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Sunish Mohanan Baker Institute for Animal Health, Department of Biomedical Sciences, School of Veterinary Medicine, Cornell University, Ithaca, NY, USA Sonia Mohinta Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University, School of Medicine, Springfield, IL, USA

lxxii

Contributors

Jan Mollenhauer Molecular Oncology Group, University of Southern Denmark, Odense, Denmark Michael B. Møller Department of Pathology, Odense University Hospital, Odense, Denmark Bruno Mondovì Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy Alessandra Montecucco Istituto di Genetica Molecolare CNR, Pavia, Italy Ruggero Montesano International Agency for Research on Cancer, Lyon, France Wolter J. Mooi Department of Pathology, VU Medical Center, Amsterdam, The Netherlands Amy C. Moore Georgia Cancer Coalition, Atlanta, GA, USA Malcolm A. S. Moore Department of Cell SloanKettering Cancer Center, New York, NY, USA

Biology,

Memorial-

Cesar A. Moran Department of Pathology, MD Anderson Cancer Center, Houston, TX, USA Jan S. Moreb Department of Medicine, Division of Hematology/Oncology, College of Medicine, University of Florida, Gainesville, USA Sergio Moreno Instituto de Biología Molecular y Celular del Cáncer, CSIC/ Universidad de Salamanca, Salamanca, Spain Fabiola Moretti Institute of Cell Biology and Neurobiology, National Council Research of Italy, Rome, Italy Eiichiro Mori Department of Radiation Oncology, School of Medicine, Nara Medical University, Kashihara, Nara, Japan Akira Morimoto Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan Pat J. Morin Laboratory of Molecular Biology and Immunology, National Institute on Aging, Baltimore, MD, USA Department of Pathology, Oncology and Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, MD, USA American Association for Cancer Research, Philadelphia, PA, USA Christine M. Morris Cancer Genetics Research, University of Otago, Christchurch, New Zealand Cynthia C. Morton Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA Gabriela Möslein Helios Klinik, Allgemein- und Viszeralchirurgie, Bochum, Germany

Contributors

lxxiii

Justin L. Mott Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Spyro Mousses Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA Pavlos Msaouel Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA Sebastian Mueller Centre of Alcohol Research (CAR), University of Heidelberg, Heidelberg, Germany Susette C. Mueller Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA Subhajit Mukherjee Albert Einstein College of Medicine, New York, NY, USA Hans K. Müller-Hermelink Institute of Pathology, University of Würzburg, Würzburg, Germany Gabriele Multhoff Klinikum rechts der Isar, Department Radiation Oncology, TU München and CCG – “Innate Immunity in Tumor Biology”, Helmholtz Zentrum München, Munich, Germany Julia Münzker Division of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, Graz, Austria Ramachandran Murali Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA Kenji Muro Department of Neurological Surgery, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Mandi Murph Department of Pharmaceutical and Biomedical Sciences, Georgia Cancer Coalition Distinguished Cancer Scholar, University of Georgia and College of Pharmacy, Athens, GA, USA Edward L. Murphy University of California, School of Medicine, San Francisco, CA, USA Paul G. Murray CRUK Institute for Cancer Studies, Molecular Pharmacology, Medical School, University of Birmingham, Birmingham, UK Ruth J. Muschel Radiation Oncology and Biology, University of Oxford, Oxford, UK Markus Müschen Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA Antonio Musio Institute for Genetic and Biomedical Research, National Research Council, Pisa, Italy Istituto Toscano Tumori, Firenze, Italy

lxxiv

Akira Naganuma Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Shigekazu Nagata Osaka University Medical School, Osaka, Japan Christina M. Nagle Cancer and Population Studies, Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, QLD, Australia Rita Nahta Department of Pharmacology, Emory University, Atlanta, GA, USA Akira Nakagawara Saga Medical Center KOSEIKAN, Tosu, Japan Tetsuya Nakatsura Division of Cancer Immunotherapy, Explonatory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa City, Chiba Prefecture, Japan Hariktishna Nakshatri IU Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA Patrizia Nanni Laboratory of Immunology and Biology of Metastasis, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Zvi Naor Department of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Mohd W. Nasser Department of Pathology, Comprehensive Cancer Centre, The Ohio State Medical Centre, Columbus, OH, USA Christian C. Naus Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, BC, Canada Tim S. Nawrot Division of Lung Toxicology, Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.), Division of Hypertension and Cardiovascular Rehabilitation, Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium David F. Nellis Biopharmaceutical Development Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD, USA Kenneth P. Nephew School of Medicine, Indiana University, Bloomington, IN, USA David M. Neskey Department of Otolaryngology and Head and Neck Surgery, Medical University of South Carolina, Charleston, SC, USA Klaus W. Neuhaus School of Dental Medicine, Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland Kornelia Neveling Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Brad Neville Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA

Contributors

Contributors

lxxv

Calvin S. H. Ng Division of Cardiothoracic Surgery, Chinese University of Hong Kong, Hong Kong, China Irene O. L. Ng Department of Pathology, The University of Hong Kong, Hong Kong, China Duc Nguyen Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Carole Nicco Faculté de Médecine Paris – Descartes, UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul, Paris, France Santo V. Nicosia H. Lee Moffitt Cancer Center, Tampa, FL, USA Anne T. Nies Dr. Margarete Fischer-Bosch-Institut Pharmakologie, Stuttgart, Germany

für

Klinische

M. Angela Nieto Instituto de Neurociencias de Alicante CSIC-UMH, Sant Joan d’Alacant, Spain Omgo E. Nieweg Melanoma Institute Australia, North Sydney, NSW, Australia Jonas Nilsson Department of Clinical Chemistry and Transfusion Medicin, Institute of Biomedicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Ewa Ninio INSERM UMRS, Université Pierre et Marie Curie-Paris, Paris, France Douglas Noonan University of Insubria, Varese, Italy Larry Norton Breast Cancer Medicine Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Francisco J. Novo Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain Ruslan Novosiadly Department of Cancer Immunobiology, Eli Lilly and Company, New York, NY, USA Noa Noy Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic and Case Western Reserve University, Cleveland, OH, USA Hala H. Nsouli Department of Epidemiology and Biostatistics, The George Washington University School of Public Health and Health Services, Washington, DC, USA Lauren M. Nunez Department of Biological Science, College of Pharmacy, Touro University-CA, Vallyo, CA, USA John P. O’Bryan Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Jesse Brown VA Medical Center, Chicago, IL, USA

lxxvi

James P. B. O’Connor Institute of Cancer Sciences, University of Manchester, Manchester, UK Sarah T. O’Dwyer Colorectal and Peritoneal Oncology Centre, The Christie NHS Foundation Trust, University of Manchester, Manchester, UK John O’Leary Departments of Obstetrics and Gynaecology/Histopathology, Trinity College Dublin, Trinity Centre for Health Sciences, Dublin, Ireland Ruth M. O’Rega Winship Cancer Institute, Emory University, Atlanta, GA, USA Sharon O’Toole Departments of Obstetrics and Gynaecology/Histopathology, Trinity College Dublin, Trinity Centre for Health Sciences, Dublin, Ireland André Oberthür Department of Pediatric Oncology and Hematology, Children’s Hospital, University of Cologne, Cologne, Germany Takahiro Ochiya Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan Stefan Offermanns Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany Anat Ohali Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Takeo Ohnishi Department of Radiation Oncology, School of Medicine, Nara Medical University, Kashihara, Nara, Japan Hitoshi Ohno Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan Kevin R. Oldenburg MatriCal, Inc., Spokane, WA, USA Magali Olivier Group of Molecular Mechanisms and Biomarkers, International Agency for Research on Cancer, World Health Organization, Lyon, France Egbert Oosterwijk Laboratory of Experimental Urology, University Medical Centre Nijmegen, Nijmegen, The Netherlands Gertraud Orend Department of Clinical and Biological Sciences, Institute of Biochemistry and Genetics, Center for Biomedicine, DKBW, University of Basel, Basel, Switzerland Makoto Osanai Department of Pathology, Kochi University School of Medicine, Kochi, Japan Eduardo Osinaga Departamento de Inmunobiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay German Ott Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Stuttgart, Germany

Contributors

Contributors

lxxvii

Christian Ottensmeier CRC Wessex Oncology Unit, Southampton General Hospital and Tenovous Laboratory, Southampton University Hospital Trust, Southampton, UK Sai-Hong Ignatius Ou Chao Family Comprehensive Cancer Center, University of California, Irvine, CA, USA Iwata Ozaki Health Administration Center, Saga Medical School, Saga University, Saga, Japan Shuji Ozaki Department of Hematology, Tokushima Prefectural Central Hospital, Tokushima, Japan Mónica Pérez-Ríos Department of Preventive Medicine and Public Health, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Helen Pace Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA Simon Pacey Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK Mabel Padilla Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, NM, USA Sumanta Kumar Pal Department of Medical Oncology and Experimental Therapeutics, City of Hope Comprehensive Cancer Center, Duarte, CA, USA Viswanathan Palanisamy Department of Oral Health Sciences, Medical University of South Carolina, Charleston, SC, USA Pier Paolo Pandolfi Division of Genetics, Beth Israel Deaconess Medical Center, Boston, MA, USA Klaus Pantel Universitäts-Krankenhaus Eppendorf, Hamburg, Germany Melissa C. Paoloni National Cancer Institute, Center for Cancer Research, Comparative Oncology Program, Bethesda, MD, USA Evangelia Papadimitriou Laboratory of Molecular Pharmacology, Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece Philippe Paparel Department of Urology, Lyon Sud University Hospital, Pierre Benite, France Athanasios G. Papavassiliou Department of Biological Chemistry, Medical School, University of Athens, Goudi, Athens, Greece Sabitha Papineni Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA

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Benoit Paquette Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada Ben Ho Park The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA Geoff J. M. Parker Centre for Imaging Sciences, University of Manchester, Manchester, UK Sarah J. Parsons University of Virginia, Charlotteville, VA, USA Eddy Pasquier Centre for Research in Oncobiology and Oncopharmacology, INSERM U911, Marseille, France Metronomics Global Health Initiative, Marseille, France Children’s Cancer Institute, Randwick, NSW, Australia Oneel Patel Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia Rusha Patel Otolaryngology, Medical University of South Carolina, Charleston, SC, USA Shyam Patel Standford University, Palo Alto, CA, USA Patrizia Paterlini-Bréchot Faculté de Médecine Necker Enfants Malades, INSERM Unit 1151, Team 13, Paris, France Yvonne Paterson Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Konan Peck Institute of Biomedical Sciences, Academia Sinica Taipei, Taiwan, Republic of China Florence Pedeutour Laboratory of Solid Tumors Genetics, Faculty of Medicine, Nice University Hospital, Nice, France Dan Peer Laboratory of Precision NanoMedicine, Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Department of Materials Science and Engineering, The Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel Tobias Peikert Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA Miguel A. Peinado Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain Angel Pellicer Department of Pathology, New York University School of Medicine, New York, NY, USA

Contributors

Contributors

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Juha Peltonen Department of Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland Sirkku Peltonen Department of Dermatology, University of Turku, Turku, Finland Josef M. Penninger Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Richard T. Penson Division of Hematology Oncology, Massachusetts General Hospital, Boston, MA, USA Maikel P. Peppelenbosch Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The Netherlands Carlos Perez-Stable Geriatric Research, Education, and Clinical Center Research Service, Bruce W. Carter Veterans Affairs Medical Center, Miami, FL, USA Francisco G. Pernas National Institute on Deafness and Other Communication, Disorders and National Cancer Institute, NIH, Bethesda, MD, USA Silverio Perrotta Department of Pediatrics, Second University of Naples, Naples, Italy Godefridus J. Peters Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Marleen M. R. Petit Department of Human Genetics, University of Leuven, Leuven, Belgium Peter Petzelbauer Department of Dermatology, Division of General Dermatology, Medical University of Vienna, Vienna, Austria Claudia Pföhler Department of Dermatology, Saarland University Medical School, Homburg/Saar, Germany Michael Pfreundschuh Klinik für Innere Medizin I, Universität des Saarlandes, Homburg, Germany Philip A. Philip Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA Marco A. Pierotti Molecular Genetics of Cancer, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy Paola Pietrangeli Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy Torsten Pietsch Institut für Neuropathologie, Kinderchirurgie, Universitätskliniken Bonn, Bonn, Germany Sreeraj G. Pillai Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA Lorenzo Pinna Department of Biological Chemistry, University of Padua, Padua, Italy

lxxx

Michael Pishvaian Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Ellen S. Pizer Laboratory of Cellular and Molecular Biology, National Institute on Aging, NIH, Baltimore, MD, USA Kristjan Plaetzer Laboratory of Photodynamic Inactivation of Microorganisms, Division of Physics and Biophysics, University of Salzburg, Salzburg, Austria Christoph Plass German Cancer Research Center (DKFZ), Heidelberg, Germany Jeffrey L. Platt Departments of Microbiology and Immunology and Department of Surgery, University of Michigan, Ann Arbor, MI, USA Mark R. Player Johnson & Johnson Pharmaceutical Research and Development, Spring House, PA, USA Isabelle Plo INSERM, U1170, Hématopoièse et cellules souches, Gustave Roussy–PR1, Villejuif, France Stephen R. Plymate Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA, USA Klaus Podar Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Heidelberg, Germany Beatriz G. T. Pogo Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Jeffrey W. Pollard MRC Centre for Reproductive Health, Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, NY, USA Simona Polo University of Milan, Medical School, Milan, Italy Satyanarayana R. Pondugula Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, AL, USA Auburn University Research Initiative in Cancer, Auburn University, Auburn, AL, USA Sreenivasan Ponnambalam Endothelial Cell Biology Unit, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK Mirco Ponzoni Experimental Therapies Unit, Laboratory of Oncology, Istituto Giannina Gaslini, Genoa, Italy Beatrice L. Pool-Zobel Nutritional Toxicology, Friedrich-Schiller-University of Jena, Jena, Germany Annemarie Poustka Division of Molecular Genome Analysis, DKFZ, Heidelberg, Germany

Contributors

Contributors

lxxxi

Marissa V. Powers Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton, London, UK Garth Powis NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Graziella Pratesi Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy George C. Prendergast Department of Pathology, Anatomy and Cell Biology, Jefferson Medical School, Lankenau Institute for Medical Research, Wynnewood, PA, USA Victor G. Prieto Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Sharon Prince Department of Human Biology, Health Science Faculty, Division of Cell Biology, University of Cape Town, Rondebosch, South Africa Kevin M. Prise Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, UK Kathy Pritchard-Jones Institute of Cancer Research/Royal Marsden Hospital, Sutton, Surrey, UK Tassula Proikas-Cezanne Autophagy Laboratory, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Faculty of Science, Eberhard Karls University Tübingen, Tübingen, Germany Ching-Hon Pui St. Jude Children’s Research Hospital, Memphis, TN, USA Karen Pulford Nuffield Division of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK Teresa Gómez Del Pulgar Instituto de Investigaciones Biomedicas, CSIC, Madrid, Spain Vinee Purohit The Eppley Institute for Research in Cancer and Allied Diseases, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Keith R. Pye Cell ProTx, Aberdeen, UK Chao-Nan Qian Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China Jiahua Qian Qiagen, Frederick, MD, USA Liang Qiao Storr Liver Centre, Westmead Millennium Institute for Medical Research, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia Hartmut M. Rabes Institute of Pathology, University of Munich, Munich, Germany Bar-Shavit Rachel Department of Oncology, Hadassah-University Hospital, Jerusalem, Israel

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Ronny Racine Department of Urology, University of Miami – Miller School of Medicine, Miami, FL, USA Dirk Rades Department of Radiation Oncology, University Hospital Schleswig-Holstein, Campus Luebeck, Germany Jerald P. Radich Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Norman S. Radin Department of Psychiatry, University of Michigan, Ann Arbor, MI, USA Fulvio Della Ragione Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy Ryan L. Ragland Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA Gilbert J. Rahme Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Nino Rainusso Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Texas Children’s Cancer and Hematology Centers, Houston, TX, USA Ayyappan K. Rajasekaran Nemours Center for Childhood Cancer Research, Alfred I duPont Hospital for Children, Wilmington, DE, USA Jayadev Raju Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, Ottawa, ON, Canada Sundaram Ramakrishnan Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Kota V. Ramana Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA Pranela Rameshwar Medicine-Hematology/Oncology, Rutgers, New Jersey Medical School, Newark, NJ, USA Santiago Ramón y Cajal Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Giorgia Randi Department of Epidemiology, Institute for Farmacological Research Mario Negri, Milan, Italy Ramachandran Rashmi Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA Mariusz Z. Ratajczak Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA Anke Rattenholl Applied Biotechnology Division, Department of Engineering and Mathematics, University of Applied Sciences Bielefeld, Bielefeld, Germany

Contributors

Contributors

lxxxiii

Cocav A. Rauwerdink Lahey Center for Hematology/Oncology at Parkland Medical Center, Salem, NH, USA Alberto Ravaioli Department of Oncology, Instituto Scientifico Romagnolo per lo s, Infermi Hospital, Rimini, Italy Mira R. Ray The Prostate Centre at Vancouver General Hospital, University of British Columbia, Vancouver, BC, Canada Roger Reddel Children’s Medical Research Institute, The University of Sydney, Westmead, NSW, Australia May J. Reed Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA, USA Eduardo M. Rego Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Reuven Reich Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Jean-Marie Reimund Université de Strasbourg, Faculté de Médecine, INSERM U1113 and Fédération de Médecine Translationnelle de Strasbourg (FMTS), and, Hôpitaux Universitaires de Strasbourg, Hôpital de Hautepierre, Service d’Hépato-Gastroentérologie et d’Assistance Nutritive, Strasbourg, France Celso A. Reis Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal Ling Ren Pediatric Oncology Branch, National Cancer Institute, Center for Cancer Research, Bethesda, MD, USA Andrew G. Renehan Colorectal and Peritoneal Oncology Centre, The Christie NHS Foundation Trust, University of Manchester, Manchester, UK Marcus Renner Division of Molecular Genome Analysis, DKFZ, Heidelberg, Germany Paul S. Rennie The Prostate Centre at Vancouver General Hospital, University of British Columbia, Vancouver, BC, Canada Domenico Ribatti Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy Raul C. Ribeiro Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA Des R. Richardson Department of Pathology, University of Sydney, Sydney, NSW, Australia Victoria M. Richon Merck Research Laboratories, Boston, MA, USA Justin L. Ricker Merck Research Laboratories, Boston, MA, USA Thomas Ried Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

lxxxiv

Jörg Ringel Department of Medicine A, University of Greifswald, Greifswald, Germany Carrie Rinker-Schaffer Department of Surgery, Section of Urology, The University of Chicago, Chicago, IL, USA Francisco Rivero Centre for Cardiovascular and Metabolic Research, The Hull York Medical School, University of Hull, Hull, UK Tadeusz Robak Department of Hematology, Medical University of Lodz, Lodz, Poland Rita Roberti Department of Experimental Medicine, University of Perugia, Perugia, Italy Fredika M. Robertson The University of Texas MD Anderson Cancer Center, Houston, TX, USA Angelo Rodrigues Department of Pathology, Portuguese Oncology InstitutePorto, Porto, Portugal Delvys Rodriguez-Abreu Hospital Universitario Insular, Las Palmas de Gran Canaria, Spain Jose Luis Rodríguez-Fernández Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Madrid, Spain Carlos Rodriguez-Galindo Dana-Farber Cancer Institute, Boston, MA, USA Florian Roka Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Cleofé Romagosa Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Ze’ev Ronai Signal Transduction Program, Burnham Institute for Medical Research, La Jolla, CA, USA Luca Roncucci Department of Diagnostic and Clinical Medicine, and Public Health, University of Modena and Reggio Emilia, Modena, Italy Igor B. Roninson Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Columbia, SC, USA Jatin Roper Tufts Medical Center, Boston, MA, USA Rafael Rosell Instituto Oncológico Dr. Rosell, Quiron-Dexeus University Hospital, Barcelona, Spain Pangaea Biotech, Barcelona, Spain Cancer Biology and Precision Medicine Program, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain Molecular Oncology Research (MORe) Foundation, Barcelona, Spain

Contributors

Contributors

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Eliot M. Rosen Department of Oncology, Georgetown University School of Medicine, Washington, DC, USA Department of Biochemistry, Molecular and Cellular Biology, Georgetown University School of Medicine, Washington, DC, USA Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC, USA Carol L. Rosenberg Boston Medical Center and Boston University School of Medicine, Boston, MA, USA Steven A. Rosenzweig Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA Angelo Rosolen Department of Pediatrics, Hemato-oncology Unit, University of Padua, Padova, Italy Jeffrey S. Ross Albany Medical College, Albany, NY, USA Theodora S. Ross Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX, USA Catalina A. Rosselló University of the Balearic Islands, Palma de Mallorca, Spain Anita De Rossi Viral Oncology Unit and AIDS Reference Center, Section of Oncology and Immunology, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy Alberto Ruano-Ravina Department of Preventive Medicine and Public Health, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Tami Rubinek Tel Aviv Medical Center and Tel Aviv University, Tel Aviv, Israel Luca Rubino Department of Oncology, Humanitas Research Hospital, Humanitas Cancer Center, Rozzano, Milan, Italy Marco Ruggiero Dream Master Laboratory, Chandler, AZ, USA Francisco Ruiz-Cabello Osuna UGC Laboratorio Clínico Hospital Universitario Virgen de las Nieves Facultad de Medicina, Universidad de Granada, Granada, Spain María Victoria Ruiz-Pérez Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden Zoran Rumboldt Department of Radiology and Radiological Science, Medical University of South Carolina, Charleston, SC, USA Erkki Ruoslahti Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

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Center for Nanomedicine and Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA Dario Rusciano Friedrich Miescher Institute, Basel, Switzerland Giandomenico Russo Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Roma, Italy Irma H. Russo Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA, USA Jose Russo Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA, USA James T. Rutka The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, The University of Toronto, Toronto, ON, Canada James Ryan Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Venkata S. Sabbisetti Renal Division, Department of Medicine, Brigham and Women’s Hospital, The Harvard Clinical and Translational Science Center, Boston, MA, USA Anne Thoustrup Saber National Institute of Occupational Health, Copenhagen, Denmark Gauri Sabnis University of Maryland School of Medicine, Baltimore, MD, USA Mohamad Seyed Sadr Montreal, QC, Canada Guillermo T. Sáez Department of Biochemistry and Molecular Biology, Faculty of Medicine and Odontology-INCLIVA, University of Valencia, Valencia, Spain Service of Clinical Analysis, Dr. Peset University Hospital, Valencia, Spain Stephen Safe Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA Xavier Sagaert Department of Pathology, University Hospitals of K.U. Leuven, Leuven, Belgium Asim Saha University of Cincinnati and The Barrett Cancer Center, Cincinnati, OH, USA Emine Sahin Institute for Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria Kunal Saigal National Institute on Deafness and Other Communication, Disorders and National Cancer Institute, NIH, Bethesda, MD, USA

Contributors

Contributors

lxxxvii

Toshiyuki Sakai Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan Bodour Salhia Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ, USA Helmut Rainer Salih Department of Internal Medicine II, University Hospital of Tübingen, Eberhard-Karls-University, Tübingen, Germany Beth A. Salmon Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA Howard W. Salmon Department of Radiation Oncology, North Florida Radiation Oncology, Gainesville, FL, USA Raed Samar Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Julian R. Sampson Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK Nianli Sang Department of Biology, Drexel University College of Arts and Sciences, Philadelphia, PA, USA Manoranjan Santra Neurology Research, Henry Ford Health System, Detroit, MI, USA Ehsan Sarafraz-Yazdi Division of Gynecologic Oncology, Department of OB/GYN, State University of New York, Downstate Medical Center, New York, NY, USA Frank Saran Department of Radiotherapy and Paediatric Oncology, Royal Marsden Hospital NHS Foundation Trust, Sutton, Surrey, UK Devanand Sarkar Department of Human and Molecular Genetics, Virginia Commonwealth University, VCU Medical Center, School of Medicine, Richmond, VA, USA Fazlul H. Sarkar Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA Debashis Sarker Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK Ken Sasaki Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA Hiroyuki Sasaki Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Tomikazu Sasaki Department of Chemistry, University of Washington, Seattle, WA, USA A. Kate Sasser Department of Pediatrics, Columbus Children’s Research Institute, The Ohio State University, Columbus, OH, USA

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Aaron R. Sasson Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA Robert L. Satcher Orthopaedic Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Leonard A. Sauer Bassett Research Institute, Cooperstown, NY, USA Christobel Saunders School of Surgery and Pathology, QEII Medical Centre, University of Western Australia, Crawley, WA, Australia Constance L. L. Saw Department of Pharmaceutics, Rutgers, The State University of New Jersey, Ernest Mario School of Pharmacy, Piscataway, NJ, USA Anurag Saxena Department of Pathology and Laboratory Medicine, Royal University Hospital, Saskatoon Health Region/University of Saskatchewan, Saskatoon, SK, Canada Reinhold Schäfer Comprehensive Cancer Center, Charité Universitätsmedizin Berlin, Berlin, Germany Amanda Schalk University of Illinois at Chicago, Chicago, IL, USA Manfred Schartl Physiologische Chemie I, Biozentrum, Universität Würzburg, Würzburg, Germany Huub Schellekens Department of Innovation Studies, Department of Pharmaceutical Sciences, Utrecht University, TD Utrecht, The Netherlands Detlev Schindler Department of Human Genetics, Biozentrum University of Würzburg, Würzburg, Germany Peter M. Schlag Comprehensive Cancer Center, Charité Campus Mitte, Berlin, Germany Peter Schlosshauer Department of Pathology, The Mount Sinai School of Medicine, New York, NY, USA Martin Schlumberger Department of Nuclear Medicine and Endocrine Oncology, Referral Center for Refractory Thyroid Tumors, Institut National du Cancer, Institut Gustave Roussy, Villejuif, France Peter Schmezer Division Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Annette Schmitt-Graeff Department of Pathology, University hospital Freiburg, Freiburg, Germany Marc Schmitz Institut für Immunologie, Technische Universität Dresden, Dresden, Germany Dominik T. Schneider Clinic of Pediatrics, Klinikum Dortmund, Dortmund, Germany Katrina J. Schneider Research Service, Veterans Administration Medical Center, Omaha, NE, USA

Contributors

Contributors

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Stefan W. Schneider Hauttumorzentrum Mannheim (HTZM), Universitätsmedizn Mannheim, Mannheim, Germany Maria Schnelzer Department of Radiation Protection and Health, Bundesamt für Strahlenschutz (Federal Office for Radiation Protection), Oberschleissheim, Germany Nathalie Scholler Center for Cancer, SRI Biosciences, Menlo Park, CA, USA Axel H. Schönthal University of Southern California, Keck School of Medicine, Los Angeles, CA, USA Bart H. W. Schreuder Department of Orthopaedics, Radboud University Medical Centre, Nijmegen, The Netherlands Morgan S. Schrock Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA Laura W. Schrum Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC, USA Wolfgang A. Schulz Department of Urology, Heinrich Heine University, Düsseldorf, Germany Manfred Schwab German Cancer Research Center (DKFZ), Heidelberg, Germany Markus Schwaiger Department of Nuclear Medicine, Technical University of Munich, Munich, Germany Edward L. Schwartz Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY, USA Julie K. Schwarz Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA Rony Seger Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel Gail M. Seigel Center for Hearing and Deafness, University at Buffalo, Buffalo, NY, USA Hiroyuki Seimiya Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan Paule Seite UMR CNRS 6187 Pôle Biologie Santé, University of Poitiers, Poitiers cedex, France Helmut K. Seitz Centre of Alcohol Research (CAR), University of Heidelberg, Heidelberg, Germany Department of Medicine, Salem Medical Center, Heidelberg, Germany Periasamy Selvaraj Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA

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Wolfhard Semmler Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany Subrata Sen Department of Molecular Pathology (Unit 951), The University of Texas MD Anderson Cancer Center, Houston, TX, USA Suvajit Sen Department of Obstetrics and Gynecology, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA Vitalyi Senyuk Department of Medicine (M/C 737), College of Medicine Research Building, University of Illinois at Chicago, Chicago, IL, USA Nedime Serakinci Medical Genetics, Near East University, Nicosia, Northern Cyprus Christine Sers Institute of Pathology, University Medicine Charité, Berlin, Germany Marta Sesé Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain Vijayasaradhi Setaluri Department of Anatomy, Kasturba Medical College, Manipal University, Manipal, Karnataka, India John F. Seymour Haematology Department, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia University of Melbourne, Parkville, VIC, Australia Girish V. Shah Department of Pharmacology, University of Louisiana College of Pharmacy, Monroe, LA, USA Rabia K. Shahid Department of Medicine, University of Saskatchewan, Saskatoon, SK, Canada Sharmila Shankar Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, KS, USA Anand Sharma Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Narinder Kumar Sharma Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Medical Center, Kansas City, KS, USA Jerry W. Shay University of Texas Southwestern Medical Center, Dallas, TX, USA Shijie Sheng Department of Pathology and Oncology, Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit, MI, USA James L. Sherley Asymmetrex, LLC, Boston, MA, USA Donna Shewach Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

Contributors

Contributors

xci

Ie-Ming Shih Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Kentaro Shikata Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Yosef Shiloh Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Hyunsuk Shim Department of Hematology/Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA Yutaka Shimada Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Masahito Shimojo School of Medicine, Osaka Medical College, Takatsuki, Osaka, Japan Yong-Beom Shin BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Republic of Korea Toshi Shioda Massachusetts General Hospital Center for Cancer Research, Charlestown, MA, USA Janet Shipley The Institute of Cancer Research, Sutton, Surrey, UK Girja S. Shukla Department of Surgery, Vermont Comprehensive Cancer Center, College of Medicine, University of Vermont, Burlington, VT, USA Arthur Shulkes Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia Antonio Sica Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy Gene P. Siegal Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA Dietmar W. Siemann Department of Radiation Oncology, University of Florida, Gainesville, FL, USA Christine L. E. Siezen National Institute of Public Health and Environment, Bilthoven, The Netherlands Alexandra Silveira Ocular Molecular Genetics Institute, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, USA Martin J. Simard Le Centre de recherche du CHU de Québec-Université Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Diane M. Simeone Department of Physiology, University of Michigan Medical Center, Ann Arbor, MI, USA Hans-Uwe Simon Department of Pharmacology, University of Bern, Bern, Switzerland

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Bryan Simoneau Le Centre de recherche du CHU de Québec-Université Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Ajay Singh Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA Amrik J. Singh Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA Harprit Singh De Montfort University, Leicester, UK Kamaleshwar Singh The Institute of Environmental and Human Health (TIEHH), Texas Tech University, Lubbock, TX, USA Narendra P. Singh Departments of Bioengineering, University of Washington, Seattle, WA, USA Pankaj K. Singh The Eppley Institute for Research in Cancer and Allied Diseases, and Department of Pathology and Microbiology, and Department of Biochemistry and Molecular Biology, and Department of Genetic Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA Shalini Singh Department of Surgery, McMaster University, Hamilton, ON, Canada Shree Ram Singh Basic Research Laboratory, National Cancer Institute at Frederick, Frederick, MD, USA Vineeta Singh School of Surgery and Pathology, QEII Medical Centre, Sir Charles Gairdner Hospital, Nedlands, WA, Australia Lillian L. Siu Department of Medical Oncology and Hematology, Robert and Maggie Bras and Family New Drug Development Program, Princess Margaret Hospital, Toronto, ON, Canada Anita Sjölander Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital, Malmö, Sweden Judith Skoner Head and Neck Tumor Program, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Keith Skubitz Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, Minneapolis, MN, USA Christopher Slape Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Keiran S. M. Smalley The Wistar Institute, Philadelphia, PA, USA Lubomir B. Smilenov Department of Radiation Oncology, Columbia University, New York, NY, USA Bruce F. Smith Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL, USA

Contributors

Contributors

xciii

Russell Spencer Smith Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Josef Smolle Department of Dermatology, Medical University Graz, Graz, Austria Jimmy B. Y. So Department of Surgery, National University of Singapore, National University Hospital, Singapore, Singapore Robert W. Sobol University of South Alabama Mitchell Cancer Institute, Mobile, AL, USA Alexander S. Sobolev Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Eric Solary Inserm Unité Mixte de Recherche (UMR) 1009, Institut Gustave Roussy, University Paris-Sud 11, Villejuif, France Graziella Solinas Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy Toshiya Soma Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Guru Sonpavde Texas Oncology and Veterans Affairs Medical Center and the Baylor College of Medicine, Houston, TX, USA Anil K. Sood Departments of Gynecologic Oncology and Reproductive Medicine and Cancer Biology and The Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Henrik Toft Sørensen Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus C, Denmark Pavel Soucek Toxicogenomics Unit, Center for Toxicology and Health Safety, National Institute of Public Health, Prague, Czech Republic Lorenzo Spaggiari University of Milan School of Medicine, Milan, Italy Ulrich Specks Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA David W. Speicher The Wistar Institute, Philadelphia, PA, USA Valerie Speirs Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK Dietmar Spengler Max-Panck-Institut für Psychiatrie, Munich, Germany Phillippe E. Spiess Department of Genitourinary Oncology, Moffitt Cancer Center, Tampa, FL, USA Melanie Spotheim-Maurizot Centre de Biophysique Moleculaire, CNRS, Orleans, France

xciv

Cynthia C. Sprenger Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA, USA Lakshmaiah Sreerama Department of Chemistry and Biochemistry, St. Cloud State University, St. Cloud, MN, USA Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar Rakesh Srivastava Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, KS, USA Satish K. Srivastava Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA M. Sharon Stack Northwestern University Medical School, Chicago, IL, USA Jan A. Staessen Division of Lung Toxicology, Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.), Division of Hypertension and Cardiovascular Rehabilitation, Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium Eric Stanbridge Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA Barry Staymates Department of Pathology, Henry Mayo Newhall Memorial Hospital, Valencia, CA, USA Stacey Stein Center for Advanced Biotechnology and Medicine, UMDMJ – Robert Wood Johnson Medical School, Piscataway, NJ, USA Martin Steinhoff UCD Charles Institute of Dermatology, University College Dublin, Belfield, Ireland Department of Dermatology School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland Alexander Steinle Institute for Molecular Medicine, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Carsten Stephan Department of Urology, Charité, Universitätsmedizin, Campus Charité Mitte, Berlin, Germany Peter L. Stern Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK William G. Stetler-Stevenson Extracellular Matrix Pathology Section, Cell and Cancer Biology Branch, National Cancer Institute, Bethesda, MD, USA Richard G. Stevens University of Connecticut Health Center, Farmington, CT, USA Freda Stevenson CRC Wessex Oncology Unit, Southampton General Hospital and Tenovous Laboratory, Southampton University Hospital Trust, Southampton, UK

Contributors

Contributors

xcv

William P. Steward Department of Cancer Studies, University of Leicester, Leicester, UK Constantine A. Stratakis Program on Developmental Endocrinology of Genetics, NICHD, NIH, Bethesda, MD, USA Alex Y. Strongin Burnham Institute for Medical Research, La Jolla, CA, USA Deepa S. Subramaniam Georgetown University Hospital, Washington, DC, USA Garnet Suck Health Sciences Authority, Centre for Transfusion Medicine, Singapore, Singapore Paul H. Sugarbaker Washington Cancer Institute, Washington Hospital Center, Washington, DC, USA Baocun Sun Department of Pathology, Tianjin Cancer Hospital and Tianjin Cancer Institute, Tianjin, People’s Republic of China Duxin Sun Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA Shi-Yong Sun School of Medicine and Winship Cancer Institute, Emory University, Atlanta, GA, USA Zhifu Sun Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN, USA Saul Suster Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA Russell Szmulewitz The University of Chicago Medicine, Chicago, IL, USA Thomas Tüting Laboratory for Experimental Dermatology, Department of Dermatology, University of Bonn, Bonn, Germany Dirk Taeger Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Ruhr-University Bochum, Bochum, Germany Masatoshi Tagawa Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan Stanley Tahara Keck School of Medicine, Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA Yoshikazu Takada UC Davis School of Medicine, Sacramento, CA, USA Akihisa Takahashi Heavy Ion Medical Center, Gunma University, Maebashi, Gunma, Japan Tsutomu Takahashi Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

xcvi

Yoshimi Takai Faculty of Medicine, Osaka University Graduate School of Medicine, Suita, Japan Tamotsu Takeuchi Department of Pathology, Kochi Medical School, Kochi, Japan Constantine S. Tam Haematology Department, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia University of Melbourne, Parkville, VIC, Australia Luca Tamagnone Department of Oncology, University of Turin, Candiolo, Italy Candiolo Cancer Center-IRCCS, University of Turin, Candiolo, Italy Harald Tammen PXBioVisioN GmbH, Hannover, Germany Masaaki Tamura Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA David S. P. Tan Department of Medical Oncology, National University Cancer Institute, Singapore (NCIS), National University Hospital, and Cancer Science Institute, National University of Singapore, Singapore, Singapore Takuji Tanaka Department of Oncologic Pathology, Kanazawa Medical University, Kanazawa, Japan Dean G. Tang Department of Carcinogenesis, Science Park-Research Division, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA Ya-Chu Tang Department of Life Sciences, College of Science, National Central University, Jhongli City, Taiwan Nizar M. Tannir Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Weikang Tao Department of Cancer Research, Merck Research Laboratories, West Point, PA, USA Chi Tarn Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA Clive R. Taylor Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Jennifer Taylor Committee on Cancer Biology, The University of Chicago, Chicago, IL, USA Andrew R. Tee Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK Ayalew Tefferi Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN, USA Bin T. Teh Cancer and Stem Cell Biology (CSCB), Duke-NUS, Graduate Medical School, Singapore, Singapore

Contributors

Contributors

xcvii

Marie-Hélène Teiten Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC), Hôpital Kirchberg, Luxembourg, Luxembourg Joseph R. Testa Fox Chase Cancer Center, Philadelphia, PA, USA John Thacker Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK Rajesh V. Thakker Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, UK Nicholas B. La Thangue Department of Oncology, University of Oxford, Oxford, UK Dan Theodorescu Department of Surgery, Urology, School of Medicine, University of Colorado Cancer Center, Aurora, CO, USA Panayiotis A. Theodoropoulos Department of Basic Sciences, The University of Crete, School of Medicine, Heraklion, Crete, Greece Frank Thévenod Private Universität Witten/Herdecke gGmbH, Witten, Germany Karl-Heinz Thierauch Berlin, Germany Megan N. Thobe University of Cincinnati College of Medicine, Cincinnati, OH, USA Natalie Thomas Clinical Network Services Pty Ltd, St Albans, UK Peter Thomas Departments of Surgery and Biomedical Sciences, Creighton University, Omaha, NE, USA Sufi M. Thomas Departments of Otolaryngology-Head and Neck Surgery, University of Kansas Medical Center, Kansas City, KS, USA Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA Sven Thoms University of Göttingen, Göttingen, Germany Magnus Thörn Department of Surgery (MT), Karolinska Institutet, Stockholm, Sweden Anna Tiefenthaller Clinic for Radiotherapy and Radiation Oncology, LMU Munich, Munich, Germany Derya Tilki Martini-Klinik, Prostatakrebszentrum, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany Donald J. Tindall Department of Urology Research, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA

xcviii

Umberto Tirelli Department of Medical Oncology, National Cancer Institute, Aviano, PN, Italy Martin Tobi Section of Gastroenterology, Detroit VAMC, Detroit, MI, USA Philip J. Tofilon Radiation Oncology Branch, National Cancer Institute, Bethesda, MD, USA Masakazu Toi Department of Surgery (Breast Surgery), Graduate School of Medicine, Kyoto University, Kyoto, Japan Amanda Ewart Toland Division of Human Cancer Genetics, The Ohio State University, Columbus, OH, USA Massimo Tommasino Infections and Cancer Biology Group, International Agency for Research on Cancer, Lyon, France Antonio Toninello Department of Biological Chemistry, University of Padua, Padua, Italy Jeffrey A. Toretsky Department of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Jorge R. Toro National Institutes of Health, Bethesda, MD, USA Manuel Torres University of the Balearic Islands, Palma de Mallorca, Spain Tibor Tot Department of Pathology and Clinical Cytology, Central Hospital Falun, Uppsala University, Falun, Sweden Mathilde Touvier UMR U1153 INSERM, U1125 INRA, CNAM, Université Paris 13, Centre de Recherche Epidémiologie et Statistique Sorbonne Paris Cité, Bobigny, France Philip C. Trackman Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA Tiffany A. Traina Breast Cancer Medicine Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Luba Trakhtenbrot Molecular Cytogenetics Laboratory, Institute of Hematology, The Chaim Sheba Medical Center, Tel Hashomer, Israel Janeen H. Trembley Minneapolis VA Health Care System and University of Minnesota, Minneapolis, MN, USA Pierre-Luc Tremblay Le Centre de recherche du CHU de QuébecUniversité Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Matthew Trendowski Department of Biology, Syracuse University, Syracuse, NY, USA Edward L. Trimble Department of Health and Human Services, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Contributors

Contributors

xcix

Jörg Trojan Universitätsklinikum Frankfurt, Medizinische Klinik 1, Frankfurt am Main, Germany Alisha M. Truman Northeastern University, Boston, MA, USA Gregory J. Tsay Department of Medicine, Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan Apostolia-Maria Tsimberidou Department of Investigational Cancer Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Kunihiro Tsuchida Division for Therapies Against Intractable Diseases, Institute for Comprehensive Medical Science (ICMS), Fujita Health University, Toyoake, Japan Nobuo Tsuchida Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Florin Tuluc Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Mehmet Kemal Tur Institute of Pathology, University Hospital, JustusLiebig-University Giessen, Giessen, Germany Greg Turenchalkb 454 Life Sciences, Branford, CT, USA Andrew S. Turnell Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Jeffrey Turner Prostate Oncology Specialists, Los Angeles, CA, USA Michelle C. Turner McLaughlin Centre for Population Health Risk Assessment, University of Ottawa, Ottawa, ON, Canada ISGlobal, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain CIBER Epidemiología y Salud Pública (CIBERESP), Madrid, Spain Guri Tzivion Cancer Institute, Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA Salvatore Ulisse Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy Nick Underhill-Day School of Biosciences, Swift Ecology Ltd, Warwickshire, UK Rosemarie A. Ungarelli Boston Medical Center and Boston University School of Medicine, Boston, MA, USA Gretchen M. Unger GeneSegues Inc., Chaska, MN, USA

c

Motoko Unoki Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Markus Vähä-Koskela Molecular Cancer Biology Research Program, University of Helsinki, Helsinki, Finland Antti Vaheri Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland Kedar S. Vaidya Global Pharmaceutical Research and Development, Abbott Laboratories, North Chicago, IL, USA Ilan Vaknin The Lautenberg Center for Immunology and Cancer Research, Israel-Canada Medical, Research Institute Faculty of Medicine, The Hebrew University, Jerusalem, Israel Anne M. VanBuskirk Takeda Oncology, Cambridge, MA, USA Wim Vanden Berghe Epigenetic Signaling Lab PPES, Department Biomedical Sciences, University Antwerp, Antwerp, Belgium Marry M. van den Heuvel-Eibrink Princess Maxima Center for Pediatric Oncology/Hematology, Utrecht, The Netherlands Michael W. Van Dyke Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA, USA Casper H. J. van Eijck Department of Surgery, Erasmus MC, Rotterdam, The Netherlands Manon van Engeland Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Hospital, Maastricht, The Netherlands Wilhelmin M. U. van Grevenstein Department of Surgery, Erasmus MC, Rotterdam, The Netherlands Ad Geurts van Kessel Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Ron H. N. van Schaik Department of Clinical Chemistry, Erasmus University Medical Center, Rotterdam, The Netherlands Viggo Van Tendeloo Vaccine and Infections Disease Institute (VAXINFECTIO) Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium Alex van Vliet Department of Cellular and Molecular Medicine, Cell Death Research and Therapy Lab, KU Leuven Campus Gasthuisberg, Leuven, Belgium Carter Van Waes National Institute on Deafness and Other Communication, Disorders and National Cancer Institute, NIH, Bethesda, MD, USA Sakari Vanharanta Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

Contributors

Contributors

ci

Roberta Vanni Department of Biomedical Science and Technology, University of Cagliari, Monserrato (CA), Italy Judith A. Varner Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA, USA Aikaterini T. Vasilaki University Department of Surgery, Royal Infirmary, Glasgow, UK Peter Vaupel Department of Radiooncology and Radiotherapy, University Medical Center Mainz, Mainz, Germany Guillermo Velasco Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain Marcel Verheij Department of Radiotherapy, The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Mukesh Verma Division of Cancer Control and Population Sciences, National Cancer Institute (NCI), National Institutes of Health (NIH), Rockville, MD, USA Rakesh Verma Prescient Healthcare Group, London, UK Srdan Verstovsek Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX, USA René P. H. Veth Department of Orthopaedics, Radboud University Medical Centre, Nijmegen, The Netherlands G. J. Villares Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Akila N. Viswanathan Brigham and Women’s/Dana-Farber Cancer Center, Boston, MA, USA Kris Vleminckx Department of Biomedical Molecular Biology and Center for Medical Genetics, Ghent University, Ghent, Belgium Israel Vlodavsky Anatomy and Cell Biology, Technion Israel Institute of Technology, Cancer and Vascular Biology Research Center, Haifa, Israel Martina Vockerodt Department of Pediatrics I, Children’s Hospital, GeorgAugust University of Gottingen, Gottingen, Germany Charles L. Vogel Sylvester Cancer Center, School of Medicine, University of Miami, Plantation, FL, USA Tilman Vogel Department Mönchengladbach, Germany

of

Surgery,

Krankenhaus

Maria

Hilf,

Ulla Vogel National Institute of Occupational Health, Copenhagen, Denmark Daniel D. von Hoff Arizona Cancer Center, Tucson, AZ, USA Silvia von Mensdorff-Pouilly Department of Obstetrics and Gynaecology, Vrije Universiteit Medisch Centrum (VUmc), Amsterdam, The Netherlands

cii

Ingo Kausch von Schmeling Klinik für Urologie und Kinderurologie, Ammerland Klinik GmbH, Westerstede, Germany Dietrich von Schweinitz Klinikum der Universität München, Kinderchirurgische Klinik im Dr. von Haunerschen Kinderspital, München, Germany Alireza Vosough Department of Radiotherapy, Royal Marsden Hospital NHS Foundation Trust, Sutton, Surrey, UK George F. Vande Woude Van Andel Research Institute, Grand Rapids, MI, USA Tom Waddell GI/Lymphoma Research Unit, Royal Marsden Hospital, Surrey, UK Christoph Wagener University Medical Center Hamburg-Eppendorf, Hamburg, Germany Sabine Wagner Department of Pediatrics, Klinik St. Hedwig, Krankenhaus der Barmherzigen Brüder, Regensburg, Germany Kristin A. Waite Genomic Medicine Institute, Lerner Research Institute, and Taussing Cancer Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Toshifumi Wakai Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Heather M. Wallace University of Aberdeen, Aberdeen, UK Håkan Wallin National Institute of Occupational Health, Copenhagen, Denmark Susan E. Waltz Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati Veteran’s Administration Hospital, Cincinnati, OH, USA Jack R. Wands Division of Gastroenterology, Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI, USA Bo Wang The Ohio State University, Columbus, OH, USA Gang Wang Feil Brain and Mind Research Institute, Weill Cornell Medicine, Cornell University, New York, NY, USA Helen Y. Wang Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA Hwa-Chain Robert Wang Molecular Oncology, Department of Biomedical and Diagnostic Sciences, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN, USA Jianghua Wang Department of Pathology, Baylor College of Medicine, Houston, TX, USA Mingjun Wang Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA

Contributors

Contributors

ciii

Rong-Fu Wang Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA Xianghong Wang Department of Anatomy, The University of Hong Kong, Hong Kong, China Xiang-Dong Wang Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA Yu Wang Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China Zhu A. Wang Department of Genetics and Development, Columbia University Medical Center, Herbert Irving Comprehensive Cancer Center, New York, NY, USA Patrick Warnat Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany Kounosuke Watabe Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University, School of Medicine, Springfield, IL, USA School of Medicine, Department of Cancer Biology, Wake Forest University, Winston-Salem, NC, USA Dawn Waterhouse Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, Canada Catherine Waters The Ohio State University College of Medicine, Columbus, OH, USA Valerie M. Weaver Department of Surgery, University of California, San Francisco, San Francisco, CA, USA Lau Weber Department of Urology, Singapore General Hospital, Singapore, Singapore Daniel S. Wechsler Pediatric Hematology-Oncology, Duke University Medical Center, Durham, NC, USA Scott A. Weed Department of Neurobiology and Anatomy, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV, USA Oliver Weigert Department of Internal Medicine III, University of Munich, Großhadern, Munich, Germany Eugene D. Weinberg Biology and Medical Sciences, Indiana University, Bloomington, IN, USA I. Bernard Weinstein Columbia University, New York, NY, USA Ellen Weisberg Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA Lawrence M. Weiss Division of Pathology, City of Hope National Medical Center, Duarte, CA, USA

civ

Danny R. Welch Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA Thilo Welsch Department of Visceral, Thoracic and Vascular Surgery, TU Dresden, Dresden, Germany Sarah J. Welsh Harris Manchester College, University of Oxford, Oxford, UK Tania M. Welzel Universitätsklinikum Frankfurt, Medizinische Klinik 1, Frankfurt am Main, Germany Tamra E. Werbowetski-Ogilvie Regenerative Medicine Program, Biochemistry and Medical Genetics and Physiology and Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada Frank Westermann DKFZ, German Cancer Research Center, Heidelberg, Germany Linda C. Whelan UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland Bruce A. White Department of Cell Biology, UConn School of Medicine, UConn Health, Farmington, CT, USA Robert P. Whitehead Nevada Cancer Institute, Las Vegas, NV, USA Theresa L. Whiteside University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Christophe Wiart University of Nottingham, Nottingham, UK Andreas Wicki Department of Medical Oncology, University Hospital, Basel, Switzerland Carol Wicking Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia Lisa Wiesmüller Department of Obstetrics and Gynaecology, University of Ulm, Ulm, Germany Edwin van Wijngaarden Department of Public Health Sciences, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Kandace Williams Department of Biochemistry and Cancer Biology, Health Science Campus, UT College of Medicine, Toledo, OH, USA Elizabeth D. Williams Australian Prostate Cancer Research Centre – Queensland (APCRC-Q), Brisbane, QLD, Australia Translational Research Institute, Institute of Health and Biomedical Innovation, Faculty of Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Elizabeth M. Wilson Department of Pediatrics and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Contributors

Contributors

cv

George Wilson Storr Liver Centre, Westmead Millennium Institute for Medical Research, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia Ola Winqvist Department of Medicine (OW), Karolinska Institutet, Stockholm, Sweden Jordan Winter Department of Surgery, Thomas Jefferson University, Philadelphia, PA, USA John Pierce Wise Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA Christian Wittekind Department für Diagnostik, Institut für Pathologie, Universitätsklinikum Leipzig, Leipzig, Germany Isaac P. Witz Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel Ido Wolf Division of Oncology, The Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Roland C. Wolf Biomedical Research Centre, University of Dundee, Dundee, UK Alice Wong University of Hong Kong, Hong Kong, China Chun-Ming Wong Department of Pathology, The University of Hong Kong, Hong Kong, China Yung H. Wong Division of Life Science, Biotechnology Research Institute, The Hong Kong University of Science and Technology, Kowloon, Hong Kong Dori C. Woods Northeastern University, Boston, MA, USA Paul Workman Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK Maria J. Worsham Department of Otolaryngology, Henry Ford Health System, Detroit, USA Thomas Worzfeld Institute of Pharmacology, University of Marburg, Marburg, Germany Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany Jie Wu Department of Molecular Oncology, SRB-3, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Mei-Yi Wu Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, USA Ray-Chang Wu Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, USA

cvi

Shiyong Wu Edison Biotechnology Institute and Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USA Wen Jin Wu Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA Xiaosheng Wu Department of Immunology, Mayo Clinic, College of Medicine, Rochester, MN, USA Xifeng Wu Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Yi-Long Wu Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China Christopher Xiao Department of Otolaryngology-Head and Neck Surgery, Medical University of South Carolina, Charleston, SC, USA Guang-Hui Xiao Fox Chase Cancer Center, Philadelphia, PA, USA Huajiang Xiong Department of Zoophysiology, Zoological Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Jianming Xu Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Tian Xu Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Zhengping Xu Zhejiang University School of Medicine, Hangzhou, China Jing Xue Stanford University School of Medicine, Stanford, CA, USA Judy W. P. Yam Department of Pathology, The University of Hong Kong, Hong Kong, China Sho-ichi Yamagishi Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan Michiko Yamamoto Department of Respiratory Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan Wei Yan Department of Cancer Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA Haining Yang University of Hawaii Cancer Center, Honolulu, HI, USA Hong Yang Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Jia-Lin Yang Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia

Contributors

Contributors

cvii

Ping Yang Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN, USA Rongxi Yang Molecular Epidemiology Unit, German Cancer Research Center, Heidelberg, Germany Libo Yao Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi’an, Shananxi, China Masakazu Yashiro Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan Nelson Yee Penn State Hershey Cancer Institute, Hershey, PA, USA Yerem Yeghiazarians Department of Medicine, Division of Cardiology, Translational Cardiac Stem Cell Program, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Cardiovascular Research Institute, University of California San Francisco (UCSF), San Francisco, CA, USA W. Andrew Yeudall Department of Oral Biology, College of Dental Medicine, Georgia Regents University, Augusta, GA, USA Maksym V. Yezhelyev Winship Cancer Institute, Emory University, Atlanta, GA, USA Ömer H. Yilmaz The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Açelya Yilmazer Aktuna Biomedical Engineering Department, Engineering Faculty, Ankara University, Golbasi, Ankara, Turkey Anthony P. C. Yim Division of Cardiothoracic Surgery, Chinese University of Hong Kong, Hong Kong, China John H. Yim Department of Surgery, City of Hope, Duarte, CA, USA Chengqian Yin Department of Biology, Drexel University College of Arts and Sciences, Philadelphia, PA, USA Helen L. Yin Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Min-Jean Yin Oncology Research, Pfizer Worldwide R&D, San Diego, CA, USA Xiao-Ming Yin Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, IN, USA Zhimin Yin College of Life Science, Nanjing Normal University, Nanjing, People’s Republic of China George Wai-Cheong Yip Department of Anatomy, National University of Singapore, Singapore, Singapore Kenneth W. Yip Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada

cviii

Harry H. Yoon Mayo Clinic Comprehensive Cancer, Rochester, MN, USA Jung-Hwan Yoon Department of Internal Medicine, Seoul National University College of Medicine, Chongno-gu, Seoul, South Korea Kazuhiro Yoshida Department of Surgical Oncology, Gifu University School of Medicine, Gifu, Japan Tatsushi Yoshida Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan Kouichi Yoshimasu Department of Hygiene, School of Medicine, Wakayama Medical University, Wakayama, Japan Anas Younes Lymphoma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA Graeme P. Young Flinders Cancer Control Alliance, Flinders University, Adelaide, SA, Australia Ken H. Young Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Dihua Yu Departments of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Jian Yu Department of Pathology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Yan Ping Yu Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Yu Yu Department of Pathology, University of Sydney, Sydney, NSW, Australia Xiao Yuan Research and Development Center, Wuhan Botanical Garden, Chinese Academy of Science, Wuhan, Hubei, People’s Republic of China Anthony Po-Wing Yuen Division of Otorhinolaryngology, Department of Surgery, The University of Hong Kong, Hong Kong, SAR, China Zhong Yun Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA Stefan K. Zöllner Department of Pediatric Hematology and Oncology, University Childrens Hospital Münster, Münster, Germany Leo R. Zacharski VA Hospital, White River Junction, VT, USA Gerard P. Zambetti Department of Biochemistry, Dana-Farber Cancer Institute, Boston, MA, USA Behrouz Zand UT MD Anderson Cancer Center, Houston, TX, USA Laura P. Zanello Department of Biochemistry, University of CaliforniaRiverside, Riverside, CA, USA

Contributors

Contributors

cix

Uwe Zangemeister-Wittke Department of Pharmacology, University of Bern, Bern, Switzerland Andrew C. W. Zannettino Myeloma Research Laboratory, School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, SA, Australia Kamran Zargar-Shoshtari Department of Urology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA Laura Zavala-Flores Redox Biology Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA Berton Zbar Laboratory of Immunobiology, NIH – Frederick, Frederick, MD, USA Herbert J. Zeh III UPMC/University of Pittsburgh Schools of the Health Sciences, Pittsburgh, PA, USA Jason A. Zell Cancer Prevention Program, Division of Hematology/Oncology and Epidemiology, Department of Medicine, School of Medicine, Chao Family Comprehensive Cancer Center, University of California, Irvine, CA, USA Danfang Zhang Department of Pathology, Tianjin Cancer Hospital and Tianjin Cancer Institute, Tianjin, People’s Republic of China Fengrui Zhang Michigan State University, East Lansing, MI, USA Hao Zhang The University of Texas MD Anderson Cancer Center, Houston, TX, USA Hong Zhang Biogen Idec, San Diego, CA, USA Hui Zhang Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, NV, USA Jinping Zhang Departments of Pathology and Immunology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA Ji-Hu Zhang Lead Discovery Center, Novartis Institute for Biomedical Research, Cambridge, MA, USA Lin Zhang Biogen Idec, San Diego, CA, USA Lin Zhang Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Ruiwen Zhang University of Alabama at Birmingham, Birmingham, AL, USA Shiwu Zhang Department of Pathology, Tianjin Cancer Hospital and Tianjin Cancer Institute, Tianjin, People’s Republic of China Yong Zhang Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

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Xin A. Zhang Departments of Medicine and Molecular Sciences, Vascular Biology Center, Cancer Institute, University of Tennessee Health Science Center, Memphis, TN, USA Xuefeng Zhang Duke Pathology, Duke University School of Medicine, Durham, NC, USA Yu-Wen Zhang Department of Oncology, Georgetown University Medical Center, Washington, DC, USA Yuesheng Zhang Roswell Park Cancer Institute, Buffalo, NY, USA Liang Zhong Le Centre de recherche du CHU de Québec-Université Laval: axe Oncologie, Le Centre de recherche sur le cancer de l’Université Laval, Québec, QC, Canada Guang-Biao Zhou State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Jerry Zhou School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW, Australia Zeng B. Zhu Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology and the Gene Therapy Center, Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, USA M. Zigler Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Margot Zoeller DKFZ, Heidelberg, Germany Massimo Zollo Department of Molecular Medicine and Medical Biotechnology, University Federico II of Naples, Naples, Italy Roberto T. Zori University of Florida, Gainesville, FL, USA Enrique Zudaire NCI Angiogenesis Core Facility, National Cancer Institute, National Institutes of Health, Advanced Technology Center, Gaithersburg, MD, USA Carsten Zwick Klinik für Innere Medizin I, Universität des Saarlandes, Homburg, Germany

Contributors

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284461-73-0 ▶ Sorafenib

85622-93-1 ▶ Temozolomide

conserved in evolution, is distributed intracellularly in many cells and also extracellularly on vascular cells, shares an epitope with motilityrelated proteins (alpha-actinin and a fast twitch skeletal muscle protein), and contains potential heparin binding and thrombin cleavage sites. Antibody and antisense studies have indicated compartment (intracellular or extracellular) specific roles for AAMP in angiogenesis, cell-cell and cell-matrix interactions, and cell migration.

17-1A ▶ EpCAM

A Disintegrin and Metalloprotease ▶ ADAM Molecules

AAMP Marie E. Beckner Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Definition Angio-associated migratory cell protein; gene maps to chromosome 2q35. AAMP has been # Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3

Characteristics The cDNA derived from mRNA encoding AAMP was originally cloned from a human melanoma cell library (A2058) in a search for migrationrelated proteins. AAMP has been found in the cytoplasm of many nucleated cells, in an extracellular mesh-like network on monolayers of endothelial and vascular-associated smooth muscle cells, and on the apical membranes of endometrial glandular cells. AAMP expression when normalized for tissue source has shown the highest levels of distribution in the esophagus (7.17% of tissue clones) (http://smd.stanford.edu/cgi-bin/source/ sourceImage?File = Hs.83347). Local homologies discovered initially to human immunodeficiency viral proteins led to identification of two immunoglobulin-like domains in AAMP. In addition to melanoma, expression of AAMP has been observed in a variety of malignant cells, including poorly differentiated colon adenocarcinoma

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within lymphatics, gastric adenocarcinoma, Jurkat lymphoma, gastrointestinal stromal tumors with mutated c-kit, breast cancer cell lines and ductal adenocarcinoma in situ with necrosis, and brain tumor cells. Co-culture of astrocytes with endothelial cells (without physical contact) led to increased amounts of extracellular AAMP associated with the endothelial cells. Stimulation of T lymphocytes and monocytes by a phorbol ester led to greatly increased AAMP expression, 1.6 kb message, and 52 kDa protein. Hypoxia increased expression of the AAMP gene in a breast carcinoma cell line. AAMP has demonstrated compartment-specific effects on endothelial cell migration. Affinitypurified antibodies, which interacted with the extracellular form of AAMP on nonpermeabilized endothelial cells, inhibited cell migration and endothelial tube formation. However, antisense oligonucleotides, which decreased total AAMP expression, paradoxically increased cell migration, presumably via loss of intracellular AAMP. The structure of AAMP was initially characterized as having two immunoglobulin-like domains and six WD repeats. Now eight WD repeats have been identified in AAMP, UniProt KB/Swiss-Prot Q13685. AAMP has been conserved in evolution. Comparisons of reference sequences for human AAMP (433 aa) with related forms in mouse (434 aa), rat (471 aa), chicken (419 aa), frog (438 aa), and zebrafish (408 aa) have shown 99.5, 98.9, 86.7, 76.5, and 69.0% identity, respectively (UniGene, NCBI, NIH). An acid box (short contiguous run of glutamic or aspartic acid residues) has been identified in the amino terminal regions of several AAMP homologs. They are comprised of seven glutamic acids in human, eight glutamic acids in mouse and rat, and six aspartic acid residues in the zebrafish forms of AAMP. AAMP contains a strongly immunoreactive ESESES epitope at its amino terminal end that has been used to generate an antipeptide antibody. Under normal reducing conditions, the epitope is immunoreactive for AAMP only in lysates of human brain and activated T lymphocytes. AAMP (52 kDa) shares this epitope with nonskeletal alpha-actinin (100 kDa) and an unidentified fast twitch skeletal muscle fiber

AAMP

protein (23 kDa), as demonstrated with antiRRLRRMESESES (anti-P189) and related antipeptide antibodies. The ESESES epitope is linear in AAMP but is discontinuous or conformational (formed by secondary structure) in alpha-actinin. The fast twitch skeletal muscle fiber protein with immunoreactivity for antiP189 was found in the periodic bands (Z discs). An alternatively spliced, slightly longer form of AAMP (452 aa) includes coding sequence upstream from MESESES. The immediate upstream sequence, RRLRR, potentially functions as a heparin binding site. In addition to an alternative initiating methionine, the upstream human coding sequence differs by only two of 17 codons when compared to an even longer form of AAMP in rat. The coding sequence of AAMP in rat includes the sequence GRFRRMESESES that corresponds to RRLRRMESESES in the alternative form of human AAMP. In peptide studies, the bipolar RRLRRMESESES sequence was strongly self-aggregating, sensitive to thrombin digestion, and displayed binding to heparin and cells as either an immobilized, single peptide or as an aggregated peptide, without affecting cell viability or adhesion to collagen. Peptide sequencing verified the presence of RLRR in recombinant AAMP translated in Escherichia coli following thrombin digestion that cleaved the first R. Although anti-P189 (RRLRRMESESES) did not demonstrate reactivity with the RRLRR epitope in tissue that displayed reactivity with ESESES, the lack of reactivity for RRLRR could have been due to interference by strongly adherent glycosaminoglycans. Thus initial studies of AAMP’s distribution and structure are supportive of a role for this protein in cell migration and angiogenesis.

References Adeyinka A, Emberley E, Niu Y et al (2002) Analysis of gene expression in ductal carcinoma in situ of the breast. Clin Cancer Res 8:3788–3795 Allander SV, Nupponen NN, Ringner M et al (2001) Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res 61:8624–8628 Beckner ME, Krutzsch HC, Stracke ML et al (1995) Identification of a new immunoglobulin superfamily protein

AAV expressed in blood vessels with a heparin-binding consensus sequence. Cancer Res 55:2140–2149 Beckner ME, Krutzsch HC, Klipstein S et al (1996) AAMP, a newly identified protein, shares a common epitope with alpha-actinin and a fast skeletal muscle fiber protein. Exp Cell Res 225:306–314 Beckner ME, Jagannathan S, Peterson VA (2002) Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture. Microvasc Res 63:259–269

See Also (2012) Alpha-Actinin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 143. doi:10.1007/978-3-642-16483-5_203 (2012) Amino Terminal End. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 156. doi:10.1007/978-3-642-16483-5_224 (2012) Domain. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1150. doi:10.1007/978-3-642-16483-5_1702 (2012) Epitope. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1297. doi:10.1007/978-3-642-16483-5_1966 (2012) Glycosaminoglycans. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2453 (2012) Phorbol Ester. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2865. doi:10.1007/978-3-642-16483-5_4522 (2012) Secondary Structure. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3348. doi: 10.1007/978-3-642-16483-5_5205 (2012) WD Repeats. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3945. doi:10.1007/978-3-642-16483-5_6233

AAPC ▶ APC Gene in Familial Adenomatous Polyposis

AAV Dirk Grimm BIOQUANT, Cluster of Excellence Cell Networks, University of Heidelberg, Heidelberg, Germany

Definition Adeno-associated viruses (AAV) are small DNA-containing viruses that belong to the family

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of Parvoviridae. Thus far, 11 serotypes of adenoassociated viruses (AAV-1 to AAV-11) have been cloned from humans and primates, and multiple further isolates were identified in various other species, including birds, bovines, mice, rats, and goats. According to current knowledge, none of these naturally occurring viruses are pathogenic in humans. AAV type 2 (AAV-2) has been studied for over 40 years and is the best characterized AAV isolate, hence its frequent referral as the AAV prototype. All AAV serotypes are currently being developed and evaluated as gene transfer vectors for the human ▶ gene therapy of various inherited or acquired diseases, including different types of cancer.

Characteristics As typical members of the Parvovirus family, AAV are characterized by nonenveloped, icosahedral capsids of about 18–24 nm in diameter. These capsids carry linear single-stranded DNA genomes of ~4.6–4.8 kb. The genomes of all known AAV serotypes have been cloned and sequenced. With the exception of AAV-4 and -5, which are distinct (>30%) from the other serotypes at both the nucleotide and amino acid level, all human and primate AAV genomes are related and highly homologous (>80%). Accordingly, their genomic structure and organization are also very similar. AAV Genome Structure As an example, the organization of the 4,681 nucleotide AAV-2 prototype genome is described (Fig. 1). The AAV-2 genome consists of two large open reading frames (orf), one at the left end encoding the nonstructural proteins (replication, rep orf), and one at the right end encoding the structural proteins (capsid, cap orf). In addition, a single intron sequence is found in the center of the genome, where the rep and cap orfs overlap. The AAV-2 rep gene encodes four closely related proteins (Rep proteins) with partially shared amino acid sequences. On the basis of their molecular weights, these proteins were designated Rep78, Rep68, Rep52, and Rep40. Unspliced and spliced

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AAV ITR

ITR p5

p19

p40 PolyA rep cap

AAV, Fig. 1 Structure of the AAV-2 genome. The 4,681 nucleotide single-stranded genome is depicted as a solid line; by convention, AAV genomes are drawn in 30 –50 orientation. Shown are the locations of the rep and cap orfs and the single intron (caret), as well as the position of

the three promoters (p5, p19, p40) and the polyA signal, which is used for polyadenylation of all AAV-2 transcripts. Further depicted at the ends of the genome are the palindromic inverted terminal repeat (ITR) sequences in their hairpin configuration

transcripts originating from a promoter located at map unit 5 (p5) are translated into the two large Rep proteins, Rep78 and Rep68. Rep52 and Rep40 are expressed from similarly spliced mRNAs that initiate from a second promoter, p19. The third AAV-2 promoter, p40, controls transcription of the cap gene. Translation of differentially spliced cap mRNAs results in expression of the three proteins that form the AAV-2 capsid: VP1, VP2, and VP3 (in a 1:1:10 ratio). The two viral genes are flanked by short (AAV-2: 145 nucleotides) inverted terminal repeats (ITR), palindromic sequences, that are able to fold into T-shaped stem loop structures. The ITRs are necessary and sufficient for replication and encapsidation of the viral genome during a productive infection of cells. Moreover, they are important for integration and rescue of the AAV DNA into, or from, the genome of latently infected cells, respectively. Thereby, the ITRs serve as minimal cis-acting sequences during the two different AAV life cycles (see also below).

including herpes simplex virus, vaccinia virus, and cytomegalovirus. In the case of adenovirus, one of the major helper functions is to stimulate AAV gene expression, by trans-activating the AAV-2 promoters. Additional help for the AAV life cycle is mediated at the posttranscriptional level, where adenoviral proteins and RNAs help to facilitate the cytoplasmic transport of AAV-2 mRNAs. Concurrently, adenoviral functions help to stabilize replicated AAV-2 genomic DNA later in the AAV infection. Notably, once expressed in the infected cell, AAV-2 Rep proteins subsequently further regulate and coordinate gene expression from the AAV promoters. They also play important roles for AAV DNA replication, as well as for packaging of viral genomes into empty new capsids (assembled from AAV-2 VP proteins). To mediate these diverse functions, Rep proteins bind to the AAV-2 ITRs and to sequences located in the AAV-2 promoters. They also interact with various cellular proteins, e.g., the TATA-box binding protein (TBP), as well as with each other and the AAV-2 VP proteins. The final step in a productive AAV-2 infection is the helpervirus-mediated lysis of the infected cell. This results in cell death and release of both new AAV-2 and helpervirus particles. In contrast to this productive (or lytic) phase, AAV-2 can establish latency in the absence of any helpervirus. Rather than replicating, the AAV-2 DNA then integrates into the target cell genome, where it stably persists as a so-called provirus. Important to point out, wildtype AAV-2 integration is not random, as is the case for retroviruses

AAV Life Cycles AAV serotypes belong to the Parvovirus genus Dependovirus, indicative of their dependence on an unrelated helpervirus to undergo a productive infection of cells. In fact, AAV genomes can only express their genes, replicate, and become encapsidated if the cell is simultaneously coinfected by one of these helperviruses. The typical helpervirus for AAV-2 is human ▶ adenovirus type 2 or 5, but many other human viruses can also provide full or partial helper functions,

AAV

(▶ Retroviral Insertional Mutagenesis) and other integrating viruses. Instead, it is targeted to a specific region on the long arm of human chromosome 19 (19q13.3-ter). The large Rep proteins (albeit only weakly expressed in the absence of a helpervirus) mediate this site-specific integration through binding to the AAV-2 ITRs, as well as to homologous sequences (AAVS1) located in chromosome 19. However, if a latently AAV-infected cell is later superinfected with a helpervirus, AAV-2 gene expression is induced and the AAV-2 genome is rescued from its integrated state. From this point on, a typical productive AAV-2 infection will occur. Thus, the helpervirus can act as an efficient switch between the two different phases that characterize the AAV-2 life cycle, lytic and latent. Clinical Relevance In theory, due to its inherent antitumor properties (see below), wildtype AAV-2 (and probably other serotypes alike) could be used as a therapeutic agent for the treatment of human cancers. However, more widely studied and applied are recombinant vectors derived from wildtype AAVs. Typically, these vectors are generated by replacing the two viral genes (rep and cap) with a foreign gene expression cassette, encoding RNAs or proteins that mediate an antitumor effect (if used for cancer therapy). The general clinical relevance of wildtype and recombinant AAVs is briefly discussed below; for more depth, the reader is referred to excellent reviews on the use of AAV for the treatment of human disease (see “References” below). Are Wildtype AAVs Pathogenic in Humans? According to the bulk data available, wildtype AAV serotypes are believed to be nonpathogenic in humans. In fact, despite estimates that up to 80% of adults are seropositive for AAV-2, no human disease has ever been causally linked to infection with the wildtype virus. This is even more remarkable considering that AAV-2 can infect a large variety of cells from diverse organs and tissues. Yet, although without gross pathological consequences for the cell, a latent AAV-2 infection can induce subtle changes in the cell

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phenotype. Examples are an increased ability to respond to stress factors, or a perturbation of the cell cycle, resulting in retarded cell growth. Most probably, these various effects are mediated by the large AAV-2 Rep proteins, even at the low expression levels typical for the latent stage. Is There a Natural Connection Between AAV Infection and Cancer? One frequently reported observation is that AAV-2-infected cells exhibit an increased resistance to ▶ oncogene- or tumorvirus-induced transformation. It is moreover known that AAV-2 infection can inhibit the proliferation of cultured cells derived from human cancers, e.g., melanomas. Cumulatively, these data strongly suggest that wildtype AAV-2 is not only nonpathogenic, but in fact has oncosuppressive properties. Moreover, certain human cancer cell lines become more sensitive to gamma irradiation (▶ Ionizing Radiation Therapy) and chemotherapeutic drugs (▶ Chemotherapy) upon experimental infection with wildtype AAV-2, as compared to noninfected controls. From a clinical point of view, these findings are of particular interest, since a major limitation of cancer chemotherapies is increasing resistance of transformed cells towards the drugs used. The observations of AAV-2-mediated cell sensitization therefore suggest that wildtype AAV might help to improve cancer chemotherapy, when applied in combination with conventional drugs. What Are Recombinant AAV Vectors? Recombinant AAV (rAAV) vectors are derivatives of wildtype AAV which lack the rep and cap genes, and instead carry a foreign gene expression cassette inserted between the two viral ITRs. By definition, AAV vectors are thus “gutless” or “gutted” (i.e., devoid of any viral genes). The generation of rAAV vectors is technically feasible and simple, due to the wide availability of molecular clones of the various wildtype viruses. These clones are easily modified using standard molecular laboratory techniques. Particularly beneficial is that wildtype and recombinant AAV are very small as compared to all other viruses developed as vectors, which aids in their

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experimental manipulation. Except for the replacement of the wildtype genes with a recombinant DNA, AAV vectors are identical in structure and organization to wildtype viruses and thus also function alike. In fact, AAV vectors will infect the target cell via the same molecular and cellular pathways as the wildtype virus. Ultimately, this will lead to expression of the encapsidated recombinant gene in the cell and thus to the intended therapeutic effect. As gene transfer vehicles, AAV vectors hold enormous promise for therapeutic intervention for a multitude of human acquired or innate genetic diseases, including cancer. Is AAV Unique as a Human Gene Therapy Vector? AAV vectors possess a multitude of advantages over all other virus-derived gene transfer vectors currently in (pre-)clinical development. One asset already mentioned is the lack of pathogenicity of the wildtype virus, which is in stark contrast, e.g., to adenovirus, another commonly used virus for gene therapy. Consequently, the production and handling of AAV vectors requires the lowest biosafety levels (S1, i.e., causing minimal risks for humans and the environment). The safety of AAV vectors is further increased by their “gutted” nature, precluding the expression of viral gene products which could cause cellular immune responses in the treated patient (a frequent adverse reaction to adenoviral vectors). A third unique asset, and a further difference to other viral vectors, is the availability of a wide spectrum of human, mammalian, and nonmammalian natural serotypes. These isolates typically differ in their tropism, i.e., the range of cells and tissues they can infect. Fortunately, it is technically very simple to generate recombinant AAV vectors which carry the same expression cassette, but differ in the viral capsid. This process is called “pseudotyping” and allows for the targeted delivery of a given recombinant DNA to virtually any desired cell or tissue, provided it can be infected by a known wildtype AAV (or a mutant thereof, see below). A plethora of reports have already demonstrated the power of this approach, to use AAV vectors for therapeutic and specific gene transfer to all clinically relevant

AAV

target organs, including liver, muscle, lung, eye, and brain. Last but not least, AAV vectors also differ from all other viral vectors by their capability to mediate persistent and long-term gene expression, both in actively dividing and in quiescent (i.e., nondividing) cells, and most importantly, without integrating into the host chromosome. Instead, the vector forms stable but extra-chromosomal DNA molecules, which are not capable of perturbing chromosome structures and thus do not pose a mutational risk. This is clinically most pertinent, as many gene therapy applications will require stable gene expression, ideally for the life-span of the patient. The only other viral vectors able to mediate long-term gene expression (and in nondividing cells) are derived from retroviruses or lentiviruses (HIV). However, these vectors are associated with drastically higher concerns about biosafety, due to the inherent pathogenic nature of the parental wildtype virus as well as due to their propensity for integration into the human genome. The latter can readily result in insertional mutagenesis, i.e., activation of endogenous oncogenes, or vice versa, inactivation of ▶ tumor suppressor genes. In both cases, the result is malignant transformation of the infected cell. This potentially serious adverse event from the use of retroviral vectors has indeed been observed in a clinical study, where multiple children developed leukemias, and some even died. Likewise, adenoviral vectors and the associated immune response have been blamed for the death of a patient in an early gene therapy trial in 1999. In striking contrast, thus far, none of the over 30 clinical trials using AAV vectors has yielded any evidence for a tumorigenic or lethal potential of this particular vector system. What Are Advances in AAV Vector Technology? In the early years, AAV vectors have been criticized for their small size (preventing packaging and therapeutic transfer of recombinant DNA >5 kb in length), their relatively slow transduction kinetics (resulting from the single-stranded DNA genome and its need for conversion into a transcriptionally active DNA duplex), and their

AAV

restricted cell and tissue tropism (based on the sole availability of the AAV-2 capsid in the early phase of AAV vector development). Nonetheless, even with those presumed limitations, AAV-2 vectors have been tested successfully in various large animal models and in human patients, addressing diverse diseases such as cystic fibrosis or hemophilia B. Most importantly, all three initial limitations of the AAV vector system have now been overcome, leading to the rapid expansion of AAV-based human gene therapy, especially for cancer treatment. First of all, the issue of limited packaging capacity has been solved with the creation of “split” AAV vectors which exploit the virus’ natural propensity for concatamerization. In an infected cell, rAAV genomes frequently recombine with each other, resulting in large “head-to-tail” concatamers (i.e., multiple copies of an rAAV genome in the same orientation). This can be exploited experimentally by splitting a large recombinant DNA (e.g., a gene and its promoter) into two halves, each of which is then delivered by a separate rAAV vector. This strategy effectively doubles the packaging limit of AAV vectors to up to 10 kb, which is sufficient even for large DNAs such as the factor VIII gene (encoding a blood clotting factor missing or defect in hemophilia A patients). Secondly, the inherently slow transduction kinetics of AAV have been overcome with the development of selfcomplementary or double-stranded vectors. In these, two copies of a foreign gene expression cassette are cloned and packaged in an inverted format, only separated by a minimal version of an AAV ITR. In the transduced cell, these two inverted copies then rapidly anneal with each other without the need for conversion into a duplex AAV DNA molecule. This results in an extremely rapid onset as well as maximum efficacy of gene expression, both far superior to what is obtained with conventional single-stranded AAV vectors, or most other viral vector systems. Thirdly, the limited host range of AAV-2 was readily overcome with the engineering of the over 100 alternative naturally occurring AAV serotypes as vectors. This approach has not only substantially broadened the range of cells and tissues that can now be infected with AAV

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vectors, but it has also alleviated concerns over the prevalence of neutralizing antibodies against the AAV-2 prototype in the human population. In fact, a wealth of studies have shown that AAV vectors derived from non-type-2 serotypes are functional in many tissues that are refractory to AAV-2 infection, and most importantly, transduction readily occurred in the (experimentally induced) presence of anti-AAV-2 antibodies, mimicking the situation in most humans. Moreover, very recent work demonstrated the feasibility to create synthetic AAV capsids which are further unique from the AAV-2 prototype, as well as from any of the naturally occurring isolates. Multiple strategies are currently being pursued, including the random mutagenesis of the AAV(2) cap gene, the insertion of peptide pools into exposed regions of the AAV-2 capsid (hoping the peptides will mediate re-targeting to unknown cellular receptors), or the creation of libraries of “shuffled” viruses, in which capsid genes from several parental viruses are mixed and recombined. Most importantly, all of these new approaches and designs remain fully compatible with already established AAV vector technology, allowing for their rapid and straight-forward preclinical evaluation. In fact, current AAV vector production methodologies are highly advanced and permit the generation of high titer stocks (>1  1014 recombinant particles per batch) in a very short amount of time (~10 days) (Fig. 2). As a result, AAV vectors have entered clinical evaluation and are currently being studied in about 30 ongoing trials in human patients. What Are Clinically Relevant rAAV Applications in Cancer Treatment? The sum of assets described above – safety, versatility, efficacy, and specificity – makes AAV an ideal vector for multiple and diverse therapeutic applications in humans. With particular respect to cancer, the use of AAV vectors is still in its infancy, but increasing preclinical data suggest that this vector system holds enormous potential also for this specific application. Thus far, the approaches can be divided into strategies that either target the tumor cell directly or that modify host mechanisms. In more detail, AAV vectors

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AAV

vectors and thus be used to effectively and specifically suppress, for instance, expression of cellular or virally encoded oncogenes. RNAi will likely become a valuable and crucial aspect of AAV-based cancer therapy in the near future and will complement or perhaps even replace many of the currently existing strategies.

Time required (days) 1

Seeding of cells Co-transfection of cells with 2 plasmids:

1 Foreign gene

rep

cap

2

Inclubation of cells

1

Harvesting of rAAV (freeze-thaw cycles)

1−2

Purification of rAAV (density gradient centrifugation, affinity chromatography)

1−3

Quantification of rAAV (various methods)

Ad

AAV, Fig. 2 Streamlined protocol for rAAV production. Cultured cells are transfected with two plasmids: the vector plasmid containing the foreign gene to be packaged into the viral particles, flanked by the AAV-2 ITRs, and the helper plasmid carrying the AAV-2 rep and cap genes to supply the Rep and VP proteins, respectively. In addition, the helper contains all adenoviral (Ad) genes which encode proteins with supportive function for AAV vector production, but it does not yield adenovirus after transfection. Helpervirus infection is thus superfluous, and the resulting AAV-2 vectors are free of contaminating adenovirus. Following a 2-day incubation of the transfected cells, the rAAV particles are harvested, purified, and quantified. Note that there are numerous modifications to this basic protocol, e.g., in the number of plasmids (1–3, depending on the arrangement of AAV and adenoviral sequences)

have been employed in the following major categories: Anti-angiogenesis, ▶ immunotherapy, tumor suppressors, suicide gene therapy, drug resistance, repair strategies, and, last but not least, purging of tumor cells. For many of those categories, a currently emerging therapeutic modality which is also still in its infancy is RNA interference or RNAi (▶ RNA interference). This term describes the natural phenomenon of gene silencing mediated by short double-stranded RNAs. The latter can be expressed from AAV

Anti-Angiogenesis The efficacy of ▶ angiogenesis inhibitors to undermine tumor neovascularization and to block cancer progression as well as formation of metastases (▶ metastasis) has been established in many animal models. However, this cancer therapy requires that the inhibitors are chronically administered as recombinant proteins, which is usually associated with severe problems. Therefore, AAV vectors with their unique ability to mediate sustained gene expression should prove particularly useful for this type of tumor therapy. Especially promising will be the future combination with synthetic AAV capsids that have been evolved to target the vasculature. Thus far, mostly AAV-2-based vectors have been used to deliver and express various anti-angiogenesis factors in small animals, typically mice. A first important example is angiostatin, which has been expressed from AAV-2 in multiple mouse models of human cancers, including gliomas (Glioblastoma Multiforme) and liver cancers (▶ Hepatocellular Carcinoma Molecular Biology). In all reported cases, this led to suppression of in vivo tumor growth and to substantial improvements in tumor-free survival rates. Similarly impressive are results with the related anti-angiogenic peptide ▶ endostatin, whose expression from AAV-2 vectors inhibited the establishment or growth of various human cancers in mice, including liver, ovarian (▶ Ovarian Cancer), pancreatic, and colorectal (Colon Cancer) tumors. Even better results have been obtained with the co-expression of both angiostatin and endostatin from a single or from two separate AAV vectors, exemplifying the potential for synergistic effects from combinatorial AAV therapies. Other examples for antiangiogenic AAV therapies already evaluated include the expression of a truncated form of the ▶ vascular endothelial growth factor receptor

AAV

(renal tumors), or of tissue inhibitors of ▶ matrix metalloproteases. Immunotherapy Failure of the immune system to recognize cancer antigens can substantially contribute to tumor manifestation and progression. Although tumors can illicit strong immune responses in the early stages, this effect is frequently lost in later phases, eventually allowing for aggressive and metastatic tumor growth. Gene transfer protocols involving AAV (or other viral) vectors have thus been developed which aim to potentiate the patient’s antitumor responses, by either targeting the tumor cells directly or by transducing hostderived immune effector cells. Examples for already reported tumor cell-directed therapies include AAV-mediated delivery of interferon genes to ex vivo cultured cancer cells or via intra-tumoral injection (gliomas). Likewise, AAV-2 has been used to express tumor necrosis factor-related ▶ apoptosis-inducing ligand (TRAIL) in colorectal, lung, and liver tumor models, resulting in significantly inhibited tumor growth and, in some cases, even in regression. Targeting cells of the host immune system, on the other hand, is a promising alternative approach and could eventually be developed into a vaccination therapy. Already, AAV-2 vectors have been used to deliver dominant tumor epitopes to antigen-presenting cells, such as CD40 ligand which was expressed in B-cells from ▶ chronic lymphocytic leukemia (CLL) patients, leading to specific proliferation of ▶ HLA Class I-matched allogeneic T-cells. Another potential vaccine could be AAV vectors expressing a HPV16 E7 CTL (cytotoxic T cell) epitope/heat shock fusion protein, based on reports that infected mice became immunized against E7-expressing tumor cells. Last but not least, encouraging studies have identified ▶ dendritic cells (DC), the most potent antigen-presenting cells, as an attractive target for AAV-based cancer immunotherapies. For instance, DCs transduced with AAV vectors encoding HPV16 E6 or E7 genes caused a stark CTL response against cervical cancer cell lines, while in another study, DCs transduced with CD80-expressing AAVs induced high levels of

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CD8+ T-cells. Together, these findings suggest that AAV can be used to trigger strong antitumor CTL responses, and that AAV-based immunotherapy has substantial clinical potential for cancer treatment. Tumor Suppressors Highly attractive targets for AAV-mediated cancer therapy are oncogenes and tumor suppressor genes, respectively, whose expression is frequently dysregulated in malignant human cancers. An important example for a tumor suppressor involved in cellular checkpoint control is p53 (p53 Protein, Biological and Clinical Aspects), which normally prevents passage of cells with DNA damage through the cell cycle. Consequently, expression of p53 from AAV vectors was consistently found to block the growth of cancer cells in vitro and in vivo and to mediate apoptosis and cytotoxicity. Similar results were obtained after expression of the fragile histidine triad tumor suppressor (FHIT), which delayed the growth of human pancreatic tumor xenografts and extended long-term animal survival. In a third example, delivery of the gene encoding the monocyte chemoattractant protein MCP-1 from AAV vectors suppressed expression of the HPV E6 and E7 proteins in cervical cancer cell lines as well as in tumors derived from these cells. Suicide Gene Therapy This approach is based on the idea to bioactivate a pro-drug within tumor cells to a toxic species, triggered by the tumor-directed delivery of the activating enzyme from AAV vectors. The best studied example for this category is the Herpes simplex virus-encoded enzyme thymidine kinase (tk) in combination with gancyclovir. This system has already been used successfully from AAV vectors to inhibit tumor growth in a variety of human xenograft models, including liver cancer, gliomas, and oral squamous carcinomas. Notably, the specificity of this approach can be enhanced by the use of tissue- and/or tumor-specific promoters, such as those only active in liver or melanoma cells. Moreover, the overall efficacy of the AAV/tk vectors was shown to increase following

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treatment of transduced cells with irradiation or topoisomerase inhibitors, both known to enhance AAV infection (in addition to their direct effects on cells). Drug Resistance Development of multiple drug resistance (MDR) is a major issue with cancer chemotherapies and is often associated with over-expression of the ▶ P-glycoprotein (an ATPase that pumps chemotherapeutic drugs out of the cancer cell). One reported, highly effective approach to reverse the MDR phenotype is to use double-stranded AAV vectors to express anti-P-glycoprotein short hairpin RNAs (effectors of RNAi). In human ▶ breast cancer and oral cancer cells, this led to a substantial sensitization to chemotherapy, suggesting a high potential to overcome the MDR obstacle with this approach. Another application is expression of the MDR1 gene from AAV vectors in hematopoietic progenitors. This should confer myeloprotection in patients undergoing high-dose chemotherapy for advanced tumors and thus prevent myelosuppressive effects (▶ Myelosuppression) from the chemotherapeutic regimen, such as infection or hemorrhaging. However, this strategy has not been fully explored in animal models yet. Repair Strategies ▶ Telomerase (the enzyme maintaining and stabilizing the integrity of telomeres, i.e., chromosome ends) is an example for a therapeutically relevant target for repair strategies. Its activity is often elevated in tumor cells, and it was shown that delivery of telomerase antisense molecules [Antisense DNA Therapy] via AAV vectors (in this particular case hybrids with adenoviral vectors) can reduce tumor cell proliferation as well as induce apoptosis. Purging of Tumor Cells from Autologous Transplants Autologous grafts (▶ Graft Acceptance and Rejection), e.g., peripheral blood progenitor cells, are used for treatment of many solid human cancers. However, they can be contaminated with tumor cells that give rise to relapse

AAV

after ▶ myeloablative megatherapy and graft transplantation. There is evidence that following infection of such contaminated grafts with recombinant AAV-2, the contaminating tumor cells are preferentially infected, while the hematopoietic progenitors are spared. Indeed, infection of sarcoma cells with AAV/tk vectors (see above) extended the survival of transplanted mice (over nontreated controls), while the same vector was unable to transduce and kill human peripheral blood progenitors. However, it remains to be proven that this strategy can indeed be applied to selectively purge tumor cells from autologous transplants. RNAi RNA-mediated silencing of gene expression (RNAi) will clearly become a major part of antitumor therapies in the future, as proof-ofconcept for the efficacy of this approach is already overwhelming. In combination with AAV, there have only been a few reports thus far, but this field will certainly expand. One described application is to use AAV vectors to deliver short hairpin RNAs against the hec1 gene, which is highly expressed in mitotic cells where it represents a vital component of the kinetochore outer plate. Transduction of glioma cells with anti-hec1 AAV vectors resulted in selective cell death, while mitotically inactive control cells were unaffected. Likewise, infected xenografts showed lower densities and were highly fibrotic as a result of AAV treatment. It can generally be predicted that virtually any over-expressed gene that contributes to transformation can be an AAV/RNAi target, including virally encoded (see above, e.g., HPV E6/7) or cellular oncogenes. Future Applications With the current state-of-the-art technology, the AAV vector system is already one of the most powerful and promising toolkits for development as antitumor bioreagents. In the future, the versatility of this system will further increase with the discovery and creation of new natural or synthetic capsids, respectively. Likewise, the field will benefit from the engineering of novel tumor- and tissue-specific gene expression cassettes, and

AAV

from the design of safer and more effective therapeutic sequences, e.g., for the induction of anticancer RNAi. A very important approach will be to merge the different strategies into combinatorial therapies, e.g., by mixing immunotherapies with RNAi vectors or suicide gene expression with repair approaches. Examples for such multimodality cancer therapies with AAV vectors have already been reported, and their numbers will increase in the future. Last but not least, it will also be crucial to combine AAV (or other viral) vectors with further anticancer effectors, such as new classes of compounds including proteasome (Proteasomal Inhibitors) and histone deacetylase inhibitors.

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References Grimm D (2002) Production methods for gene transfer vectors based on adeno-associated virus serotypes. Methods 28:146–157 Grimm D, Kay MA (2004) From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr Gene Ther 3:281–304 Grimm D, Pandey K, Kay MA (2005) Adeno-associated virus vectors for short hairpin RNA expression. Methods Enzymol 392:381–405 Li C, Bowles DE, van Dyke T et al (2005) Adenoassociated virus vectors: potential applications for cancer gene therapy. Cancer Gene Ther 12:913–925 Warrington KH, Herzog RW (2006) Treatment of human disease by adeno-associated viral gene transfer. Hum Genet 119:571–603

See Also

Cross-References ▶ Adenovirus ▶ Angiogenesis ▶ Apoptosis ▶ Breast Cancer ▶ Chemotherapy ▶ Chronic Lymphocytic Leukemia ▶ Colorectal Cancer ▶ Dendritic Cells ▶ Endostatin ▶ Fragile Histidine Triad ▶ Gene Therapy ▶ Graft Acceptance and Rejection ▶ Hepatocellular Carcinoma Molecular Biology ▶ HLA Class I ▶ Immunotherapy ▶ Ionizing Radiation Therapy ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Myeloablative Megatherapy ▶ Myelosuppression ▶ Oncogene ▶ Ovarian Cancer ▶ P-Glycoprotein ▶ Retroviral Insertional Mutagenesis ▶ Telomerase ▶ TNF-Related Apoptosis-Inducing Ligand ▶ Tumor Suppressor Genes ▶ Vascular Endothelial Growth Factor

(2012) Concatamerization. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 965. doi:10.1007/978-3-642-16483-5_1296 (2012) FHIT. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1394. doi:10.1007/ 978-3-642-16483-5_2168 (2012) Gene Expression Cassette. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1522. doi:10.1007/978-3-642-16483-5_2366 (2012) Hematopoietic Progenitors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1645. doi:10.1007/978-3-642-16483-5_2618 (2012) Interferon. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1888. doi:10.1007/978-3-642-16483-5_3090 (2012) Kinetochore Outer Plate. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1944. doi:10.1007/978-3-642-16483-5_3225 (2012) Neovascularization. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2474. doi:10.1007/978-3-642-16483-5_4016 (2012) Open Reading Frame. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2642. doi:10.1007/978-3-642-16483-5_4241 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Parvovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2791. doi:10.1007/978-3-642-16483-5_4398 (2012) Phenotype. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2856. doi:10.1007/978-3-642-16483-5_4514 (2012) Promoter. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4768 (2012) Receptor for TNF-Related Apoptosis-Inducing Ligand. In: Schwab M (ed) Encyclopedia of Cancer,

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12 3rd edn. Springer Berlin Heidelberg, p 3198. doi:10.1007/978-3-642-16483-5_4981 (2012) Recombinant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3205. doi:10.1007/978-3-642-16483-5_4991 (2012) Seropositive. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3389. doi:10.1007/978-3-642-16483-5_5261 (2012) Serotypes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3389. doi:10.1007/978-3-642-16483-5_5263 (2012) TBP. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3620. doi:10.1007/978-3-642-16483-5_5694 (2012) Tropism. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3785. doi:10.1007/978-3-642-16483-5_5990 (2012) Vector. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3906. doi:10.1007/978-3-642-16483-5_6173 (2012) Xenograft. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3967. doi:10.1007/978-3-642-16483-5_6278

Ab (Latin: Away) -Scopus (Greek: Target) Effects ▶ Abscopal Effects

Ab (Latin: Away) -Scopus (Greek: Target) Effects

of transmembrane proteins that use ATP-derived energy to transport various substances over cell membranes. Primary-active transporters, driven by energy released from ATP by inherent ATPase activity, that export substrates from the cell against a chemical gradient. Based on the arrangement of the nucleotide-binding domain and the topology of its transmembrane domains, human ABC transporters are classified into seven distinct families (ABC-A to ABC-G), including ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (cMOAT, MRP2), ABCC4 (MRP4), and ABCG2 (ABCP, MXR, BCRP). Structural characteristics based on their Walker motif (ATP-binding domain) and their nucleotidebinding folds across the membrane are responsible for their classification into this superfamily. Their localization pattern over the body suggests that they have an important role in the prevention of absorption as well as the excretion of potentially toxic metabolites and xenobiotics, both on a systemic and a cellular level. ABC drug transporters (may) show substrate overlap. Examples of mammalian ABC transporters include ▶ P-glycoprotein, MRP (▶ multidrug resistance protein), ▶ cystic fibrosis transmembrane conductance regulator (CFTR), and transporter associated with antigen processing (TAP).

ABC (ATP-Binding Cassette) Superfamily ▶ ABC Drug-Transporters

ABC Drug-Transporters Synonyms ABC (ATP-binding cassette) superfamily; ABC transporter

Definition The adenosine triphosphate (ATP)-binding cassette (ABC) transporters form the largest family

Cross-References ▶ Cystic Fibrosis ▶ Fluoxetine ▶ Glutathione Conjugate Transporter RLIP76 ▶ Irinotecan ▶ Major Vault Protein ▶ Pharmacogenomics in Multidrug Resistance ▶ P-Glycoprotein

See Also (2012) Multidrug resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2393. doi:10.1007/978-3-642-16483-5_3887 (2012) Walker A Motif. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3941. doi:10.1007/978-3-642-16483-5_6228

ABCC Transporters

ABC Transporter ▶ ABC Drug-Transporters

ABCC Transporters Rodrigo Franco and Laura Zavala-Flores Redox Biology Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA

Synonyms ATP-binding-cassette transporters sub-family C; MRP; Multidrug resistance-associated proteins

Definition The ATP-binding cassette transporters from the sub-family C (encoded by ABCC genes) are plasma membrane ATP-dependent efflux transporters with broad substrate specificity for endogenous and xenobiotic anionic substances.

Characteristics Members and Functional Properties The human ABCC subfamily of transporters contains 13 members from the ATP binding cassette (ABC) superfamily with sizes from 1,325 to 1,545 amino acids. The ABCC subfamily includes the cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), two sulfonylurea receptors SUR1 (ABCC8) and SUR2A/B (ABCC9), and nine MRPs. ABCC proteins are energydependent transporters, except for CFTR which acts as channel gated by ATP binding and The entry “ABCC Transporters” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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hydrolysis, and SURs which act as ATP-dependent potassium channel regulators. All ABCC proteins share structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC proteins. CFTR 7 and MRPs 4, 5, 8, 9, and 10 (ABCC4, 5, 11, 12, and 13) have a typical ABC transporter structure with two polytropic membrane spanning domains (MSD1 and 2) containing six transmembrane a-helices and two nucleotide binding domains (NBD1 and 2). SURs and MRPs 1, 2, 3, 6, and 7 (ABCC1, 2, 3, 6, and 10) have an additional N-terminal MSD0 domain (Fig. 1). MSD1 and MSD2 domains form the translocation pathway by which substrates cross the plasma membrane. In humans, ABCC13 gene is incapable of encoding a functional transporter. A single polypeptide can encode all four of these domains (NH2-MSD-NBD-MSD-NBDCOOH) or functional transporters may be formed of homo- or heterodimer of polypeptides, each contributing an MSD and an NBD. The NBDs contain Walker A and B motifs essential for ATP binding and hydrolysis and a “C” signature motif that has the core sequence LSGGQ. Only MSD0 of SUR1 has been shown to have clear functional role by its interaction between Kir6.2 potassium channels (Chen and Tiwari 2011; Deeley et al. 2006). Multidrug Resistance-Associated Proteins (MRPs) MRP members are ATP-dependent efflux pumps with broad substrate specificity for the transport of endogenous and xenobiotic anionic substances. MRP proteins mediate the efflux of conjugates, often generated in phase II reactions of drug metabolism in the pathway of detoxification of many xenobiotics and some endogenous metabolites (Gillet and Gottesman 2010; Keppler 2011). MRP1 (ABCC1) – The MRP1 is present in many human cell types and tissues such as lung, testis, kidney, skeletal, blood–tissue barriers, and cardiac muscles, placenta, and macrophages, while normal human hepatocytes lack detectable amounts of MRP1. It localizes predominantly in the plasma membrane and selectively to the basolateral component in polarized cells. MRP1 is a high-affinity transporter for many

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ABCC Transporters

ABCC Transporters, Fig. 1 Domain organization of multidrug-resistance proteins ABCC Transporters, Fig. 2 MRPs in chemotherapy, redox homeostasis, and cell death

amphipathic organic anions including conjugates with glutathione (leukotriene C4 or LTC4) sulfate and/or glucuronate. MRP1 is found highly expressed in leukemias, esophageal carcinomas, and non–small cell lung cancer, which seems to correlate with clinical outcome. In tumor cells, MRP1 confers resistance to a wide variety of toxic agents such as doxorubicin, MTX, daunorubicin, vincristine, etoposide, and tyrosine kinase inhibitors. Glutathione (GSH) is an important

regulator of MRP1 transport. Four distinct mechanisms have been proposed for the efflux of organic anions in a GSH-dependent manner (Fig. 2). (a) Chemotherapeutic agents such as etoposide and Vinca alkaloids (vincristine) appears to be co-transported with GSH. (b) MRP1 has also been shown to mediate transport of etoposide and doxorubicin GSH-conjugates generated by the action of glutathione-S-transferases (GSTs). (c) Transport of the conjugates

ABCC Transporters

4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanolO-glucuronide and estrone sulfate and probably etoposide-glucuronide is enhanced by GSH, but GSH is not transported by MRP1. Interestingly, although the biological activity of GSH is attributed to the reducing capacity of the cysteine sulfhydryl moiety, efflux transport by MRP1 can be stimulated in nonreducing GSH analogs (S-methyl GSH) and ophthalmic acid. (d) MRP1 has low affinity for GSH. However, the presence of xenobiotics such as verapamil and dietary flavonoids such as apigenin stimulate GSH transport without being transported themselves. (e) On the other hand oxidized glutathione (GSSG) is a physiological substrate for MRP1 with relatively high affinity compared to GSH, suggesting a protective role of MRP1 against oxidative stress by preventing the accumulation of GSSG. MRP2/CMOAT (ABCC2) – MRP2 is found in distinct tissues including liver, kidney, small intestine, colon, gallbladder, placenta, and lung. It is consistently found in apical membranes and its traffic requires the presence of the MSD0 domain. MRP2 contains a PDZ-domain located at its COOH terminus which suggests an interaction with scaffolding proteins that could target MRP2 to the F-actin cytoskeleton, but conflicting results have been described about this. The substrate specificities of MRP2 and MRP1 are similar. MRP2 transports LTC4 and mediates low-affinity transport of GSH and also of GSSG. MRP2 is found expressed in lung, gastric, renal, and colorectal tumor cell lines and in cells from patients with acute myelogenous leukemia. MRP2 is also expressed in kidney, colon, breast, lung, and ovary tumors. MRP2 transports a variety of anticancer drugs, including MTX, cisplatin, irinotecan, paclitaxel, and vincristine, and increased MRP2 levels are associated with resistance to cisplatin and doxorubicin. MRP3/CMOAT2 (ABCC3) – MRP3 is expressed in the adrenal gland, kidney, small intestine, colon, pancreas, gut, gall bladder, and placenta. MRP3 is also found at the basolateral membranes of polarized cells such as hepatocytes and cholangiocytes. MRP3-mediated transport does not require GSH and has a reduced capacity to transport GSH and GSH conjugates. It transports a variety of

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amphipathic anions including glucuronate conjugates. MRP3 is overexpressed in human hepatocellular carcinoma, primary ovarian cancer, and adult acute lymphoblastic leukemia cells, and it is also predicted to be a prognostic factor in acute lymphoblastic and myeloid leukemia. MRP3 transports etoposide, teniposide, and MTX. Interestingly, knock-out animal studies have demonstrated that Mrp2 and Mrp3 provide compensatory efflux pathways for etoposide glucuronide. MRP4/MOATB (ABCC4) – Except for prostate, MRP4 is present at low levels in normal tissues, and can be localized in both basolateral and apical membranes in polarized cells. MRP4 mediates the transport of endogenous metabolites including nucleoside and nucleotide analogs such as cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP), which are involved in signaling transduction. Although the affinity of MRP4 for cAMP and cGMP is low, it is proposed that MRP4 might be involved in regulating local microdomain levels of these signaling molecules. Eicosanoids such as prostaglandin E1 and E2 are also substrates of MRP4. MRP4 also transports GSH, sulfated bile acids, GSH-conjugated leukotriene B4 (LTB4), and LTC4. MRP4 has been implicated in the high proliferative growth of prostate tumors and neuroblastoma, and also confers resistance to anticancer agents including thiopurine analogs, MTX, and topotecan. MRP5/MOATC (ABCC5) – MRP5 is highly expressed in skeletal muscle and cardiac and cardiovascular myocytes. MRP5 is also found on the apical side of brain capillary endothelial cells and is also present in astrocytes and pyramidal neurons. In polarized epithelial cells, MRP5 is located to the basolateral membrane. MRP5 is also involved in the extrusion of cGMP and cAMP. It acts as a high-affinity transporter for cGMP and a low-affinity transporter of cAMP. MRP5 mediates the efflux of other organic anion molecules such as S-(2,4-dinitrophenyl) glutathione and GSH. Elevated levels of MRP5 are found in lung, colon, pancreatic, and breast cancer samples. Interestingly, exposure to cisplatin and doxorubicin increases MRP5 levels in non–small cell lung cancer cells. MRP5 confers resistance to cisplatin, purine analogs (such as

A

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6-mercaptopurine and 6-thioguanine), pyrimidine analogues such as (gemcitabine, cytosine arabinoside, and 5-fluorouracil), doxorubicin, and MTX, but not to vincristine. MRP6/MOATE (ABCC6) – MRP6 is expressed in the liver and kidney, and at low levels in most other tissues. Relatively high levels of MRP have been found in skin keratinocytes, intestinal mucosa, tracheal, bronchial and corneal epithelium, as well as endothelial and smooth muscle cells of the cardiovascular system. MRP6 mediates the transport of GSH-conjugated organic anions including LTC4-GSH and S-(2,4-dinitrophenyl) glutathione conjugates, but not glucuronated substrates or cyclic nucleotides. MRP6 confers resistance to etoposide, teniposide, doxorubicin, daunorubicin, actinomycin D, and cisplatin. MRP7 (ABCC10) – MRP7 mRNA is highly expressed in the colon, skin, and testes. MRP7 mediates the transport of glucuronate conjugates such as estradiol glucuronide and to a lesser extent GSH conjugates such as LTC4. MRP7 mediates resistance to docetaxel, paclitaxel, vincristine, and vinblastine in vitro, to nucleoside-based agents such as cytosine arabinoside and gemcitabine, and to the microtubule-stabilizing agent epothilone B. Significant levels of MRP7 expression have been detected in non–small cell lung cancer cells after exposure to paclitaxel or vinorelbine. MRP8 (ABCC11) – Conflicting reports exist regarding whether the expression of MRP8 is widespread or limited, being highest in the liver, brain, placenta, breasts, and testes. MRP8 transports a wide range of compounds, including cGMP and cAMP, lipophilic anions including glutathione conjugates such as LTC4 and S-(2,4-dinitrophenyl) glutathione, estradiol glucuronide, sulfate conjugates such as dehydroepiandrosterone 3-sulfate and estrone sulfate, glucuronidated steroids, and folic acid. Significant levels of MRP8 have been reported in breast cancer samples. MRP8 confers resistance to antimetabolites including such as 9-(2-phosphonylmethoxyethyl) adenine, MTX, cytosine arabinoside, and 5-fluorouracil. MRP8 is also significantly associated with low prognosis in acute myeloid leukemia patients.

ABCC Transporters

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7)

CFTR(ABCC7) belongs to the same family as MRPs. However, despite the structural similarity with MRPs, CFTR is a chloride-channel. Genetic variations in the CFTR gene have been associated with increased risk of lung cancer. Because ion channels are reported to regulate growth and proliferation of cancer cells, it is hypothesized that impaired CFTR might regulate the survival/ proliferative and/or cell death pathways of cancer cells, but no experimental evidence exists supporting this idea. GFTR has also been proposed to mediate GSH efflux but its role in cancer progression has not been studied (Li et al. 2010). Sulfonylurea Receptors SUR1 (ABCC8) and SUR2A/B (ABCC9)

ATP-sensitive potassium (KATP) channels are constituted by the association of four pore-forming Kir6.x subunits (Kir6.1 and Kir6.2) and four regulatory SUR subunits (SUR1, SUR2A, and SUR2B), which are present in excitable cells, where they couple membrane electrical properties to intracellular metabolism. SUR proteins are the site of action of numerous drugs that either close (blockers including sulfonylureas like glibenclamide) or open the Kir6.x potassium pore. SURs’ only known function is that of a channel regulator although they present strong sequence homologies with other ABC transporters. Glibenclamide has been reported to exert antitumor activity in human gastric cancer cells by inducing oxidative stress and programmed cell death (Qian et al. 2008). Glutathione Transport, Redox Signaling, and Apoptosis Regulation of GSH/GSH-conjugate transport is of great relevance for both the carcinogenic process and antitumorigenic therapies. When antineoplastic or chemotherapeutic drugs enter cancer cells, they are conjugated to glutathione and are excreted through GSH-pumps of the MRP family of transporters (Fig. 2). Increased expression of g-glutamylcysteine ligase that mediates de novo GSH synthesis is found in cancer cells. Thus,

ABC-Transporters

depletion of intracellular GSH can be used to impair multidrug resistance of transformed cells. In addition, GSH homeostasis is an important regulator of apoptosis or programmed cell death. GSH depletion is a hallmark of the progression of cell death and GSH efflux contributes not only to GSH depletion but also to oxidative stress, redox signaling, and the activation of cell death pathways (apoptosis). However, the molecular identity of the GSH-transporters involved in GSH depletion during apoptosis is unclear. As mentioned before, GSH is a poor substrate for MRPs, while GSSG is more efficiently transported by these transporters. Conflicting results exist regarding the role of MRP1 in GSH efflux during apoptosis. However, stimulation of MRP1-mediated GSH efflux sensitizes transformed cells to apoptotic cell death induced by both extrinsic (death receptormediated) and intrinsic (mitochondria-mediated) pathways and also overcomes the effect of antiapoptotic oncogenes such as Bcl-2 (Franco and Cidlowski 2009).

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ABC-Transporters Hermann Lage Institute of Pathology, Charité Campus Mitte, Berlin, Germany

Synonyms ATP-binding cassette-transporters; Multidrug resistance transporters; Traffic ATPases

Definition ABC (ATP-binding cassette)-transporters are membrane-embedded proteins with a characteristic ABC domain that utilize the energy from ATP hydrolysis for the transport of their substrates across a cellular membrane.

Characteristics References Chen ZS, Tiwari AK (2011) Multidrug resistance proteins (MRPs/ABCCs) in cancer chemotherapy and genetic diseases. FEBS J 278(18):3226–3245 Deeley RG, Westlake C, Cole SP (2006) Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 86(3):849–899 Franco R, Cidlowski JA (2009) Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ 16(10):1303–1314 Gillet JP, Gottesman MM (2010) Mechanisms of multidrug resistance in cancer. Methods Mol Biol 596:47–76 Keppler D (2011) Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy. Handb Exp Pharmacol 201:299–323 Li Y, Sun Z, Wu Y, Babovic-Vuksanovic D, Li Y, Cunningham JM, Pankratz VS, Yang P (2010) Cystic fibrosis transmembrane conductance regulator gene mutation and lung cancer risk. Lung Cancer 70(1):14–21 Qian X, Li J, Ding J, Wang Z, Duan L, Hu G (2008) Glibenclamide exerts an antitumor activity through reactive oxygen species-c-jun NH2-terminal kinase pathway in human gastric cancer cell line MGC-803. Biochem Pharmacol 76(12):1705–1715

The superfamily of ABC-transporters comprises one of the most abundant protein families in nature. These transporters are believed to date back in evolutionary time more than 3 billion years and are distributed in all three kingdoms of living organisms, archaea, eubacteria, and eukaryotes. Archaea are a unique group of microorganisms classified as bacteria (Archaeobacteria) but genetically and metabolically different from all other known bacteria. They appear to be living fossils, the survivors of an ancient group of organisms that bridged the gap in evolution between bacteria and the eukaryotes. ABC-transporters have to be distinguished from ABC-proteins. Both types of proteins are defined by the presence of a highly conserved ~215 amino acids consensus sequence designated as ABC domain or nucleotide-binding domain (NBD). The domain contains two short peptide motifs, a glycine-rich Walker A and a hydrophobic Walker B motif, both involved in ATP binding and commonly present in all nucleotide-binding proteins. A third consensus sequence is named

A

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ABC-Transporters

a

TMD Membrane

C

N NBD

b

TMD1

TMD2 Membrane

N

C NBD1

c

N TMD0

TMD1

NBD2 TMD2 Membrane C

NBD1

NBD2

ABC-Transporters, Fig. 1 Schematic representation of the predicted domain arrangement of (a) half-size transporters having only one TMD fused to one NBD (TMD-NBD), e.g., ABCG2 (BCRP); and (b, c) full-size transporters (TMD-NBD)2, whereby (b) shows the predicted structure of ABCB1 (MDR1), and (c) the structure of ABCC1 (MRP1) containing an additional TMD (TMD0) of unknown function. Half-size transporters

probably dimerize to form a biological active ABC-transporter. These three ABC-transporters are the most important drug extrusion pumps in multidrugresistant cancers. TMD transmembrane domain consisting of six a-helices, NBT nucleotide-binding domain. It should be noted that the orientation of ABCG2 is reverse to that of ABCB1 and ABCC1

ABC signature and is unique in ABC domains. ABC-containing proteins couple the phosphate bond energy of ATP hydrolysis to many cellular processes and are not necessarily restricted to transport functions. However, the proper meaning of the term ABC-transporter is satisfied when the ABC-protein is in addition associated with a hydrophobic, integral transmembrane domain (TMD) forming a translocation path. TMDs are usually composed of at least six transmembrane (TM) a-helices. They are believed to determine the specificity for the substrate molecules transported by the ABC-transporter. The minimal structural requirement for a biological active ABC-transporter seems to be two TMDs and two NBDs (TMD-NBD)2 (Fig. 1). In full-size transporters, this structural arrangement may be formed by a single polypeptide chain and in

multiprotein complexes by more than one polypeptide chain. In prokaryota, ABC transport systems are often half-size transporters having only one TMD fused to one NBD (TMD-NBD). Half-size transporters probably dimerize to form a full-size transporter (TMD-NBD)2 to mediate mainly the influx of essential compounds such as sugars, vitamins, and metal ions into the cell. Eukaryotic ABC-transporters commonly function as exporters mediating the efflux of compounds from the cytosol to the extracellular space or to the inside of intracellular membranebound compartments, i.e., endoplasmic reticulum, mitochondria, peroxisomes, or vacuoles. The range of physiologically transported compounds includes lipids and sterols, ions, diverse small molecules, oligopeptides, and polypeptides.

ABC-Transporters

19

ABC-Transporters, Table 1 Family of human ABC-transporters Subfamily ABCA

ABCB

ABCC

ABCD

ABCE ABCF

HUGOnomenclature ABCA1 ABCA2 ABCA3 ABCA4

Size (AA) 2,261 2,436 1,704 2,273

ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA12 ABCA13 ABCB1 ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11 ABCC1

MDR1, PGY1 TAP1 TAP2 MDR3, PGY3 MTABC3 ABC7 MABC1 TABL MTABC2 BSEP, SPGP MRP1, MRP

Location 9q31.1 9q34 16p13.3 1p22.1p21 17q24.3 17q24.3 19p13.3 17q24 17q24.2 17q24 2q34 7p12.3 7q21.1 6p21.3 6p21.3 7q21.1 7p15.3 2q36 Xq12-q13 7q36 12q24 1q42 2q24 16p13.1

ABCC2

MRP2, cMOAT

10q24

1,545

ABCC3 ABCC4 ABCC5 ABCC6 ABCC7 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12 ABCD1 ABCD2 ABCD3 ABCD4 ABCE1

MRP3 MRP4 MRP5 MRP6 CFTR SUR1 SUR2 MRP7 MRP8 MRP9 ALD, ALDP ALDL1, ALDR PXMP1, PMP70 PXMP1L, P70R RNASELI, OABP ABC50

17q22 13q32 3q27 16p13.1 7q31.2 11p15.1 12p12.1 6p21.1 16q12.1 16q12.1 Xq28 12q11-q12 1p22-p21 14q24.3 4q31

1,527 1,325 1,437 1,503 1,480 1,581 1,549 1,464 1,382 1,359 745 740 659 606 402

6p21.33 7q36 3q27.1

807 623 709

ABCF1 ABCF2 ABCF3

Common names ABC1 ABC2 ABC3, ABCC ABCR

ABCX

1,642 1,617 2,146 1,581 1,624 1,543 2,595 5,058 1,280 808 653 1,279 842 752 718 723/766 738 1,321 1,531

Function Cholesterol-, PS transport Surfactant production N-retinylidene-PE transport

MDR Peptide transport Peptide transport PC transport Iron transport Iron-, Sulfur- cluster transport

Bile salt transporter MDR, organic anion transporter MDR, organic anion transporter Organic anion transporter Organic anion transporter Organic anion transporter Chloride transport Regulation Regulation

FA-, FA AcylCoA transport FA-, FA AcylCoA transport FA-, FA AcylCoA transport FA-, FA AcylCoA transport

(continued)

A

20

ABC-Transporters

ABC-Transporters, Table 1 (continued) Subfamily ABCG

HUGOnomenclature ABCG1 ABCG2 ABCG4 ABCG5

Common names ABC8, White BCRP, MXR White2 White3

Location 21q22.3 4q22 11q23.3 2p21

Size (AA) 638 655 627 651

Function Cholesterol transport MDR Sterol transport

AA amino acids, FA fatty acids, MDR multidrug resistance, PC phosphatidylcholine, PE phosphatidylethanolamine, PS phosphatidylserine

Human ABC-Transporters In humans, 48 ABC-transporters distributed to seven subfamilies have been identified (Table 1). Although the number of human ABC-transporters is much smaller than found in bacteria, many of them are of clinical significance. Currently, 18 human genes encoding ABC-transporters have been associated with genetic diseases. Even though the majority of the members of the human ABC-transporter family are active transporters, there are some exceptions in which the energy of ATP hydrolysis is utilized to control alternative biological processes. Thus, ABCC7 (CFTR), well known as mutated in patients suffering on ▶ cystic fibrosis, appears as a chloride ion channel; ABCC8 (SUR1) and ABCC9 (SUR2) are both regulatory subunits of the regulatory sulfonylurea receptor (SUR). Other members of the ABC-transporter family couple ATP binding and hydrolysis to the control of translation or ▶ DNA repair. Although the active transporters have dedicated functions involving the transport of specific substrates, the complex physiological network of ABC-transporters may also have an important role in host ▶ detoxification and protection against ▶ xenobiotics. This general function is revealed by their tissue distribution. ABC-transporters are highly expressed in important pharmacological barriers, such as the epithelium that contributes to the blood–brain barrier (BBB), the brush border membrane of intestinal cells, the biliary canalicular membrane of hepatocytes, or the lumenal membrane in proximal tubules of the kidney. Anyway, this xenobiotics pump function is the basis for the pivotal role of ABC-transporters in multidrug resistance (MDR) of cancer.

ABC-Transporters and Multidrug Resistance of Cancer MDR is defined as the simultaneous resistance of a tumor against a variety of antineoplastic agents with different chemical structure and mode of action. Thus, MDR is a major obstacle in clinical management of cancer by ▶ chemotherapy. Although various mechanisms have been identified to mediate a multidrug-resistant phenotype to malignant diseases, the enhanced drug extrusion activity of the ABC-transporter ABCB1 or ▶ P-glycoprotein (MDR1; PGY1) was the first mechanism that was demonstrated to be the reason for MDR. The substrates of ABCB1 include first and foremost natural product-derived anticancer drugs, such as ▶ Anthracyclines, epipodophyllotoxins, taxans, and vinca alkaloids, but not clinically important drugs like platinumcontaining compounds or antimetabolites. Besides ABCB1, in particular, ABCC1 (MRP1) and ABCG2 (BCRP) were found to be associated with a multidrug-resistant phenotype, but also alternative ABC-transporters can pump drugs from the inside to the outside of a cancer cell, e.g., ABCC2 (MRP2) is a platinum drug transporter. ABCB1, ABCC1, and ABCG2 have partial overlapping but not identical substrates. ABC-Transporters as Anticancer Drug Targets Following the identification of ABCB1 as a pivotal MDR-mediating factor, tremendous efforts were undertaken to identify ABCB1-interacting agents that inhibit its pump activity and, therewith, reverse the MDR phenotype. Such drugs are commonly designated as chemosensitizers or MDR modulators. Although many compounds, e.g., verapamil and cyclosporin derivatives, were

Abscopal Effect

identified as ABCB1 inhibitors or inhibitors of alternative MDR-mediating ABC-transporters, so far all of them failed in clinical trials.

21

Abraxas Definition

Cross-References ▶ Anthracyclines ▶ Chemotherapy ▶ Cystic Fibrosis ▶ Detoxification ▶ P-Glycoprotein ▶ Repair of DNA ▶ Xenobiotics

References Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2:48–58 Higgins CF (1993) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8:7–113 Holland IB, Cole SPC, Kuchler K, Higgins CF (eds) (2003) ABC proteins from bacteria to man. Academic Press, an Imprint of Elsevier Science, London/San Diego Lage H (2003) ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int J Antimicrob Agents 22:188–199

See Also (2012) Antimetabolite. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 216. doi: 10.1007/978-3-642-16483-5_326 (2012) Ciclosporin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 857. doi: 10.1007/978-3-642-16483-5_1167 (2012) DNA Repair. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1141. doi: 10.1007/978-3-642-16483-5_1687 (2012) Epipodophyllotoxins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1291. doi: 10.1007/978-3-642-16483-5_1953 (2012) Multidrug Resistance. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2393. doi: 10.1007/978-3-642-16483-5_3887 (2012) Taxane. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3614. doi: 10.1007/978-3-642-16483-5_5689 (2012) Verapamil. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3906. doi: 10.1007/978-3-642-16483-5_6179 (2012) Vinca Alkaloids. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3908. doi: 10.1007/978-3-642-16483-5_6187

BRCA1-A complex subunit Abraxas. Component of the BRCA1-A complex, a complex that specifically recognizes “Lys-63”-linked ubiquitinated (see “▶ Ubiquitination”) histones H2A and gamma-H2AX at ▶ DNA damage sites, leading to target the BRCA1-▶ BARD1 heterodimer to sites of DNA damage at double-strand breaks (DSBs). The BRCA1-A complex also possesses deubiquitinase activity that specifically removes “Lys-63”-linked ubiquitin on histones H2A and H2AX. In the BRCA1-A complex, it acts as a central scaffold protein that assembles the various components of the BRCA1-A complex and mediates the recruitment of BRCA1. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response.

Cross-References ▶ BARD1 ▶ DNA Damage ▶ Ubiquitination

See Also (2012) BRCA1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 481. doi: 10.1007/978-3-642-16483-5_6868 (2012) Double Strand Break. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1156. doi: 10.1007/978-3-642-16483-5_1718 (2012) GammaH2AX. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1494. doi: 10.1007/978-3-642-16483-5_2576 (2012) Histones. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1706. doi: 10.1007/978-3-642-16483-5_2762 (2012) RAP80. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3173. doi: 10.1007/978-3-642-16483-5_6873

Abscopal Effect ▶ Bystander Effect

A

22

Abscopal Effects Gabriele Multhoff Klinikum rechts der Isar, Department Radiation Oncology, TU München and CCG – “Innate Immunity in Tumor Biology”, Helmholtz Zentrum München, Munich, Germany

Synonyms ab (Latin: away) -scopus (Greek: target) effects; Away from the target effects; Distant bystander effects; Non-targeted effects; Out-of-field effect; Tumor rejection of non-irradiated tumor areas

Definition Abscopal effects describe nontargeted, radiotherapy (RT)-induced tumor regression in lesions or tumor or metastatic regions distant from the irradiated site.

Characteristics More than 50% of patients with solid tumors are treated with radiotherapy either alone or in combination with chemotherapy. Apart from direct cytotoxic effects of radiation therapy which are predominantly caused by the deposition of low and high LET (▶ Radiosensitization) energy to the nucleus and DNA (▶ DNA Damage; ▶ DNA Damage Response; ▶ DNA Damage-Induced Apoptosis; ▶ Repair of DNA), non-(DNA)targeting radiation effects that result in tumor regression in lesions distant from the irradiated tumor have been described. These so-called abscopal effects, firstly described by Mole in 1953 (Whole body irradiation; radiobiology or medicine? Br J Radiol 26, 234–41. Doi:10.1259/ 0007-1285-26-305-234), are most likely mediated by an activation of the immune system (Demaria et al. 2004). For the last 20 years, the role of the immune system to fight solid tumors was a matter

Abscopal Effects

of debate. Nowadays it is generally accepted that at least in vitro and in immunocompetent tumor mouse models, an active immune system can monitor, edit, and destroy malignantly transformed cells. After irradiation of a primary tumor in a mouse tumor, responses can be seen at distant lesions outside of the radiation field. It is also known that irradiation can induce non-immunogenic (▶ apoptosis) and immunogenic cell death of tumor cells such as necrosis (▶ Tumor Necrosis Factor), necroptosis, and mitotic catastrophe which in turn causes an increased presentation of ER-derived molecules (i.e., calreticulin), heat shock protein 70 (Hsp70), classical (classes I and II) and nonclassical (MICA/B, H60) MHC molecules, retinoic acid early antigen 1 (RAE-1), UL16-binding protein1-3 (ULBP1-3) molecules, and death receptors (i.e., CD95) on the cell surface of tumor cells and the release of pro-inflammatory cytokines/chemokines, danger-associated molecular patterns (DAMPs) such as adenosine tri-phosphate (ATP), high-mobility group box protein 1 (HMGB1), phosphatidylserine (PS), and heat shock proteins (HSPs) with different molecular weights ranging from approximately 20–90 kDa which act as immune adjuvants to stimulate the adaptive and innate immune system. Abscopal effects often display nonlinear dose relationships (Rödel et al. 2013) and are rarely seen in the clinical situation apart from few cases of highly immunogenic tumor entities such as melanomas (Postow et al. 2012). Therefore, a combination of radiotherapy with active and passive immunotherapies, such as cytokine therapies, vaccines, T-cell modulation, and co-stimulation using immune checkpoint (cytotoxic T-lymphocyte antigen-4, CTLA-4; programmed death-1, PD-1; PD-1 ligand, PD-L1; LAG-3) and T-cell checkpoint (CD137, CD134, GITR, CD27, CD40) inhibitors, antibody- or cell-based (T; NK; dendritic cells, DCs; ▶ tumor-associated macrophages, TAMs) therapies, toll-like receptor 9 (TLR-9) activation, chimeric antigen receptor (CAR; www.sciencedaily.com/releases/2015/01/ 150114140039.htm), T/NK cell therapies, and inhibition of immunosuppressive but tumorigenic

Abscopal Effects

23

Bystander Effect Local Tumor Irradiation Abscopal Effect

Abscopal Effects, Fig. 1 Schematic representation of nontargeted effects in a tumor mouse model. ▶ Bystander Effect. Effect on nonirradiated tumor regions in close proximity to irradiated tumor or tumor micromilieu which can induce genomic instability in later cell generations. Abscopal Effect. Immunological effects (see below) on nonirradiated tumor lesions locally distant from irradiated tumor. Immunological Effects. Secretion of pro-inflammatory responses (i.e., IL1b, TNFa, IL15, M-CSF), Activation of tumor suppressor proteins (i.e., ATM, CHK1), Activation of transcription factor p53, Expression of classical and nonclassical MHC molecules, Expression of tumor-associated antigens (CEA, CpG, La), Expression of death receptors (CD95, FAS, La autoantigen), Expression of adhesion molecules on tumor endothelial cells to recruit effector cells (Selectin, ICAM1, VCAM1), Inhibition of the migratory capacity of regulatory T cells (Tregs), Expression of ligands for activatory NK receptors (MICA/B, ULBPs, HMBG1, Hsp70), Release of danger-associated molecular patterns (DAMPS) and chemokines (CXCL16) to attract T cells

metabolites, has the potential to improve clinical outcome (Tang et al. 2015; Vatner et al. 2014). Another approach to improve abscopal effects of irradiation is the modulation of the tumor microenvironment. Regulatory T cells (Tregs), ▶ tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), high concentrations of anti-inflammatory cytokines, and metabolites such as immunosuppressive adenosine inhibit antitumor immune responses by blocking the cytolytic function of NK and CD8+ T cells and increase tumor cell survival, progression, and angiogenesis. These tumorpromoting parameters could be antagonized by changing the radiation dose, fractionation, site of irradiation, and timing or by combined radiation with chemotherapeutic regimens. Furthermore, the definition of the appropriate clinical endpoint of abscopal effects should also be considered with care. It appears that the Wolchok immune-related response criteria (2009) are superior in defining antitumor immune responses compared to the classical RECIST response criteria. Conclusion Abscopal effects describe nontargeted, radiotherapy (RT)-induced tumor regression in lesions or tumor areas locally distant from the irradiated i.e. tumor site (Fig. 1). Abscopal effects are best understood in mouse models and are very rarely seen in clinical practice. In order to augment RT-induced abscopal effects, a combination of RT with modern active and/or passive immunotherapeutic approaches appears to be a promising strategy to treat immunogenic tumors such as malignant melanoma, renal cell carcinomas (RCCs), and ▶ non-small cell lung cancer (NSCLC). Presently more than 50 clinical trials are ongoing that combine RT and immunotherapy in the treatment of solid tumors.

Cross-References ▶ Apoptosis ▶ Bystander Effect ▶ DNA Damage

A

24

▶ DNA Damage Response ▶ DNA Damage-Induced Apoptosis ▶ Non-Small-Cell Lung Cancer ▶ Radiosensitization ▶ Repair of DNA ▶ Tumor Necrosis Factor ▶ Tumor-Associated Macrophages

References Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L et al (2004) Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Radiat Oncol Biol 58:862–870. doi:10.1016/ j.ijrobp.2003.09.012 Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S, Mu Z, Raslan T, Adamow M, Ritter E, Sedrak C, Jungbluth AA, Chua R, Yang AS, Roman RA, Rosner S, Benson B, Allison JP, Lesokhin AM, Gnjatic S, Wolchok JD (2012) Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med 366:925–931. doi:10.1056/NEJMoa1112824 Rödel F, Frey B, Multhoff G, Gaipl US (2013) Contribution of the immune system to bystander and non-targeted effects of ionizing radiation. Cancer Lett. doi:10.1016/ j.canlet.2013.09.015 Tang C, Wang X, Soh H, Cortez MA, Krishnan S, Massarelli E et al (2015) Combining radiation and immunotherapy: a new systematic therapy for solid tumors. Cancer Immunol Res 2:831–837 Vatner RE, Cooper BT, Vanpouille-Box C, Demaria S, Formenti SC (2014) Combinations of immunotherapy and radiation in cancer therapy. Front Oncol 4:325. doi:10.3389/fonc2014.00325 Wolchok JD, Hoos A, O’Day S, Weber JS, Hamid O, Lebbe C et al (2009) Guidelines for the evaluation of immune therapy activity in solid tumors: immunerelated response criteria. Clin Cancer Res 15:7412–7420. doi:10.1158/1078-432.CCR-09-1624

ABVD Anas Younes Lymphoma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Definition Doxorubicin, bleomycin, vinblastine, and dacarbazine combination chemotherapy used for

ABVD

the treatment lymphoma.

of

patients

with

Hodgkin

Characteristics ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) is the most widely used regimen for the treatment of early and advanced stage Hodgkin lymphoma (HL). Treatment of patients with early stage classical HL evolved over the last three decades. Radiation therapy alone as the single treatment modality is no longer practiced. Today, the most widely used approach is combined modality therapy (chemotherapy plus involved field radiation therapy). In general, two (for favorable early stage) to four (for unfavorable early stage) cycles of ABVD plus 20 to 30 Gy of involved field radiation therapy is the most widely used standard of care approach. Using this approach, more than 90% of the patients are expected to be cured of their disease. Functional imaging is used to guide therapy aiming at eliminating the need for radiation therapy. Patients with bulky stage II disease (especially with bulky mediastinal mass) or stage II with B-symptoms are usually treated similar to those with advanced stage HL with six to eight cycles of ABVD followed by involved field radiation therapy to the bulky area. Use of chemotherapy alone has been proposed for a selected group of patients with early stage classical HL. The rationale for this approach is to reduce radiation-induced morbidity and mortality, including second malignancies and cardiac complications. While this approach is appealing, it will need to be further examined after prolonged follow-up. For now, it seems appropriate to treat young female patients with nonbulky early stage classical HL (especially those with mediastinal or axillary adenopathy) with chemotherapy alone to reduce the risk for breast cancer. The risks and benefits of combined modality versus chemotherapy alone should be discussed with patients before making a final treatment recommendation. Based on several randomized studies comparing ABVD with other multidrug regimens, ABVD became the most widely used combination regimen for

Acetylsalicylic Acid

the treatment of patients with advanced HL. Chemotherapy alone (six to eight cycles) is usually considered sufficient for treating patients with advanced stage classical HL. However, involved field radiation therapy is frequently added at the end of chemotherapy to areas of bulky disease. This combined modality approach has been compared with chemotherapy (MOPP/ ABV) alone in a randomized trial in patients with advanced stage classical HL, and showed no survival advantage, especially in those who achieved complete remission after the completion of chemotherapy. Newer treatment programs such as Stanford V and BEACOPP have shown successful results but remain less widely used compared with ABVD. Although BEACOPP has been shown to be superior to ABVD-like regimens in large-scale randomized trials, efficacy as Stanford V has similar ABVD has not yet been established. Because ABVD may cure only 50–65% of patients with poor risk advanced stage HL, more intensive programs such as BEACOPP may add benefit, despite the increased toxicity. Patients with good risk features have a high cure rate with ABVD, so the use of more intensive and more toxic regimens in this patient population should be used with caution and preferably within a clinical trial. In fact, a published randomized study demonstrated that early intensification with autologous stem cell transplantation after four cycles of ABVD-like chemotherapy did not improve the outcome in patients with advanced stage HL compared with conventional chemotherapy, perhaps because many patients did not have poor risk features as identified by the international prognostic score for HL.

25 Diehl V, Thomas RK, Re D (2004) Part II: Hodgkin’s lymphoma – diagnosis and treatment. Lancet Oncol 5:19–26 Meyer RM, Gospodarowicz MK, Connors JM et al (2005) Randomized comparison of ABVD chemotherapy with a strategy that includes radiation therapy in patients with limited-stage Hodgkin’s lymphoma: National Cancer Institute of Canada Clinical Trials Group and the Eastern Cooperative Oncology Group. J Clin Oncol 23:4634–4642 Straus DJ, Portlock CS, Qin J et al (2004) Results of a prospective randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) versus ABVD alone for stages I, II, and IIIA nonbulky Hodgkin disease. Blood 104:3483–3489

AC1L50CF ▶ Sorafenib

ACDC ▶ Adiponectin

2-Acetoxybenzenecarboxylic Acid ▶ Aspirin

2-Acetoxybenzoic Acid References Bonadonna G, Bonfante V, Viviani S et al (2004) ABVD plus subtotal nodal versus involved-field radiotherapy in early-stage Hodgkin’s disease: long-term results. J Clin Oncol 22:2835–2841 Canellos GP (1996) Is ABVD the standard regimen for Hodgkin’s disease based on randomized CALGB comparison of MOPP, ABVD and MOPP alternating with ABVD? Leukemia 10(Suppl 2):s68

▶ Aspirin

Acetylsalicylic Acid ▶ Aspirin

A

26

Achneiform Rash

Achneiform Rash

Activated Natural Killer Cells

Definition

Norimasa Ito1, Herbert J. Zeh III2 and Michael T. Lotze3 1 Departments of Surgery and Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA 2 UPMC/University of Pittsburgh Schools of the Health Sciences, Pittsburgh, PA, USA 3 Department of Surgery and Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA

Is a pustular rash with usual distribution over the face, scalp, and upper trunk.

Cross-References ▶ Erlotinib

Synonyms

Acral Metastasis

K cells; K lymphocyte; Killer cells; LAK; Large granular lymphocyte; Lymphokine activated killer

▶ Bone Metastasis

Definition

ACRP30 ▶ Adiponectin

Actinic Keratosis Definition Scaly, erythematous patches found on the skin in sun-exposed areas. Radiation induced keratosis (hornification) of the skin. It represents a precancerous lesion also known as solar keratosis or senile keratosis. May undergo malignant progression to form squamous cell carcinoma.

Cross-References ▶ Photodynamic Therapy ▶ Squamous Cell Carcinoma

White blood cells that kill tumor and virusinfected cells as part of the body’s immune system (Unified Medical Language System). A type of white blood cell that contains granules with enzymes that can kill tumor cells or microbial cells (National Cancer Institute). A circulating cellular biosensor, regulating immunity through release of cytokines, maturation of dendritic cells, and recognition and lysis of stressed cells, allowing sampling of cellular contents for delivery to phagocytic cells (our definition).

Characteristics Biology of NK Cells ▶ Natural killer cells comprise 10–15% of circulating lymphocytes in normal adults and are also found in peripheral tissues, including the liver, peritoneal cavity, lymph nodes, and placenta. NK cells were first reported by Wunderlich, Herberman, and Sendo and others in the early 1970s. They were first discovered on the basis of their nonspecific killer activity, disturbing attempts to generate tumor-specific,

Activated Natural Killer Cells

MHC-restricted cytotoxic T lymphocytes (CTLs). NK cell belongs to the innate immune system, bridging ▶ adaptive immunity in concert with ▶ dendritic cells. NK cells play a major role in the host defense against tumors and infected cells. NK cells mediate cytolysis of cultured tumor cells, and when lymphokine activated (LAK activity) against freshly acquired tumor cells. “Natural killer” suggests the initial notion that they do not require activation in order to kill target cells. NK cells are large granular lymphocytes (LGLs). The targets of NK cells are stressed cells expressing either “nonself” or “the self that changed in quality,” prompting their recognition. NK cells, when activated, can recognize cells which fail to express cognate self MHC molecules and simultaneously express (stress-induced) ligands recognized by activating NK receptors. These ligands include MICA/MICB ULPBs, PVR, and Nectin-2 in humans or Rae-1 in mice. NK cytolytic activity is almost nonexistent at birth, increases until 15 years of age, and then gradually reduces through old age. Natural killer cells (NK cells) lack the ability to destroy tumor cells at the time of birth, acquiring cytolytic capacity following recognition. Given their ready acquisition from the peripheral blood, multiple studies have evaluated their activity in various clinical studies; for example, chronic mental stress, fatigue, and physical exertion suppress NK activity. Reduced NK activity may be related to increasing cancer risk. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection. Many studies indicate that NK activity is reduced in patients with advanced cancer. Tumor infiltrating NK cells of pediatric cancer are significantly less in number than that observed in adult cancers, prompting the notion that this creates a major nosologic difference of adult and pediatric neoplasms. Role of NK Cells in Human Cancer NK cells induce tumor cell death when NK cells recognize tumor cells with NK cell activating receptors. NK cells produce many cytokines including IFNs and TNF-a and suppress proliferation of tumor and cells and drive type 1 immunity. NK cells help dendritic cells to mature into DC1.

27

NK cells have some suppressive roles against cancer. NK cells have inhibitory receptors. They become tolerant to tumor cells when inhibitory receptors are stimulated with their ligands (Fig. 1). Markers of NK Cells NK cells express CD16 (FcgRIII), CD56, CD57, CD94, or CD158a. They do not express T cell receptor (TCR) or the pan T cell marker CD3 or surface immunoglobulins (Ig) B cell receptor (CD20). NK cells recognize specific polysaccharide on target cells with NK receptor (CD161; NKR-P1) and expression of MHC class I molecules. NK Cell Receptors There are two main types of receptors for MHC class I on NK cells including the KIR (killer cell immunoglobulin-like receptors, one of the immunoglobulin superfamily) and NKG2 receptor (CD94, type C lectin family). In both, there are activating and suppressing forms that accelerate or suppress NK activity. Two explanations for NK cell self-tolerance have been proposed: first, NK cells from MHC-class-I-deficient hosts have a lower activation potential, owing to decreased activating-receptor expression and/or function; or second, NK cells are kept self-tolerant by interactions between non-MHC-dependent receptor–ligand pairs CD94:NKG2, a C-type lectin family receptor, is conserved in both rodents and primates and identifies nonclassical (also nonpolymorphic) MHC I molecules including HLA E. Though indirect, this is a means to survey the levels of classical (polymorphic) HLA molecules. Expression of HLA E at the cell surface is dependent upon the presence of classical MHC class I leader peptides. Ly49 is a relatively ancient, C-type lectin family receptor. Humans have only one pseudogenic Ly49, the receptor for classical MHC I molecules. KIRs belong to a multigene family of evolved Ig-like extracellular domain receptors. They are present in nonrodent primates and are the primary receptors for both classical MHC I (HLA A, HLA B, HLA C) and nonclassical HLA G in primates. KIRs are specific for certain HLA subtypes. ILT or LIR (leucocyte inhibitory receptors) are discovered members of

A

28

Activated Natural Killer Cells

Th

Attack

Antigen processing and presentation

Tumor

Viral infected cell

Attack NK CTL DC Antigen release HMGB1 IFN-Y TNF-α

Maturation iDC → DC1 Stimulation

Nonself IFN-Y TNF-a FAS-L

I. Apoptosis

Allo II. Autophagy

Xeno Perforin/granzymes MHC class I

III. Necrosis

NK

Inflammation

Activation

NK receptors

Inhibitory

Tolerance

Activated Natural Killer Cells, Fig. 1 Role of NK cells in tumor immunity. NK cells play multiple roles in tumor immunity. They recognize stressed cells or those failing to express cognate Class I major histocompatibility molecules, both lysing targets and serving as a source of cytokines important in initiation and perpetuation of the inflammatory response is carried out by them. They serve as helper cells, promoting immune interaction with both T and dendritic cells, critically being required for initiation of the TH1 response. Their absence may also be important in limiting autoimmunity as revealed by their critical absence in the NOD mouse strain, susceptible to

autoimmune diabetes. When lysing cells, normal cells capable of undergoing apoptotic or autophagic00128 death, Types I and II death, do so. Many virally infected or transformed cells fail to undergo such death because of block of these pathways, and when lysed, undergo necrotic cell death causing DC maturation and promoting recruitment of additional inflammatory cells. In the absence of viral or bacterial pathogen signals, such chronic necrotic cell death is associated with inhibition of immune effectors and promotion of a wound repair phenotype with angiogenesis and stromagenesis, characteristic of many tumors

the Ig receptor family. ▶ Carcinoembryonic antigen related cell adhesion molecule 1 (▶ CEACAM1 Adhesion Molecule) is an inhibitory receptor and its ligands are CEACAM1 itself and CEACAM5, known as CEA. Sialic acid binding immunoglobulin-like lectins (SIGLECs) have a V-set immunoglobulin domain, which binds sialic acid, and varying numbers of C2-set immunoglobulin domains. IRp60, KLRG1, and LAIR1 are other inhibitory receptors discovered (Table 1).

NK Cell and Cytokines NK cells are capable of producing many cytokines including IFN-g (Interferon-fg), IFN-a, IFN-b, and TNF-a. They suppress proliferation of tumor and virally infected cells and regulate immune responses. IFN-g (Interferon-fg) increases NK activity as a positive feedback mechanism. NK cytolytic activity is increased by IFN-a, IFN-b, and IFN-g (Interferon-fg) (produced by T and NK cells); IL-2(produced by T cells); and IL-10, IL-12, and IL-15 (produced by B cell, monocyte/

Activated Natural Killer Cells Activated Natural Killer Cells, Table 1 Inhibitory and activating NK cell receptors and their ligands activating NK cell receptors Receptors Ligands 2B4 CD48 NKp44 Influenza/unknown NKp30 NKp46 Influenza/unknown CD16 IgG NKG2D02396 MICA, MICB NKp80 DNAM CD112/CD155 Inhibitory NK cell receptors ILT2 MHC01094-A, B, G KIR3DL2 MHC01094-A KIR3DL1 MHC01094-B KIR2DL4 MHC01094-A, B, G KIR2DL1,2,3 MHC01094-C CD94 MHC01094-C CEACAM102441 CEACAM102441, CEACAM5 IRp60 Unknown KLRG1 Unknown LAIR1 Unknown SIGLEC7 Sialic acid SIGLEC9 Sialic acid

macrophage, or dendritic cells). NK cytolytic activity is inhibited by IL-4 (▶ Interleukin-4). IL-15 induces NK cell proliferation. IL-12 induces IFN-g (Interferon-fg) production by NK cells. IFN-a, IFN-b, IFN-g (Interferon-fg), and TNF-a produced by NK cells activate monocytes/macrophages, vascular endothelial cells, neutrophils, and induce a local inflammation response. Cytotoxicity of NK Cells Against Tumor or Infected Cells NK cells release perforin from intracellular granules when they bind to target cells, along with granules containing serine proteases known as granzymes. Perforin attaches to the membrane inducing an autophagic (▶ Autophagy) repair process, inducing uptake of vesicles containing granzymes and associated molecules that can target cells for lysis, with perforin allowing escape through pore formation once intracellular.

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Granzyme induce apoptosis to the target cells utilizing various intracellular pathways. NK cells also induce ▶ apoptosis to target cells by expressing apoptosis-inducing molecules such as FAS ligands or TRAIL on the cell surface. The distinction between apoptosis and ▶ necrosis is important in cancer immunology – necrotic cells release danger/damage associated molecular pattern molecules (DAMPs) such as high-mobility group box 1 (HMGB1) protein, whereas apoptosis leads to retention of HMGB1 within the cells or apoptotic nuclei. NK Cells and Cancer Immunotherapy Their rapid cytolytic action and broad target range suggest that NK cells may be promising candidates for cancer cell therapy. The clinical application of ex vivo manipulated cells, including NK cells, is referred to as ▶ adoptive immunotherapy (AIT). The first clinical AIT trial exploited autologous ex vivo expanded and interleukin 2 (IL-2) stimulated lymphokine activated killer (LAK). Although this approach produced nearly 15–20% partial and complete responses in initial trials, subsequent studies showed that a similar antitumor effect could be achieved with administration of high dose IL-2 alone. Purification and enrichment of NK cells on a clinical scale may improve therapeutic outcomes. Alternatively, stimulation of LAK cells with IL-15 or IL-21 instead of IL-2 might increase efficacy. Myeloid ▶ dendritic cells (mDCs) support the tumoricidal activity of NK cells, while cytokinepreactivated NK cells activate DCs and induce their maturation and cytokine production. NK–DC interactions promote the subsequent induction of tumor-specific responses of CD4+ and CD8+ T cells, allowing NK cells to act as nominal “helper” cells in the development of the desirable type-1 responses to cancer. NK–DC interaction provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer. Clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK–DC therapy of melanoma and other cancers.

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Cross-References ▶ Natural Killer Cell Activation

References Arnon TI, Markel G, Mandelboim O (2006) Tumor and viral recognition by natural killer cells receptors. Semin Cancer Biol 16:348–358 DeMarco RA, Fink MP, Lotze MT (2005) Monocytes promote natural killer cell interferon gamma production in response to the endogenous danger signal HMGB1. Mol Immunol 42(4):433–444 Ito N, Demarco RA, Mailliard RB et al (2007) Cytolytic cells induce HMGB1 release from melanoma cell lines. J Leukoc Biol 81(1):75–83 Lotze MT, Line BR, Mathisen DJ et al (1980) The in vivo distribution of autologous human and murine lymphoid cells grown in T cell growth factor (TCGF): implications for the adoptive immunotherapy of tumors. J Immunol 125(4):1487–1493 Moretta A, Bottino C, Vitale M et al (1996) Receptors for HLA-class I-molecules in human natural killer cells. Annu Rev Immunol 14:619–648

Activation-Induced Cytidine Deaminase Xiaosheng Wu and Diane F. Jelinek Department of Immunology, Mayo Clinic, College of Medicine, Rochester, MN, USA

Synonyms AICDA; AID; ARP2; CDA2

Definition Activation-induced cytidine deaminase (AID) (EC 3.5.4.5) is a 198-amino acid polypeptide enzyme that is primarily expressed in germinal center (GC) B cells of the secondary lymphoid organs. Its physiological function is to introduce point mutations into the variable and switch regions of immunoglobulin (Ig) genes during the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in GC B cells,

Activation-Induced Cytidine Deaminase

respectively, leading to a highly diversified antibody affinity repertoire and alternative use of different constant regions of Ig. Patients with defective AID due to germline mutations develop type 2 hyper-IgM syndrome (HIGM2), a type of immunodeficiency resulting in high levels of serum IgM and lack of other post-switch Ig isotypes. Given its potent mutation-inducing property, deregulated expression of AID in the wrong place or at the wrong time is often associated with various cancers.

Characteristics Identification of AID There are almost an infinite number of antigens that exist in our environmental surroundings, and our immune systems could theoretically produce a specific antibody to each one of these antigens if appropriately stimulated. In the early years, it was far beyond comprehension how this vast antibody diversity could possibly be generated from the very limited genomic resources that we now know consists of only about 30,000 proteincoding genes in humans. In the premolecular genetic era, without any experimental proof there had been many theories proposed, including the prophetic “somatic randomization” theory put forward by Nobel laureate Frank Burnet in 1957 (Ganesh and Neuberger 2011). Burnet’s proposal was eventually proven in part by the work of Susumu Tonegawa, another Nobel laureate, demonstrating that much of the diversity resulted from random rearrangement of Ig heavy chain variable (V), diversity (D), and joining (J) genes and Ig light chain VJ genes during B cell development in the bone marrow. Although imprecise V(D)J joining is an additional source of Ig diversity, it was soon realized that V(D)J recombination only generates a very limited antibody repertoire, with these antibodies typically possessing low antigen binding affinity. These observations suggested that an additional fine-turning somatic diversification mechanism accounted for the generation of better antigen fitting antibodies. This mechanism, now known as SHM, gained solid footing after protein sequencing of monoclonal light chains

Activation-Induced Cytidine Deaminase

present in multiple myeloma patients and DNA sequencing of Ig genes. Both sequencing strategies revealed missense mutations in the variable region of Ig. The precise mechanism underlying these mutations, however, remained mysterious until the discovery of the mutation introducing enzyme, AID. In 1999, Honjo’s group identified AID from a mouse lymphoma cell line CH12F3 through subtractive hybridization (Muramatsu et al. 1999) and demonstrated that AID is required for both SHM and CSR. AID Deamination Mechanism When it was first cloned, AID was thought to be an RNA deaminase based on the similarity of its domain structure with a previously known RNA deaminase, APOBEC-1. After a flurry of work focused on how this enzyme works, it quickly became clear that AID actually is a DNA cytidine deaminase. AID deaminates deoxycytidine (dC) in DNA to generate deoxyuracil (dU), which is then further processed by one of several mechanisms. First, without any DNA repair, dU can be directly copied as deoxythymidines (dT) during DNA replication leading to a transition mutation of C:G to T:A. Secondly, dU can also be excised by the base excision repair (BER) component uracil DNA glycosylase resulting in abasic sites, which can then be replicated by error-prone translesion DNA polymerases. Through this repair mechanism, transversion mutations are added to dU sites. Furthermore, abasic sites can also be excised by another downstream BER repair component AP endonuclease, leading to single stranded DNA breaks (SSBs) or double stranded DNA breaks (DSBs) if two abasic sites are in close proximity on opposite strands. Finally, AID mediated U:G mismatch is also a perfect substrate for the DNA mismatch repair (MMR) system. Together with error-prone DNA polymerases used during DNA resynthesis, MMR can introduce even more mutations in the neighborhood of dU sites, including mutations on A:T sites which are not direct targets of AID (Stavnezer 2011). It is not known how these different repair pathways are coordinately utilized, and if any of these pathways are used preferentially in SHM versus CSR. However, AID-mediated DSBs are required for CSR.

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Given its hyper mutagenic activity, it is conceivable that deregulated AID expression and/or specificity would have detrimental consequences. Strict AID expression in GC B cells is regulated transcriptionally by the concerted action of various transcriptional activators and repressors. It has also been shown that the function of AID is also regulated by posttranslational modifications. In addition to AID expression, downstream DNA repair systems play an indispensible role in minimizing the adverse effects of AID. Seminal work by Schatz’s group showed that up to 25% of genes in our genome are subjected to AID-mediated mutagenesis when the MMR gene MSH2 is absent (Liu et al. 2008). This work was later complemented by the discovery of the somatic hyperrepair (SHR) process, which results in the elevated expression of select DNA repair genes to counterbalance the adverse potential off-target effects of AID expression (Wu et al. 2010). Despite these safeguarding mechanisms, more than 75% of all hematological malignancies originate from mature B lineage cells that have gone through AID-mediated GC reactions suggesting that having a history of AID expression poses a greater risk of developing cancer. Therefore, mechanisms limiting AID’s mutagenic activity to Ig variable region genes and expression to GC B cells would be advantageous. AID Targeting How exactly AID targets V and switch (S) regions of Ig genes remains a contentious issue. Extensive studies have revealed that AID deaminates dC on single stranded DNA by recognizing the hotspot WRCY (where W = C or T, R = A or G, and Y = T or C) or RGYW (in reverse complimentary configuration) motifs. Either Crick or Watson strands can be targeted as long as they are in single-stranded conformation. Furthermore, active transcription of the target gene is required for AID to function, and AID preferentially targets the unprotected nontranscribing strand since the transcribing strand is occupied by the complimentary RNA product forming an R-loop. However, these DNA cis-acting features remain insufficient in ensuring AID targeting specificity since they are not unique to Ig genes. Therefore,

A

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the specificity seems to be largely determined by AID binding proteins in trans. There have been several reports showing that AID could bind to many intracellular proteins; yet, none of those proteins could serve as bona fide bridging factors between AID and Ig sequences. However, a study using the chicken DT40 cell line provided some tantalizing results showing that the RNA splicing factor, SRSF1, might be the missing link. Finally, AID targeting specificity is possibly determined by downstream DNA repair systems since mutations became widespread when the MMR gene MSH2 is absent as previously mentioned. AID Expression in Cancer Given its potent mutagenic property, it is conceivable that deregulated AID expression may be associated with cancer development and/or progression. The first proof that AID expression may increase the risk of cancer came from a study using an AID transgenic mouse model. In those mice, the expression of a ubiquitous promoterdriven AID transgene alone was sufficient to drive the development of thymic lymphoma, B cell lymphoma, and various others tumors of nonlymphoid origin. Subsequently, it was found that constitutive AID expression is associated with various human cancers.

Activation-Induced Cytidine Deaminase

identification of somatic mutations in various proto-oncogenes including PIM1, MYC, RhoH/ TTF, and PAX5 at the WRCY hotspots in these malignancies, further signifies the direct involvement of AID. Non-B Cell Lineage Malignancies

It is conceivable that AID, a GC-B cell specific gene, is involved in various B lineage malignancies. AID expression is also associated with other non-B lineage cancers. The best-exemplified study is on the Helicobacter pylori infection-induced gastric cancer. Here, infection of mice with “cag” pathogenicity island (cagPAI)-positive H. pylori, a gastric cancer–causing strain of bacterial, induces aberrant expression of AID in gastric epithelial cells. The expression of AID is induced by the activation of NFkB pathway, which leads to acquisition of somatic mutations in the tumor suppressor gene, p53, thereby predisposing those cells to develop cancer. Similarly, aberrant AID expression has also been detected in other cancers including hepatitis virus infection-induced hepatoma, colitis-associated colorectal cancer, bile duct inflammation-associated cholangiocarcinoma, bile acid reflux-related Barrett oesophageal adenocarcinoma, as well as some breast and prostate cancer cell lines.

B Cell Lineage Malignancies

AID Is Required for Recurrent Chromosomal Translocations

Under physiological conditions, AID is only transiently expressed in GC B cells while pre- and post-GC B lineage cells are free of AID expression. However, constitutive AID expression is often readily detectable at various levels in many B cell malignancies, including follicular lymphoma, Burkitt lymphoma, Hodgkin lymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, mediastinal B cell lymphoma, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and multiple myeloma. It seems that the variable AID levels in CLL samples were mainly attributed to the size of the AID-expressing cell pool rather than the level of AID in the entire cancer cell population. To add more complexity, it is known that AID is differentially spliced into different functional variants in some of those disease subsets. The

Chromosomal translocations are a hallmark feature of many cancers, and recurrent translocations are found in about 40% of all human tumors by creating new tumor-promoting proteins or by disrupting tumor suppressing systems. For example, the t(9;22)(q34;q11) translocation, also known as the Philadelphia chromosome, is observed in 90% of patients with chronic myelogenous leukemia (CML), and this leads to a novel BCR-ABL gene fusion that is capable of inducing oncogenic transformation in vitro. Reciprocal translocations between IgH and a number of different oncogenes including c-myc, bcl-2, bcl-6, and FGFR are characteristic of the human B cell malignancies Burkitt lymphoma, follicular lymphoma, diffuse large cell lymphoma, and multiple myeloma, respectively. Systemic studies have shown that the vast majority of these

Active Specific Immunization

translocations, if not all, require the direct function of AID for the generation of chromosomal translocation intermediates, i.e., DNA double strand breaks (DSB). AID Expression Associated with Cancer Progression and Poor Prognosis

The ongoing AID expression in cancer cells may not necessarily suggest its direct involvement in initial cancer development. However, it may play an important role in future disease events such as cancer progression, therapy effectiveness, and cancer prognosis. AID positivity has been shown to be associated with the prognosis of CLL and increased risk of transformation of indolent lymphomas. The transition from the chronic phase of CML to B lymphoid blast crisis is often accompanied by the expression of AID, which renders the disease resistant to otherwise effective BCR-ABL inhibitor imatinib therapy. One of the possible drug resistance mechanisms is the acquisition of new mutations in the kinase domain of BCR-ABL where imatinib binds. Therefore, it is possible that AID could constitute a new cancer therapy target. AID as a DNA Demethylase DNA cytosine methylation in the sequence context of CpG is a key epigenetic mark in vertebrates. It is critically important in various cell functions including cell differentiation, cell programming, parental imprinting, retroelement suppression, etc. DNA methylation is mediated by the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. However, it was not known how the 5-methyl group is removed. AID contributes to DNA demethylation in some cells during early development. Specifically, AID initiates demethylation through a DNA damage-coupled DNA repair process very similar to that in SHM. First, AID deaminates 5-methylcytosine (5-mC) to yield thymidine (T) which is then removed by the T:G mismatch DNA glycosylases TDG and MBD4. The resulting abasic site is then replaced with unmethylated deoxycytosine by the BER system. The net change is the removal of the methyl group from the cytosine residue while the primary DNA sequence is faithfully preserved. A report also showed that AID is also required

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for effectively maintaining a low methylation status in mouse primordial germ cells at embryonic day 13.5. This observation suggests that AID-mediated erasion of DNA global methylation may be important in maintaining transgenerational epigenetic inheritance, as well as in epigenetic reprogramming. Given many cancer cells possess stem cell-like properties, altered epigenetics, and aberrant expression of AID, it remains to be seen if AID plays any role in changing the epigenetic landscape in cancers in addition to its role in altering genomic stability.

References Ganesh K, Neuberger MS (2011) The relationship between hypothesis and experiment in unveiling the mechanisms of antibody gene diversification. FASEB J 25(4):1123–1132 Liu M, Duke JL, Richter DJ et al (2008) Two levels of protection for the B cell genome during somatic hypermutation. Nature 451:841–845 Muramatsu M, Sankaranand VS, Anant S et al (1999) Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J Biol Chem 274:18470–18476 Stavnezer J (2011) Complex regulation and function of activation-induced cytidine deaminase. Trends Immunol 32:194–201 Wu X, Tschumper RC, Gutierrez A Jr et al (2010) Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells. PLoS One 5(12):e15549

Active Cell Death ▶ Apoptosis

Active Specific Immunization Synonyms ASI

Definition Refers to various strategies to induce an effective cellular immune response against tumor cells.

A

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Activin

Cross-References

βA

βA

Activin A

βB

βB

Activin B

βC

βC

Activin C

βA

βB

Activin AB

α

βA

Inhibin A

α

βB

Inhibin B

▶ Colorectal Cancer Vaccine Therapy

See Also (2012) Immune Response. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1815. doi:10.1007/978-3-642-16483-5_2977.

Activin Elspeth Gold Department of Anatomy, Otago School of Medical Sciences, Dunedin, New Zealand

Synonyms Activin A; Activin B; Activin C; Activin E; EDF; Erythroid differentiation factor; INHBA; INHBB; INHBC; INHBE; Inhibin-b chain

Definition Dimeric protein complex that enhances biosynthesis of follicle-stimulating hormone (FSH) and secretion from the pituitary.

Characteristics Activins are members of the TGF-b superfamily of proteins, and in addition to stimulating FSH release from the pituitary they are involved in regulation of a diverse range of physiological processes including embryonic development, reproduction, and fertility, and are implicated in the development and progression of cancers, especially of the testis, ovary, and adrenal gland. There are five known b-subunits (designated bA through bE) that can form homodimers or heterodimers, for example activin A (= bAbA), activin B (=bBbB), activin C (=bCbC), activin AB

Activin, Fig. heterodimers

1 Activin/inhibin

homodimers

and

(=bAbB), and activin AC (=bAbC, Fig. 1). Two of these subunits (bC and bE) were discovered only in the last decade and we know little about them, mainly because the activin-bC and bE knock-out mice were normal. However, overexpression of activin-bC leads in mice to male infertility, liver, and prostate disease, while overexpression of activin-bE leads to abnormalities in the pancreas. Signaling Cascade Activin exerts it actions by binding to one of two Type II receptors (ActRIIA/ActRIIB), which in turn recruit and phosphorylate ALK-4, a Type I activin receptor. ALK4 activates intracellular signaling molecules called Smads (Smad-2 or Smad3). Activated Smad-2/3 complexes with Smad-4 and moves to the nucleus leading to activation or repression of target genes (Fig. 2). Functions Activin A has been most extensively investigated due to the existence of recombinant protein and specific assays. Activin A is a potent growth and differentiation factor, is secreted in an active form, and can elicit overt biological action at low (pg/ml)

Activin Activin, Fig. 2 The activin signaling cascade

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βA

βA

A

ActRI P

P

P

Smad2/3

ActRII

P

Smad2/3 Smad4

Smad4

P

Smad2/3

Activin responsive gene transcription

concentrations – therefore its synthesis and activity must be tightly regulated. Follistatin binding or inhibin-a subunit heterodimerization are the two most well characterized activin A antagonists. Activin Antagonists Follistatin binds activin ligands with high affinity to form biologically inactive complexes. There are two follistatin isoforms which block the activity of activin A by different mechanisms. One form binds to the cell surface and is considered to be a local regulator which diverts activin A to a pathway for degradation. The other form is present in the blood where it binds to and inactivates circulating activin A. The activin-bA and bB subunits can heterodimerize with the inhibin-a subunit to form inhibin A (a-bA) or inhibin B (a-bB) (Fig. 1). Inhibins oppose the actions of activins, particularly in the reproductive axis where they inhibit, while activin stimulates FSH release from the pituitary, inhibins also oppose the local actions of activins in the testis and ovary.

The Role of Activin A in Cancer Development and Progression Like other TGF-b super-family members, the role of activin A in cancer biology is complex and involves aspects of tumor suppression as well as tumor promotion. The ability of activin A to inhibit proliferation is central to the tumorsuppressive mechanism. However, as tumors evolve, they often become refractory to the growth inhibitory effects of activin A and many overexpress activin A, which in turn has a marked impact on the biology of the tumor cells themselves and creates a tumor micro-environment that is conducive to tumor growth and metastasis. For example, increased production of activin A by tumor cells that are no longer growth inhibited by activin A may lead to increased angiogenesis, decreased immune surveillance, or an increase in epithelial to mesenchymal transition of tumor cells. Collectively, these effects favor increased tumor growth and metastasis in the later stages of cancer progression.

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Perturbations in activin expression and/or the activin signaling cascade have been implicated in cancer development and progression in many organ systems: examples are liver, pancreas, prostate, ovary, testis, breast, and adrenal. Increased activin expression has also been implicated in cancer-associated weight loss (cachexia) and metastasis to bone. The overt effects of elevated activin A expression is evident in the inhibin knock-out mouse. This mouse model develops cancer of the testis and ovary and, when the testis/ovary are removed, adrenal tumors. Tumor formation is evident at 4 weeks of age in males and 6 weeks in females and leads to elevated activin A. Increased activin A level causes cell death in the liver and stomach, which leads to severe weight loss (cachexia) from 6 to 7 weeks and death by 12 weeks in males and 17 weeks in females. The severe wasting syndrome is delayed in gonadectomized inhibin knock-out mice due to removal of the gonadal source of activin A; but the castrate mice go on to develop adrenal tumors with the onset of the same lethal wasting syndrome. Overexpression of follistatin, being an activin binding protein, was predicted to block the overt effects of elevated activin A in the inhibin knock-out mice. The inhibin knock-out follistatin overexpression mouse confirmed this concept. With these mice showing no evidence of weight loss thus surviving significantly longer than the inhibin knock-outs. While tests and ovarian tumors were still evident in the double cross mice, tumor development was delayed. Conclusion Activin A normally maintains tissue homeostasis, yet numerous studies demonstrate aberrant expression of activin is associated with cancer development and progression. Understanding how cancer cells escape the growth inhibitory effects of activin A is likely to reveal new therapeutic avenues for the treatment of cancer.

Activin A Gold E, Risbridger G (2011) Activins and activin antagonists in the prostate and prostate cancer. Mol Cell Endocrinol. doi:10.1016/j.mce.2011.07.005 Harrison CA, Gray PC, Vale WW, Robertson DM (2005) Antagonists of activin signaling: mechanisms and potential biological applications. Trends Endocrinol Metab 16:73–78 Risbridger GP, Schmitt JF, Robertson DR (2001) Activins and inhibins in endocrine and other tumours. Endocr Rev 22:836–858 Robertson DM, Burger HG, Fuller PJ (2004) Inhibin/ activin and ovarian cancer. Endocr Relat Cancer 11:35–49 Stenvers KL, Findlay JK (2010) Inhibins: from reproductive hormones to tumor suppressors. Trends Endocrinol Metab 21:174–180

Activin A ▶ Activin

Activin B ▶ Activin

Activin C ▶ Activin

Activin E ▶ Activin

References Chen YG, Lui HM, Lin SL, Lee JM, Ying SY (2002) Regulation of cell proliferation, apoptosis, and carcinogenesis by activin. Exp Biol Med (Maywood) 227:75–87

Activin Receptor Type 2 ▶ Activin Receptors

Activin Receptors

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Activin Receptor Type 1 ▶ Activin Receptors

Activin Receptor-Like Kinase ▶ Activin Receptors

Activin Receptors Michael Grusch1 and Mir Alireza Hoda2 1 Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria 2 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria

There are two types of activin receptors designated as type I and type II. Activins first bind type II receptors, whereupon type I receptors are recruited into the complex, which leads to the phosphorylation of intracellular signaling mediators called Smads. In a wider sense, however, the term activin receptor-like kinase (ALK) is also used for the structurally related type I receptors that transmit signals from other cytokines of the transforming growth factor beta (TGFb) family including TGFb itself, bone morphogenetic proteins (BMP), myostatin, growth and differentiation factors (GDF), and Müllerian inhibitory substance (MIS). Confusingly, the abbreviation ALK is also used for ▶ anaplastic lymphoma kinase – a structurally unrelated receptor tyrosine kinase. Table 1 provides an overview of the different type I and type II receptors for the TGFb family and some of their ligands.

Characteristics Synonyms Activin receptor type 1; Activin receptor type 2; Activin receptor-like kinase; ActRI; ActRII; ACVR1; ACVR2; ALK

Definition Activin receptors are transmembrane serine threonine kinases that bind ▶ activins and subsequently trigger an intracellular signaling cascade.

Structure and Signaling All activin receptors consist of an extracellular domain involved in ligand binding, a single-pass transmembrane domain and an intracellular part harbouring a serine threonine kinase domain. Two type I and two type II receptors have been shown to transmit signals from activins. Activins first bind the type II receptors and subsequently recruit the type I receptors ALK4 or ALK7 into the complex. Activin A uses primarily ALK4, whereas activns B and AB prefer ALK7. The constitutively active type II receptors activate the

Activin Receptors, Table 1 Type I and type II activin/TGFb family receptor combinations used by selected TGFb family cytokines

ACVR2 ActRII ACVR2B ActRIIB TGFBR2 BMPR2 AMHR2

ALK1 ACVRL1

ALK2 ACVR1 BMPs

ALK3 BMPR1A BMPs

BMP9 BMP10

BMPs

BMPs

BMPs

BMPs

MIS

MIS

ALK4 ACVR1B Activin A Myostatin Activin A Myostatin

ALK5 TGFBR1

ALK6 BMPR1B BMPs BMPs

TGFb1-3 BMPs GDF5 MIS

ALK7 ACVR1C Activin B Nodal Activin B Nodal

A

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Activin Receptors

various lineages. In the adult organism, activin/ activin receptor signals are involved in reproductive biology, wound healing, ▶ inflammation, and tissue homeostasis.

Activin Receptors, Fig. 1 Graphic representation of activin receptors and activin receptor-associated Smad signaling

type I receptors via phosphorylation in the juxtamembrane GS (glycine-serine-rich) domain. Formation of active heterotetrameric receptor complexes further recruits R-Smad (receptoractivated Smad) proteins, which are phosphorylated by the type I receptors. Genuine activin receptors as well as TGFb receptors use R-Smads 2 and 3, whereas BMPs and GDFs use R-Smads 1, 5, and 8. Activated R-Smads complex with the common mediator Smad4 and the whole complex subsequently translocates into the nucleus to regulate gene expression in cooperation with numerous transcriptional coactivators and corepressors (Fig. 1). A number of extracellular proteins like Cripto/ TDGF1 (teratoma derived growth factor 1) and intracellular proteins like ARIPS (activin receptor-interacting protein) interact with activin receptors and modulate their signaling capacity. Function and Expression Activin receptors are expressed on most human cell types and their signals are of fundamental importance for embryonic development. Activin receptor-mediated signals are required for the differentiation of ▶ embryonic stem cells into

Activin Receptors in Cancer Biological consequences of activin receptor activation are complex and to a large degree cell- and context-dependent. Therefore, deregulated expression and activation of activin receptors can have both oncogenic and ▶ tumor suppressive effects. Mutations in activin receptors have been found in several malignancies including microsatellite instable prostate, pancreatic, and colorectal cancer. These mutations lead to receptor inactivation often caused by truncation of the protein. This indicates a tumor suppressing function of activin receptors in malignancies of these organs. Since activin receptor signals also contribute to protumorigenic activities like enhanced cell proliferation in some tissue types and enhanced cell migration and fibrotic tissue remodeling, blocking antibodies, ligand traps (soluble extracellular receptor domains), or kinase inhibitors targeting activin receptors have also been suggested for therapeutic application in cancer. Moreover, the inhibition of activin receptors could be beneficial against cancer cachexia, because both activin and myostatin contribute to this condition. The ALK1/ BMP9 axis plays an important role in angiogenesis and inhibitors specifically targeting ALK1 activation are consequently being developed as potential antiangiogenic therapies. Conclusion Activin receptors and activin receptor-like kinases transmit signals of the TGFb family that can have oncogenic as well as tumor suppressive effects. Inactivating mutations are found in some malignancies, but for some aspects of tumor therapy also inhibition of activin receptor function could prove beneficial.

References Cunha SI, Pietras K (2011) ALK1 as an emerging target for antiangiogenic therapy of cancer. Blood 117:6999–7006

Acute Lymphoblastic Leukemia Hinck AP (2012) Structural studies of the TGF-betas and their receptors – insights into evolution of the TGF-beta superfamily. FEBS Lett 586:1860–1870 Jung B, Doctolero RT, Tajima A, Nguyen AK, Keku T, Sandler RS, Carethers JM (2004) Loss of activin receptor type 2 protein expression in microsatellite unstable colon cancers. Gastroenterology 126:654–659 Tsuchida K, Nakatani M, Uezumi A, Murakami T, Cui X (2008) Signal transduction pathway through activin receptors as a therapeutic target of musculoskeletal diseases and cancer. Endocr J 55:11–21

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Definition Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from several cooperative genetic mutations in a single B- or T-lymphoid progenitor, leading to altered blast cell proliferation, survival, and maturation and eventually to the lethal accumulation of leukemic cells. Although cases can be subclassified further according to the multiple stages of T- or B-cell maturation, these distinctions are not therapeutically useful.

ACTR ▶ Steroid Receptor Coactivators

ActRI ▶ Activin Receptors

ActRII ▶ Activin Receptors

Acute Granulocytic Leukemia ▶ Acute Myeloid Leukemia

Acute Lymphoblastic Leukemia Ching-Hon Pui St. Jude Children’s Research Hospital, Memphis, TN, USA

Synonyms ALL

Characteristics ALL accounts for about 12% of all childhood and adult leukemias diagnosed in developed countries and for 60% of those diagnosed in persons younger than 20 years. It is the most common cancer in children (25% of all cases) and has a peak incidence in patients between the ages of 2 and 5 years, with a second, smaller peak in the elderly. The factors predisposing children and adults to ALL remain largely unknown. Children with certain constitutional genetic abnormalities (e.g., trisomy 21) are at increased risk of developing ALL and inherited mutations in TP53, PAX5 and ETV6 have also been described in familial (as well as sporadic) ALL. However, disease susceptibility for most patients is mainly influenced by common genetic variants (with eight risk loci discovered thus far) identified by genome-wide association studies (GWAS). A study identified germline mutations in 4.4% of children and adolescents with ALL, a finding which not only improves our understanding of leukemogenesis but also has major implications in direct patient care and genetic counseling of patients and families. Ionizing radiation and mutagenic chemicals have been implicated in some cases of ALL, but their contributions appear negligible. ALL is essentially a disease of acquired genetic abnormalities which can be found in leukemic cells in all cases of ALL, including chromosomal translocations, DNA copy number gains or losses, and epigenetic changes. On average, each case has 10–20 nonsilent coding mutations. Chromosomal

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translocations often activate transcription factor genes, which in many cases control cell differentiation, are developmentally regulated, and frequently encode proteins at the tops of critical transcriptional cascades. These “master” oncogenic transcription factors, which can exert either positive or negative control over downstream responder genes, are aberrantly expressed in leukemic cells as a single gene product or as a unique fusion protein combining elements from two different transcription factors. Activating mutations of NOTCH1, a gene encoding a transmembrane receptor that regulates normal T-cell development, and mutations of PAX5, a gene essential for B-lineage commitment and maintenance, have been identified to be the most frequent cooperative mutations in T-cell and B-cell ALL, respectively. Genome-wide studies including secondgeneration sequencing (exome, transcriptome, and whole-genome sequencing) have resulted in the revision of genetic classification of ALL by identifying new subtypes, defined the constellations of structural genetic alterations and sequencing mutations that characterize each subtype, and identified genetic targets for therapy. Although most leukemias begin in the bone marrow and spread to other parts of the body, some may arise in an extramedullary site, such as the thymus or intestine, and subsequently invade the bone marrow. The presenting features of ALL generally reflect the degree of bone marrow failure and the extent of extramedullary spread. Common signs and symptoms are:

Acute Lymphoblastic Leukemia, Fig. 1 Small regular blasts with scanty cytoplasm, homogeneous nuclear chromatin, and inconspicuous nucleoli

Acute Lymphoblastic Leukemia

• Fever • Fatigue and lethargy • Dyspnea, angina, and dizziness (older patients mainly) • Limp, bone pain, or refusal to walk (young children) • Pallor and bleeding in the skin or mouth cavity • Enlarged liver, spleen, and lymph nodes (more pronounced in children) • Anemia, low neutrophil count, and low platelet count • Metabolic abnormalities (e.g., high serum uric acid and phosphorus levels) The diagnosis of ALL is based on a morphologic examination of bone marrow cells (Figs. 1, 2, and 3) and immunophenotype of cells from the same sample. Karyotyping, fluorescence in situ hybridization (FISH), and molecular genetic analysis by RT-PCR (reverse transcriptasepolymerase chain reaction) are now routinely performed by many centers to identify subtypes of ALL with prognostic and therapeutic significance, for example: • BCR-ABL1 fusion gene due to the t(9;22), or Philadelphia chromosome – 25% of adult cases and 3–4% of childhood cases (improved outcome with tyrosine kinase inhibitor treatment) • ETV6-RUNX1 (also known as TEL-AML1) fusion gene due to a cryptic t(12;21) – 22% of childhood cases (favorable prognosis)

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Acute Lymphoblastic Leukemia, Fig. 2 Mature B-cell ALL blasts characterized by intensely basophilic cytoplasm, regular cellular features, prominent nucleoli, and cytoplasmic vacuolation

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Acute Lymphoblastic Leukemia, Fig. 3 Admixture of large blasts with moderate amounts of cytoplasm and smaller blasts. Such cases may be mistaken for acute myeloid leukemia, emphasizing the importance of immunophenotyping and genotyping to corroborate the differential diagnosis

• Hyperdiploidy (more than 50 chromosomes per cell) – 25% of childhood cases (favorable prognosis) • Hypodiploidy (fewer than 45 chromosomes per cell) – 2% of childhood cases and 2% of adult cases (unfavorable prognosis) Contemporary risk-directed treatment can cure up to 90% of children and up to 50% of adults with ALL. Cases are generally classified as standard or high risk in adults and as low, standard, and high risk in children. Factors used to determine the relapse hazard include the presenting leukocyte count, age at diagnosis, gender, immunophenotype, karyotype, molecular genetic abnormalities, initial response to therapy, and the amount of “minimal residual leukemia” upon achieving a complete remission. The level of

minimal residual during remission induction and consolidation therapy is the most important prognostic indicator because it accounts for the collective effect of leukemic cell genetics, microenvironment, host factors, and chemotherapy potency. Multidrug remission induction regimens almost always include a glucocorticoid (prednisone, prednisolone, or dexamethasone), vincristine, and at least a third agent (L-asparaginase or anthracycline), administered for 4–6 weeks. Some treatments rely on additional agents to increase the level of cell kill, thereby reducing the likelihood of the development of drug resistance and subsequent relapse. However, several studies suggest that intensive remission induction therapy may not be necessary for low or standard-risk patients, provided that they receive postinduction

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intensification therapy. Remission induction rates now range from 98% to 99% in children and from 80% to 95% in adults. Complete clinical remission is traditionally defined as restoration of normal blood cell formation with a blast cell fraction of less than 5% by light microscopic examination of the bone marrow. With this definition, some patients in complete remission may harbor as many as 1  1010 leukemic cells in their body. With sensitive and specific methods developed to measure minimal residual disease, it is now recognized that most patients actually have less than 0.01% of residual leukemia after 4–6 weeks of remission induction therapy, and they have excellent treatment outcome. By contrast, patients with 1% or more leukemic cells after remission induction treatment have a poor prognosis and may be candidates for hematopoietic stem cell transplantation. To improve treatment outcome, most protocols specify an intensification (or consolidation) phase in which several effective antileukemic drugs are administered in high doses soon after the patients attain a complete remission. Reinduction treatment, essentially a repetition of the initial induction therapy administered during the first few months of remission, has become an integral component of successful ALL treatment protocol. Regardless of the intensity of induction, consolidation, or reinduction therapy, all children require 2–2½ years of continuation treatment, usually methotrexate and mercaptopurine, with pulses of vincristine and dexamethasone for low-risk cases, and multiagent intensive chemotherapy for standard- and high-risk cases. The need for continuation therapy in adults is less clear, although in most cases it is discontinued after 2–2½ years of complete remission. The central nervous system can be a sanctuary site for leukemic cells, requiring intensive, intrathecally administered chemotherapy that begins early during the remission induction phase, extending through the consolidation phase and into the continuation phase. Once considered standard treatment, prophylactic cranial irradiation can be safely omitted in contemporary protocols featuring effective systemic and intrathecal chemotherapy. However, some protocols still use this

Acute Lymphoblastic Leukemia

treatment modality in up to 10% of patients who are at very high risk of relapse in the central nervous system. For selected high-risk cases, such as patients who require extended therapy to attain initial complete remission or those with high level or persistence of minimal residual disease after remission induction, hematopoietic stem cell transplantation is currently the treatment of choice. In light of the development of new therapeutics, the indications for transplantation should be continuously evaluated. For example, therapy with ABL1 ▶ tyrosine kinase inhibitors (▶ imatinib mesylate, dasatinib, ▶ nilotinib or ponatinib) has improved the duration of remission of patients with Philadelphia chromosomepositive ALL, and those with Philadelphia chromosome-like ALL and “ABL-class” kinase alterations, and reduced the need of transplantation for a substantial proportion of these patients. The development of chimeric antigen receptormodified autologous or allogeneic T cells promises to provide a new treatment option. Finally, the optimal clinical management of patients with ALL requires careful attention to methods for the prevention or treatment of metabolic and infectious complications, which may otherwise be fatal.

Cross-References ▶ Imatinib ▶ Nilotinib ▶ Tyrosine Kinase Inhibitors

References Pui C-H, Campana D, Pei D et al (2009) Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med 360:2730–27412 Pui C-H, Pei D, Coustan-Smith E et al (2015a) Clinical utility of sequential minimal residual disease measurements in the context of risk-directed therapy in childhood acute lymphoblastic leukaemia: a prospective study. Lancet Oncol 16:465–474 Pui C-H, Yang JJ, Hunger SP et al (2015b) Childhood acute lymphoblastic leukemia: progress through collaboration. J Clin Oncol 33:2938–2948

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Roberts KG, Mullighan CG (2015) Genomics in acute lymphoblastic leukaemia: insights and treatment implications. Nat Rev Clin Oncol 12:344–357 Zhang J, Walsh MF, Wu G et al (2015) Germline mutations in predisposition genes in pediatric cancer. N Engl J Med 373:2336–2346

lead to a novel clinically meaningful classification of the disease.

See Also

Epidemiology AMKL is diagnosed in 7–10% of infants and children with AML without Down syndrome (DS). In most pediatric cases the disease occurs de novo and subgroups can be identified based on cytogenetic features or biological features as described later. In contrast, AMKL is rare in adults, occurring in 1–2% of all AML cases and is frequently associated with antecedent hematological disorder such as myelodysplastic syndrome. Children with DS have a markedly increased risk to developing AMKL and represent up to 10% of children with AML. A large proportion of children with DS (estimated 10%) are born with a unique transient form of AMKL, often called transient myeloproliferative disorder (TMD) or transient abnormal myelopoiesis (TAM). This congenital leukemia resolves spontaneously in most of the patients. Up to 20% of those patients will relapse with a full blown AMKL by the age of 4 years. Thus the leukemia of DS represents a unique clinical entity of multistep leukemogenesis (Fig. 1).

(2012) Dasatinib. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1060. doi:10.1007/978-3-642-16483-5_1518 (2012) Extramedullary. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1366. doi:10.1007/978-3-642-16483-5_2074 (2012) Karyotype. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1941. doi:10.1007/978-3-642-16483-5_3200 (2012) Remission. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3225. doi:10.1007/978-3-642-16483-5_5020 (2012) Sanctuary site. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3334. doi:10.1007/978-3-642-16483-5_5154

Acute Megakaryoblastic Leukemia Jean-Pierre Bourquin1 and Shai Izraeli2 1 Pediatric Oncology, University Children’s Hospital Zurich, Zurich, Switzerland 2 Pediatric Hemato-Oncology, Sheba Medical Center and Tel Aviv University, Ramat Gan, Israel

Synonyms Acute megakaryoblastic leukemia M7; Acute myeloid leukemia; DS-ML; Myeloid leukemia of Down syndrome; Subtype AML-M7

Definition Acute megakaryoblastic leukemia (AMKL) is defined as a malignant clonal proliferation of immature hematopoietic cells of the megakaryocytic lineage. AMKL is a subtype of acute myeloid leukemia (AML). The biologic features of AMKL are heterogeneous, and the ongoing characterization of the disease pathogenesis is likely to

A Characteristics

Clinical and pathologic features Typical features at diagnosis include hepatosplenomegaly, anemia, thrombocytopenia, and myelofibrosis. The fibrosis is probably caused by soluble factors (such as TGF-b) secreted from the malignant megakaryoblasts. Infants with DS may exhibit marked liver failure that sometimes may be life threatening. The liver failure is secondary to liver fibrosis caused by the infiltration of leukemic cells. ▶ Flow cytometry is the preferred method for immunophenotypic characterization of AMKL, although in some cases the diagnosis can only be made from bone marrow or liver biopsies due to extensive myelofibrosis. Typically, the leukemic blasts express at least one megakaryoblastic antigen [CD41(GPIIb)/CD42b(GPIbalpha) or CD61].

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Acute Megakaryoblastic Leukemia

GATA1 mut

3rd hit

AMKL

1st hit TMD 20%

80%

– 9 months

birth

CURE

3 years

Acute Megakaryoblastic Leukemia, Fig. 1 Multistep evolution of AMKL in Down syndrome. Mutation in GATA1 is acquired during fetal liver hematopoiesis in cells carrying a germline trisomy 21 and results in congenital clonal megakaryoblastic proliferation (TMD). In almost all patients TMD resolves spontaneously leading

to cure. However in about 20% of the patients, additional postnatal acquired mutations in residual cells from the resolved TMD results in the development of full blown acute megakaryocytic leukemia (AMKL) during early childhood

Coexpression of the T-lineage marker CD7 is frequently observed, suggesting pathogenic mechanism that could lead to aberrant regulation of lymphoid genes. Expression of erythroid markers (e.g., glycophorin A) and of CD36 (thrombospondine receptor) characterize the AMKL of DS. Because AMKL blasts may display low expression levels of the pan-hematopoietic CD45 antigen, the distinction from metastatic solid tumors may be challenging.

immature fetal megakaryoblasts. The mutations occur during fetal liver hematopoiesis. The initiation of the leukemia during fetal liver hematopoiesis explains the frequent liver dysfunction observed in DS newborns with TMD. Strikingly, GATA1 is located on chromosome X and is mutated only in AMKL with trisomy 21. The precise mechanism by which trisomy 21 promotes the survival of cells with acquired mutation in GATA1 is presently unknown. One hypothesis suggests that genes on chromosome 21 code proteins enhance fetal megakaryopoiesis. This developmental pressure of megakaryopoiesis coupled with differentiation arresting mutation in GATA1 causes clonal accumulation of megakaryoblasts diagnosed at birth as TMD. GATA1 mutation is necessary and probably sufficient for the development TMD, but additional mutations are required for the occurrence of full blown AMKL in DS patients. Why TMD spontaneously resolves and which mutations cause further evolution to AMKL is largely unknown. There are several biological subgroups among patients with AMKL that do not have DS. The most frequent recurrent chromosomal aberration detected in non-DS AMKL is the translocation t(1;22), which typically occurs in infants and very young children that present with hepatosplenomegaly and pronounced myelofibrosis. This

Cytogenetic and Biological Features Increasing evidence suggest that distinct subtypes of AMKL can be identified based on genetic and molecular characteristics. Recurrent cytogenetic abnormalities are specifically associated with AMKL and at least in part convey a prognostic significance. The megakaryoblastic disorders associated with DS (both AMKL and TMD) are characterized by the presence of an acquired mutation in the transcription factor GATA1. The mutations occur in exon 2 or in the beginning of exon 3 and uniformly result in the production of a short GATA1 protein (GATA1s) that lacks the amino-terminal of the full length GATA1. GATA1 is a major regulator of normal megakaryopoiesis. GATA1s blocks terminal differentiation and enhances proliferation of

Acute Megakaryoblastic Leukemia

translocation fuses RBM15/OTT1, an RNA export factor to MKL1/MAL1, a cofactor of the transcription factor SRF (serum response factor). Less commonly, fusion translocations between the MLL gene and different partners, often AF10, have been reported in AMKL. Interestingly, a second translocation involving AF10, the translocation t(10;11) which results in the fusion of CALM (clathrin-assembly protein-like lymphoid myeloid) with AF10, was reported in several cases. This translocation was also identified in other AML subtypes and in cases of T-cell ALL. In a mouse model, infection of bone marrow cells with a retroviral vector to express CALMAF10 results in a transplantable AML, demonstrating that this fusion gene represents a fundamental leukemogenic event. By gene expression profiling, at least two distinct classes of non-DS AMKL could be discriminated based on their molecular phenotype. Approximately one third of the cases display an erythroid expression pattern coupled with expression of CD36 and higher expression levels of the transcription factor GATA1 in absence of detectable mutations. Interestingly, this gene expression signature is reminiscent to the increased expression of erythroid markers detected in AMKL from DS patients, which are characterized by increased expression levels of mutated GATA1s. The second subtype of non-DS AMKL samples include all cases with recurrent translocation t(1;22). Interestingly, samples that share similar expression profiles with the samples positive for the translocation t(1;22) are characterized by increased expression levels of another SRF cofactor, HOP, suggesting that similar regulatory pathways may be involved. This second class is associated with higher levels of expression of the surface antigen ▶ CD44, which was associated with worse outcome in other type of malignancies and coexpressed on the leukemia initiating cells from patients with AML. It is currently not possible to determine if the distinction of these two classes by expression profiling has a prognostic significance due to the small numbers of patients that were treated on different therapeutic protocols. A prospective study using selected genes from the AMKL signature will be required to

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determine if this information could be used as prognostic marker to guide selection of treatment intensity. Prognosis and Treatment Treatment results from several international study groups, including the European AML-BFM study group and UK-MRC cooperative groups, and the north american SJCRH and CCG cooperative groups show a marked difference in treatment outcome between DS and non-DS AMKL. Reduction of treatment intensity for patients with DS resulted in a marked decrease in treatment related mortality and an excellent treatment outcome (91% event-free survival at 5 years in the AML-BFM 98 study), strongly suggesting a distinct leukemia biology between DS and non-DS AMKL patients. AMKL blasts from patients with DS are extremely sensitive to the chemotherapy drug cytosine arabinoside (ARA-C), probably due to a decrease in its cellular degradation caused by an enzyme regulated by GATA1. The results for patients with AMKL excluding patients with DS are still poor, despite intensification of AML treatment regimens. The 5-year event-free survival (EFS) reported for the treatment regimen correspond to results obtained for other AML subtypes, with EFS of 42% reported for the AML-BFM93/98 trials and of 47% reported by the UK-MRC 10 and 12 clinical trials. Further research is necessary to identify new treatment modalities and biomarkers to guide treatment intensification, including the indication for bone marrow transplantation for patients at highest risk of relapse. Data in mouse models suggest that targeted therapy with antibodies directed against the surface marker CD44 may be a future therapeutic.

References Bourquin JP, Subramanian A, Langebrake C et al (2006) Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. Proc Natl Acad Sci USA 103:3339–3344 Ge Y, Stout ML, Tatman DA et al (2005) GATA1, cytidine deaminase, and the high cure rate of Down syndrome

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46 children with acute megakaryocytic leukemia. J Natl Cancer Inst 97:226–231 Izraeli S (2006) Down’s syndrome as a model of a pre-leukemic condition. Haematologica 91:1448–1452 Oki Y, Kantarjian HM, Zhou X et al (2006) Adult acute megakaryocytic leukemia: an analysis of 37 patients treated at M.D. Anderson Cancer Center. Blood 107:880–884 Reinhardt D, Diekamp S, Langebrake C et al (2005) Acute megakaryoblastic leukemia in children and adolescents, excluding Down’s syndrome: improved outcome with intensified induction treatment. Leukemia 19:1495–1496

Acute Megakaryoblastic Leukemia M7 ▶ Acute Megakaryoblastic Leukemia

Acute Myelogenous Leukemia ▶ Acute Myeloid Leukemia

Acute Myeloid Leukemia Barbara Deschler Comprehensive Cancer Center Mainfranken, Clinical Trials Office, University of Würzburg, Würzburg, Germany

Synonyms Acute granulocytic leukemia; Acute myelogenous leukemia; Acute nonlymphocytic leukemia; ANLL

Definition Acute myeloid leukemia (AML) is part of a group of hematological malignancies (▶ Hematological Malignancies, Leukemias and Lymphomas) in the bone marrow involving cells committed to the

Acute Megakaryoblastic Leukemia M7

myeloid line of cellular development. It is defined by the malignant transformation of a bone marrowderived, self-renewing stem cell or progenitor which demonstrates a decreased rate of self-destruction and aberrant differentiation. Uncontrolled growth of such cells, named blasts, is the result of clonal proliferation. Blasts accumulate in the bone marrow and other organs. As a result, mature cells of hematopoiesis are suppressed. For the leukemia to be called acute, the bone marrow must include greater than 20% leukemic blasts.

Characteristics Classification The first comprehensive morphologichistochemical classification system for AML was developed by the French-American-British (FAB) Cooperative Group. This classification system categorizes AML into eight major subtypes (M0 to M7) based on morphology and immunohistochemical detection of lineage markers. This classification of AML was revised under the auspices of the World Health Organization (WHO) (see List 1). In 2008 the World Health Organization (WHO), in collaboration with the European Association for Haematopathology and the Society for Hematopathology, published a revised and updated edition of the WHO Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The 4th edition of the WHO classification incorporates new information that has emerged from scientific and clinical studies in the interval since the publication of the 3rd edition in 2001, and includes new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others. It also adds entities-some defined principally by genetic features-that have been characterized.

List 1 Acute myeloid leukemia with recurrent genetic abnormalities AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 (continued)

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List 1 APL with t(15;17)(q22;q12); PML-RARAa AML with t(9;11)(p22;q23); MLLT3-MLLb AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA Acute myeloid leukemia with myelodysplasia-related changesc Therapy-related myeloid neoplasmsd Acute myeloid leukemia, not otherwise specified (NOS) Acute myeloid leukemia with minimal differentiation Acute myeloid leukemia without maturation Acute myeloid leukemia with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemia Pure erythroid leukemia Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis (syn.: acute myelofibrosis; acute myelosclerosis) Myeloid sarcoma (syn.: extramedullary myeloid tumor; granulocytic sarcoma; chloroma) Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis (syn.: transient myeloproliferative disorder) Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukemias of ambiguous lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia with t(9;22)(q34; q11.2); BCR-ABL1e Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged Mixed phenotype acute leukemia, B/myeloid, NOS Mixed phenotype acute leukemia, T/myeloid, NOS Provisional entity: Natural killer (NK) cell lymphoblastic leukemia/lymphoma Adopted from Arber DA, Vardiman JW, Brunning RD, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Fourth Edition. Edited by Swerdlow, S.H., Campo E., Harris N.L., Jaffe E.S., Pileri S.A., Stein H., Thiele J., Vardiman J.W (editors). Geneva, Switzerland. WHO PRESS 2008. For a diagnosis of AML, a marrow blast count of 20% is required, except for AML with the recurrent genetic

47 abnormalities t(15;17), t(8;21), inv(16) or t(16;16) and some cases of erythroleukemia a Other recurring translocations involving RARA should be reported accordingly: e.g., AML with t(11;17)(q23;q12)/ ZBTB16-RARA; AML with t(11;17)(q13;q12); NUMA1RARA; AML with t(5;17)(q35;q12); NPM1-RARA; or AML with STAT5B-RARA (the latter having a normal chromosome 17 on conventional cytogenetic analysis) b Other translocations involving MLL should be reported accordingly: e.g., AML with t(6;11)(q27;q23); MLLT4MLL; AML with t(11;19)(q23;p13.3); MLL-MLLT1; AML with t(11;19)(q23;p13.1); MLL-ELL; AML with t(10;11) (p12;q23); MLLT10-MLL c > 20% blood or marrow blasts AND any of the following: previous history of myelodysplastic syndrome (MDS), or myelodysplastic/myeloproliferative neoplasm (MDS/ MPN); myelodysplasia-related cytogenetic abnormality (see below); multilineage dysplasia; AND absence of both prior cytotoxic therapy for unrelated disease and aforementioned recurring genetic abnormalities; cytogenetic abnormalities sufficient to diagnose AML with myelodysplasia-related changes are: complex karyotype (defined as 3 or more chromosomal abnormalities) unbalanced changes: 7 or del(7q); 5 or del(5q); i(17q) or t (17p); 13 or del(13q); del(11q); del(12p) or t(12p); del (9q); idic(X)(q13); balanced changes: t(11;16)(q23;p13.3); t(3;21)(q26.2;q22.1); t(1;3)(p36.3;q21.1); t(2;11)(p21; q23); t(5;12)(q33;p12); t(5;7)(q33;q11.2); t(5;17)(q33; p13); t(5;10)(q33;q21); t(3;5)(q25;q34) d Cytotoxic agents implicated in therapy-related hematologic neoplasms: alkylating agents; ionizing radiation therapy; topoisomerase II inhibitors; others e BCR-ABL1 positive leukemia may present as mixed phenotype acute leukemia, but should be treated as BCRABL1 positive acute lymphoblastic leukemia

Epidemiology AML is infrequent but highly malignant, responsible for a large number of cancer-related deaths. AML accounts for approximately 25% of all leukemias in adults in industrialized countries and, thus, is the most frequent form of leukemia. Worldwide, the incidence of AML is highest in the USA, Australia, and Western Europe. According to the SEER database (http://seer. cancer.gov) the age-adjusted incidence rate of AML in the USA in the years 1975–2012 has been relatively stable at approximately 3.4-4 per 100,000 persons (=2.5 per 100,000 when age-adjusted to the world standard population). The American Cancer Society estimates that 11,930 individuals will be diagnosed with AML in 2006 in the USA. Patients that are newly

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0

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4 9 14 19 24 29 34 39 44 49 54 59 64 69 74 79 84 5+ to 8 5 0 to 5 to 0 to 5 to 0 to 5 to 0 to 5 to 0 to 5 to 0 to 5 to 0 to 5 to 0 to 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 Age group

Acute Myeloid Leukemia, Fig. 1 Age-specific incidence of AML (USA: 2000–2003) (Source: SEER)

diagnosed with AML have a median age of 65 years. From 2000 to 2003, the US incidence rate in people under the age of 65 was only 1.8 per 100,000, while the incidence rate in people aged 65 or over was 17 per 100,000 (Fig. 1). AML is thus primarily a disease of later adulthood with an age-dependent mortality of 2.7 to nearly 18 per 100,000. The incidence of AML varies to a small degree depending on gender and race. AML in adults is slightly more prevalent in males in most countries. In the USA in 2000, AML was more common in Whites with 3.8 per 100,000 than in Blacks (3.2 per 100,000). Etiology The development of AML has been associated with several risk factors summarized in Table 1. Generally, only a small number of observed cases can be traced back to known risk factors. These include age, antecedent hematological disease, genetic disorders as well as exposures to radiation, chemical or other hazardous substances (e.g., benzene), and previous chemotherapy (e.g., treatment with ▶ alkylating agents). Leukemogenesis, like ▶ carcinogenesis, is a multistep process that requires the susceptibility of a hematopoietic progenitor cell to inductive agents at multiple stages. The different subtypes of AML may have distinct causal mechanisms, suggesting a functional link between a particular molecular abnormality or mutation and the causal agent. Most cases of AML arise without objectifiable leukemogenic exposure.

Acute Myeloid Leukemia, Table 1 Risk factors Genetic disorders

Physical and chemical exposure

Radiation Exposure Chemotherapy

Down syndrome Klinefelter syndrome Patau syndrome Ataxia telangiectasia Shwachman syndrome Kostman syndrome Neurofibromatosis Fanconi anemia Li–Fraumeni syndrome Benzene Drugs as pipobroman Pesticides Cigarette smoking Embalming fluids Herbicides Nontherapeutic/therapeutic radiation Alkylating agents topoisomerase II inhibitors Anthracyclines Taxanes

Signs and Symptoms of AML AML can cause different uncharacteristic signs and symptoms such as weight loss, unusual fatigue, and fever. Many patients feel a loss of well-being. Most symptoms can be traced back to bone marrow insufficiency: anemia, immunodeficiency caused by neutropenia, and thrombocytopenia. Diagnostic procedures and types of specimen necessary to reach the diagnosis of AML are the following:

Acute Myeloid Leukemia

Acute Myeloid Leukemia, Fig. 2 Myeloid blasts in peripheral blood detected by light microscopy

• Blood cell counts and microscopic blood cell examination (Fig. 2) • Bone marrow aspiration and biopsy • Routine microscopic exam of bone marrow • Flow cytometry • Immunocytochemistry • Cytogenetics • Molecular genetic studies The peripheral blood count may reveal a decreased white blood cell count (leukopenia) as well as leukocytosis (increased white blood cell count). Leukemia cells do not protect against infection and may cause congestion of blood vessels (leukostasis). Thrombocytopenia, a decrease of platelets, can lead to excessive bruising, petechiae, and bleeding. When leukemia cells spread outside the bone marrow, it is called extramedullary manifestation. Small pigmented spots that look like common rashes may indicate skin involvement. A tumor-like collection of AML cells is called chloroma or granulocytic sarcoma. AML sometimes causes enlargement of the liver and spleen. Prognostic Factors AML is a curable disease; the chance of cure for a specific patient depends on a number of prognostic factors. Some of the strongest prognostic information can be obtained by cytogenetic analysis. Normal cytogenetics indicates average-risk AML. Cytogenetic abnormalities that suggest a good prognosis include translocations t(8;21) and t(15;17), as well as inv(16). Patients with

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AML that is characterized by deletions of the long arms or monosomies of chromosomes 5 or 7; by translocations or inversions of chromosome 3, t(6;9), t(9;22); or by abnormalities of chromosome 11q23 have particularly poor prognoses. Further adverse prognostic factors include central nervous system involvement with leukemia, elevated white blood cell count (>100,000/mm3), treatment-induced AML, and a history of MDS. Leukemias in which cells express the progenitor cell antigen CD34 and/or the P-glycoprotein (MDR1 gene product) have an inferior outcome. Due to a higher relapse rate, patients with AML associated with an internal tandem duplication of the FLT3 gene (FLT3/ITD mutation) have a poorer outcome. Beyond these disease-specific factors, patientspecific parameters like comorbidities and frailty have a strong impact on the course of the disease and treatment tolerability, as reflected by the age-dependent surge in mortality. Comorbidity describes any distinct additional clinical entity that has existed or may occur during the clinical course of a patient with a primary (index) disease. There is currently no consensus on how to quantify comorbidities, but several scales and indices are available. Therapy Therapeutic approaches can be differentiated as curative (aimed at long-term cure) or palliative (principally aimed at achieving best quality of life) (▶ palliative therapy). Curative intensive ▶ chemotherapy treatment for AML is considered the standard procedure, usually divided in two phases, induction and consolidation (post-remission) therapy. It is traditionally based on two substances, cytarabine (cytosine arabinoside) and anthracycline. The objective of a curative treatment approach is to rapidly eliminate the cancer cells with induction chemotherapy, called remission. Complete remission occurs in 60–80% of patients. More than 15% of adults with AML (about 25% of those who attain complete remission) can be expected to survive 3 or more years and may be cured. Remission rates in adult AML are inversely related to age, with an expected remission rate of >65% for those

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younger than 60 years. Duration of remission may be shorter in older patients. Increased morbidity and mortality during induction appear to be directly related to age. This is associated with several factors including the ability to tolerate intensive treatment approaches. Without treatment, the average life expectancy is about 3 months. Complications during treatment include relapse of the disease, severe infections, or lifethreatening bleeding. During this time, supportive care consists of patient isolation to prevent infection, antibiotics to treat infections, and transfusion of blood products. After remission is achieved, further treatment is known as consolidation and is necessary in order to achieve a permanent cure. Consolidation may consist of either further chemotherapy or a bone marrow, or stem cell transplantation. The aforementioned treatments are appropriate for all subtypes of AML except for one type of AML known as ▶ acute promyelocytic leukemia (APL). Newer treatments, especially for those patients not tolerating intensive chemotherapy, include monoclonal antibodies, demethylating agents, and experimental drugs given in clinical trials. Thus, while the diagnosis of AML in itself does not represent a therapeutic mandate for intensive chemotherapy in all cases, the latter is the only curative approach to treatment. Decisions whether to treat patients with intensive chemotherapy, new agents, or solely best ▶ supportive care should be based on a sum of patient factors (including age, previous history of MDS, comorbidity, frailty, and patients’ preferences), in addition to the blast count and the above-described prognostic factors. Careful consideration of these factors is especially relevant in older, multimorbid patients with AML.

Cross-References ▶ Acute Megakaryoblastic Leukemia ▶ Acute Promyelocytic Leukemia ▶ Alkylating Agents ▶ Carcinogenesis ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Myelodysplastic Syndromes

Acute Myeloid Leukemia

▶ Nucleoporin ▶ Palliative Therapy ▶ Supportive Care

References Brunning RD, Matutes E, Harris NL et al (2001) Acute myeloid leukaemia: introduction. In: Jaffe ES, Harris NL, Stein H (eds) Pathology and genetics of tumours of haematopoietic and lymphoid tissues, vol 3, World Health Organization Classification of Tumours. IARC Press, Lyon, pp 77–80 Deschler B, de Witte T, Mertelsmann R et al (2006) Treatment decision-making for older patients with high-risk myelodysplastic syndrome or acute myeloid leukemia: problems and approaches. Haematologica 91(11):1513–1522 Döhner H, Estey E, Amadori S, et al (2010) Diagnosis and Management of acute myeloid leukemia in adults: Report from an International Expert Panel, on Behalf of the European LeukemiaNet. Blood 115:453–74. Grimwade D, Walker H, Harrison G et al (2001) The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 98(5):1312–1320 Parkin DM, Whelan SL, Ferlay J et al (eds) (1997) Cancer incidence in five continents, vol 7. IARC Scientific Publications, Lyon Ries LAG, Harkins D, Krapcho M et al (eds) (2006) SEER cancer statistics review, 1975–2003. National Cancer Institute, Bethesda

See Also (2012) Clonal Proliferation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 884. doi:10.1007/978-3-642-16483-5_1220 (2012) Comorbidity. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 960. doi:10.1007/978-3-642-16483-5_1280 (2012) Cytogenetic Analysis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1050. doi:10.1007/978-3-642-16483-5_1468 (2012) Differentiation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1113. doi:10.1007/978-3-642-16483-5_1616 (2012) Hematopoiesis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1644. doi:10.1007/978-3-642-16483-5_2616 (2012) Myeloid. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2436. doi:10.1007/978-3-642-16483-5_3935 (2012) Petechiae. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2829. doi:10.1007/978-3-642-16483-5_4481

Acute Promyelocytic Leukemia

Acute Myeloid Leukemia 1 ▶ Runx1

Acute Nonlymphocytic Leukemia

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(NPM), nuclear mitotic apparatus (NuMA), and signal transducer and activator of transcription 5B (STAT5b). This leads to the generation of fusion genes encoding distinct fusion proteins. The sensitivity of APL to the differentiating action of all-trans retinoic acid (ATRA) is differentially mediated by the various fusion proteins (see Molecular Characterization).

▶ Acute Myeloid Leukemia

Characteristics

Acute Promyelocytic Leukemia Li-Zhen He1, Lorena L. Figueiredo-Pontes2, Eduardo M. Rego2 and Pier Paolo Pandolfi3 1 Memorial Sloan-Kettering Cancer Center, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA 2 Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil 3 Division of Genetics, Beth Israel Deaconess Medical Center, Boston, MA, USA

Definition Acute promyelocytic leukemia (APL) is a distinct subtype of ▶ acute myeloid leukemia (AML) characterized by the expansion of leukemic cells blocked at the promyelocytic stage of myelopoiesis. According to the French–American–British (FAB) classification of acute leukemia, APL corresponds to the M3 and M3-variant subtypes, and according to World Health Organization classification (2001) it corresponds to the subtype: AML associated with translocations involving chromosomes 15 and 17 [t(15;17)] and variants. APL accounts for 5–10% of adult AML patients in Caucasian populations and for 20–30% among patients with Latino ancestry. Invariably, APL leukemic cells harbor ▶ chromosomal translocations involving the retinoic acid receptor a (RARa) gene on chromosome 17 (Table 1), which may be fused to one of five possible partner genes: promyelocytic leukemia (PML), promyelocytic leukemia zinc finger (PLZF), nucleophosmin

Clinical and Laboratorial Presentation The symptoms of APL are similar to those of other subtypes of AML such as weight loss, fatigue, weakness, pallor, fever, and bleeding. These symptoms manifest acutely and are accompanied by petechiae, bruising, oral bleeding, or epistaxis as well as symptoms and signs related to specific bacterial infections. Patients with APL are particularly susceptible to disseminated intravascular coagulation (DIC) and extensive bleeding is common at onset. The most common sites of clinically overt extramedullary leukemic infiltration include superficial lymphonodes, liver, and spleen. The leukocyte counts are usually lower than those observed in other AML subtypes and the differential counts reveal a variable percentage of blasts in the majority of patients. In most cases, anemia and thrombocytopenia are present at diagnosis. Abnormal promyelocytes constitute more than 20% of marrow-nucleated cells or more than 20% of leukocytes in peripheral blood. Leukemic blasts are morphologically characterized by the presence of distinctive, large cytoplasmic granules, frequent multiple Auer rods, and a folded nucleus. The hypogranular variant (M3-variant) is characterized by the expansion of blasts containing large number of small granules that may be difficult to distinguish by light microscopy and may be wrongly classified as monoblasts. However, both in the classical and variant M3 subtypes the cells are strongly positive for myeloperoxidase staining. A more rare hyperbasophilic variant has been described. The diagnosis is usually suspected upon the morphological examination of bone marrow and peripheral blood smears. The immunophenotypic

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Acute Promyelocytic Leukemia

Acute Promyelocytic Leukemia, Table 1 Molecular genetics of acute promyelocytic leukemia Translocation t(15;17) t(11;17) t(5;17) t(11;17) t(17;17)

Fusion proteins PML–RARa PLZF–RARa NPM–RARa NuMA–RARa STAT5b–RARa

profile suggestive of APL is composed by heterogenous intensity of expression of the CD13 surface marker associated with a homogenous expression of CD33; HLA-DR is negative in the majority of cases, and the expression of CD15 and CD34 is mutually exclusive and usually dim. The genetic confirmation of gene rearrangements involving the RARa locus is mandatory and can be done by classical cytogenetics, FISH, or RT-PCR. The pattern of immunofluorescence staining using an anti-PML antibody is also useful for a rapid diagnosis of APL. In APL cells, a nuclear microspeckled pattern is observed in contrast to other subtypes of AML in which larger and less numerous dots (nuclear bodies) are evident. DIC occurs in 75% of M3 patients accompanied by secondary fibrinolysis. The cause of coagulopathy is complex, resulting from a combination of tissue factors and cancer procoagulantinduced activation of the coagulation, exaggerated fibrinolysis due predominantly to enhanced expression of annexin II on APL blasts and blast cell production of cytokines. Laboratory evidence of DIC (prolonged prothrombine time and partial thromboplastin time, decreased fibrinogen, and increased fibrin degradation products) should be examined in all APL patients. Molecular Characterization APL has been well characterized at the molecular level and has become one of the most compelling examples of aberrant transcriptional regulation in cancer pathogenesis. Due to reciprocal translocations, the RARa gene on chromosome 17 is fused to one of five distinct partner genes (for brevity, hereafter referred as X genes; Table 1). In the vast majority of cases, RARa fuses to the PML gene (originally named myl) on chromosome 15. In a few cases, RARa fuses to the PLZF gene, to the

RARa–PML RARa–PLZF RARa–NPM RARa–NuMA? RARa–STAT5b?

Response to RA Good Poor Good Good Poor?

NPM gene, to the NuMA gene, or to the STAT 5B gene located on chromosomes 11, 5, 11, or 17, respectively. The various translocations result in the generation of X–RARa and RARa–X fusion genes and the coexpression of their chimeric products in the leukemic blasts. The characterization of the genetic events of APL, and the availability of techniques such as FISH and RT-PCR, render it possible to confirm the diagnosis at the molecular level and to monitor minimal residual disease. RARa is a member of the superfamily of nuclear receptors, which acts as a retinoic acid (RA)-dependent transcriptional activator in its heterodimeric form with retinoid-X-receptors (RXR). In the absence of RA, RAR/RXR heterodimers can repress transcription through histone deacetylation by recruiting nuclear receptor corepressors (SMRT), Sin3A, or Sin3B, which in turn, form complexes with histone deacetylases (HDAC) resulting in nucleosome assembly and transcription repression. PML–RARa represses transcription not only through HDAC but also via interactions with DNA methyltransferases (DNMTs) leading to hypermethylation at target promoters. The epigenetic changes induced by PML–RARa are stable and maintained throughout cell divisions. ATRA causes the disassociation of the corepressor complex and the recruitment of transcriptional coactivators to the RAR/RXR complex. This is thought to result in terminal differentiation and growth arrest of various types of cells, including normal myeloid hematopoietic cells. The X–RARa fusion proteins function as aberrant transcriptional repressors, at least in part, through their ability to form repressive complexes with corepressors such as NCoR and HDACs. PLZF–RARa can also form, via its PLZF moiety, corepressor complexes that are less sensitive to RA than the PML–RARa corepressor complexes,

Acute Promyelocytic Leukemia

thus justifying the poorer response to RA-treatment observed in these patients (see also Therapeutics). The X–RARa oncoproteins retain most of the functional domains of their parental proteins and can heterodimerize with X proteins, thus potentially acting as doubledominant-negative oncogenic products on both X and RAR/RXR regulated pathways. It has been demonstrated that APL blasts present a marked defect in TGF-b signaling including Smad2/3 phosphorylation and nuclear translocation, which is similar to that in PML null primary cells. Remarkably, RA-treatment, which induces PML–RARa degradation, resensitizes the cells to TGF-b. It is plausible that PML–RARa may inhibit TGF-b signaling through direct inhibition of the interaction between Smad3 and the cytoplasmic form of PML (cPML). Modeling APL in Mice The transgenic approach in mice has been used successfully in modeling APL and in generating faithful mouse models harboring various APL fusion genes. In vivo, transgenic mice (TM) harboring X–RARa oncoproteins develop leukemia after a long latency suggesting that the fusion proteins are necessary but not sufficient to cause full-blown APL. In the PML–RARa TM model, mice develop a form of leukemia that closely resembles human APL, presenting blasts with promyelocytic features that are sensitive to the differentiating action of RA. A similar phenotype was observed in NuMA–RARa TM, in which leukemia was also preceded by a period of latency but displayed a higher penetrance. On the contrary, the leukemia developed by the PLZF–RARa TM lacked the distinctive differentiation block at the promyelocytic stage, morphologically resembling more a chronic myeloid leukemia (CML) type of disease, while NPM–RARa TM developed myelomonocytic leukemia. This analysis demonstrated that the X–RARa fusion protein plays a critical role in determining leukemic phenotype as well. Moreover, it is the X moiety of the X–RARa product to determine sensitivity to ATRA, since leukemia in PML–RARa, but not in the PLZF–RARa TM, is responsive to ATRA treatment. Modeling APL in

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TM contributed to the understanding of the important role of the reciprocal RARa–X fusion proteins. RARa–PML and RARa–PLZF TM do not develop overt leukemia. However, the coexpression of RARa–PML with PML–RARa increases the penetrance and the onset of leukemia development in double mutants. Strikingly, in the PLZF–RARa TM model, the coexpression of RARa–PLZF with PLZF–RARa metamorphoses the “CML-like” leukemia in PLZF–RARa TM to leukemia with classical APL features. In addition, RARa–PLZF renders the leukemic blasts even more unresponsive to the differentiating activity of RA. At the transcriptional level, RARa–PLZF acts as an aberrant transcription factor that can interfere with the repressive ability of PLZF. Therefore, RARa–X and X–RARa fusion products act in combination to dictate the distinctive phenotypic characteristics of each APL subtype disease. Modeling of APL in the mouse is thus allowing a better comprehension of the molecular mechanisms underlying the pathogenesis of APL as well as the development of novel therapeutic strategies. Therapeutics The exquisite sensitivity of APL blasts to the differentiating action of RA makes APL a paradigm for therapeutic approaches utilizing differentiating agents. This therapeutic approach conceptually differs from the treatments involving drug and/or irradiation therapies because instead of eradicating the neoplastic cells by killing them, it reprograms these cells to differentiate normally. The utilization of ATRA in APL patient management has reduced early death from DIC-related complications and dramatically improved the prognosis. However, treatment with ATRA alone in APL patients induces disease remission transiently and relapse is inevitable if remission is not consolidated with chemotherapy. Most contemporary therapy protocols incorporate an anthracycline (e.g., dauno or idarubicin) with ATRA during induction, followed by consolidation therapy with ATRA, anthracyclines, and cytarabine, followed by maintenance therapy. Leukocyte and platelet counts at diagnosis are frequently used as risk factors for relapse: patients

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presenting with more than 10,000 leukocytes/ml have high risk in contrast with those with less than 10,000/ml and platelet counts higher than 40,000/ml. In the majority of cases, relapse is accompanied by RA resistance. Unlike t(15;17)/ PML–RARa APL, t(11;17)/PLZF–RARa leukemias show a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with RA, thus defining a new APL syndrome. Up to 50% of patients treated with ATRA alone develop an “ATRA syndrome” characterized by a rapid rise in circulating polymorphonuclear leucocytes and associated with weight gain, fever, occasional renal failure, and cardiopulmonary failure, which may be life threatening in some patients. The combination of ATRA and chemotherapy in the induction and consolidation treatment phases has been proven to be an effective strategy to prevent “ATRA syndrome” and achieve long-term disease-free survival. Arsenic trioxide (As2O3), a chemical used in Chinese medicine, is also extremely effective in the treatment of APL. About 90% of APL patients treated with As2O3 alone achieve complete remission, especially in relapsed patients who are resistant to RA and/or conventional chemotherapy. RA triggers blast differentiation while As2O3 induces both apoptosis and partial differentiation of the leukemic blasts. Utilizing PML–RARa and PLZF–RARa transgenic mouse models of APL, it has been demonstrated that the association of RA and As2O3 is effective in the former but not in the latter. Considering the importance of HDACmediated transcriptional repression in APL pathogenesis, the utilization of histone deacetylase inhibitors (HDACIs) such as suberanilohydroxamic acid (SAHA) or sodium phenylbutyrate (SPB) in combination with RA may represent a promising experimental therapeutic approach. Preclinical studies in transgenic mouse models of APL suggest that in fact HDACIs work as growth inhibitors and inducers of apoptosis and that these effects are potentiated by RA.

Acute-Phase Response Factor

References Lin H-K, Bergmann S, Pandolfi PP (2005) Deregulated TGF-b signaling in leukemogenesis. Oncogene 24:5693–5700 Rego EM, Pandolfi PP (2002) Reciprocal products of chromosomal translocations in human cancer pathogenesis: key players or innocent bystanders? Trends Mol Med 8:396–405 Rego EM, Ruggero D, Tribioli C et al (2006) Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha. Oncogene 25(13):1974–1979 Sanz M (2006) Treatment of acute promyelocytic leukemia. Hematol/Am Soc Hematol Educ Program 147–155 Scaglioni PP, Pandolfi PP (2007) The theory of acute promyelocytic leukemia revisited. Curr Top Microbiol Immunol 313:85–100

Acute-Phase Response Factor ▶ STAT3

ACVR1 ▶ Activin Receptors

ACVR2 ▶ Activin Receptors

1-acyl-sn-glycerol-3-phosphate ▶ LPA

2-acyl-sn-glycerol-3-phosphate ▶ LPA

ADAM Molecules

ADAbp ▶ CD26/DPPIV in Cancer Progression and Spread

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identified in a variety of species. A large proportion (13 ADAMs) is exclusively expressed in the male reproductive system, and only a minority can be found throughout all tissues.

Characteristics

ADA-CP ▶ CD26/DPPIV in Cancer Progression and Spread

ADAM Molecules Jörg Ringel1 and Matthias Löhr2 1 Department of Medicine A, University of Greifswald, Greifswald, Germany 2 Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden

Synonyms A disintegrin and metalloprotease; Disintegrin metalloproteases; Metalloprotease disintegrin cysteine rich; MDC

Definition A disintegrin and metalloprotease (ADAM) molecules share a common domain structure: a propeptide (prodomain), a metalloproteinase domain, a disintegrin domain, a cysteine-rich region, an epidermal growth factor (EGF)-like domain, a transmembrane region, and a cytoplasmatic domain (Fig. 1). Several ADAMs exist in both membrane-bound and secreted isoforms; the functional significance of this, in most cases, is still unclear. A subset of the presently known ADAM molecules shows catalytic activity. To date, at least 40 ADAMs have been

ADAM molecules, with their unique potential to combine ▶ adhesion, proteolysis, and signaling, are involved in a variety of cellular functions. Some have been shown to play an important role in diverse biological processes such as fertilization, myogenesis, cell signaling, inflammatory response, and cell–cell/cell–matrix interactions. However, the respective key function has remained elusive for most ADAMs. Dysregulation of ADAM molecules has been shown in various diseases. However, there is a growing amount of reports about the role of ADAM molecules in malignant tumors. Metalloprotease Function To regulate biological activity, in normal as well as in malignant cells, a wide variety of proteins are synthesized as inactive precursors that are subsequently converted to their mature active forms by ADAM molecules. A well-studied member of the ADAM molecules is ADAM17/TACE, which was originally described as being responsible for the proteolytic cleavage of the soluble form of TNF-a. Subsequent studies have shown that ADAM17/TACE is also involved in the shedding of other biologically active proteins, including growth factors (erbB4/ HER-4 and transforming growth factor (TGF)-a), surface molecules (L-selectin), and interleukin (IL) receptors (IL-R, IL-1R type II, and IL-6R; Fig. 2). TACE cleavage functions in the activation of EGF receptor (EGFR) and EGFR signaling systems, which regulate the proliferation and motility of ▶ squamous cell carcinoma cells in vitro. The key role of the EGFR/EGFR ligand system for cancer development is well known. In this context, the transactivation of EGFR via

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ADAM Molecules

Prodomaine

Cysteine-rich

Metalloprotease

EGF-like

Disintegrin

Transmembrane

ADAM Molecules, Fig. 1 Domain structure of ADAMs. The ADAMs consist of a propeptide domain, a metalloprotease domain, a disintegrin domain, a cysteine-

ADAM Molecules, Fig. 2 Schematic overview about the published functions and interactions of ADAM17/ TACE

Cytoplasmatic

rich region, an EGF-like domain, a transmembrane domain, and a cytoplasmatic domain

Shedding/activation function IL-15Rα EGF receptor ligand IL-1RII HB-EGF, TGFα L-selectin, MUC1, erbB4/HER4 GPIb α

Function α5β1 integrin

TNFα; p55, p75 TNF-R

TRANCE

ADAM17

Cell-cycle accociated molecules MAD2 (mitotic arrest deficient 2)

ADAM17/TACE is of special interest. ADAMs such as ADAM9 and ADAM17/TACE regulate G protein-coupled receptor-induced cell proliferation and survival. Aberrant expression of a proteolytic active ADAM17/TACE has been reported in pancreas ▶ cancer cells. The increasing prevalence of ADAM17/TACE expression with higher pancreatic intraepithelial neoplasia (PanIN) grade as precursor lesions underlines the role of this molecule in ductal pancreatic adenocarcinoma development. Gene silencing experiments showed a critical role of ADAM17/TACE in the invasion process of pancreatic cancer cells. The aberrant expression of proteolytically active ADAM17/ TACE may result in an uncontrolled turnover of activated target molecules, such as TNF-a, TGF-a, and MUC1 (mucius).

Adhesion

TACE

Signaling function SH3-binding domain EGFR transactivation

Silencing of ADAM17 in human renal carcinoma cell lines corrects critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. In addition, these cells fail to form in vivo tumors in the absence of ADAM17. It has also been shown that ADAM17/TACE is overexpressed in mammary cancer and other cancer types (Table 1). ADAM12, which is upregulated, for example, in breast and gastric cancer (Table 1), is expressed in two splice forms, the transmembrane ADAM12-L and the soluble ADAM12-S. In a mouse breast cancer model, ADAM12 decreased tumor cell apoptosis and increased stromal cell apoptosis. The shedding of heparin-binding EGF by ADAM12 was shown to promote human glioblastoma. In addition, in liver cancers, ADAM12 and ADAM9 expressions are associated with

ADAM Molecules ADAM Molecules, Table 1 Overview about the aberrant expression of ADAM molecules in different human cancer types as published ADAM molecule ADAM2 ADAM8 ADAM9 ADAM10 ADAM11 ADAM12

ADAM15 ADAM17/ TACE ADAM19 ADAM21 ADAM22 ADAM23 ADAM28 ADAM29

Human cancer type Renal Brain, prostate, lung adenocarcinoma Prostate, colon, pancreas, liver, gastric, non-small cell lung cancer, renal Breast, colon, prostate, pheochromocytoma, neuroblastoma Glioma, breast Breast, gastric, glioblastoma, liver, aggressive fibromatosis, giant cell tumor of the bone, brain Prostate, breast, lung, ovarian, gastric, brain, bladder Pancreas, renal, breast, colon, liver, brain, squamous cell carcinoma cells Brain ADAM21-like (ADAM21-L) T-cell leukemia Brain Brain, gastric, breast (pancreas) Non-small cell lung carcinoma Chronic lymphocytic leukemia

tumor aggressiveness and progression. ADAM9 is also described to shed heparin-binding EGF. Overexpression of cytoplasmatic ADAM9 in pancreatic cancer is associated with poor differentiation and shortened survival. It is of particular interest for cancer development that ADAM molecules reported to shed cellassociated adhesion molecules such as L-selectin, MUC1, and glycoprotein (Gb) 1ba. In general, the metalloprotease protease function might be involved in various processes of cancer cells and be relevant to promote cell migration and invasion. Adhesion Function ADAM molecules are potential ligands for integrins due to the presence of binding sites within the disintegrin domain. Only one ADAM (ADAM15) contains the RGD integrin-binding motif, and it can therefore interact not only with the avb3 integrin but also with the avb5. Additional ADAM–integrin interactions have been

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reported: a large number of ADAMs (1, 2, 3, 9, 12, and 15) with a9b1, ADAM9 with a6b1 and avb5, and ADAM28 with a4b1. Considering the published data on the interaction of ADAM17/TACE with the a5b1 integrin in HeLa cells, it is also conceivable that ADAM17/TACE may influence the migration and invasion in other cancer types. We are beginning to gather insights into ADAM–integrin and ADAM–▶ extracellular matrix (ECM) interactions. The interplay with integrins and ECM compounds might promote ADAM function in malignant cells. Thus, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions. Furthermore, it was shown that the cysteine-rich domain of ADAM12 supports tumor cell adhesion through syndecan. ADAM23 with its inactive metalloprotease domain is exclusively involved in cell adhesion. It was demonstrated that the interaction between the disintegrin loop of ADAM23 and the avb3 integrin promotes the adhesion of ▶ neuroblastoma and ▶ astrocytoma cells. In contrast to the described overexpression or de novo expression in various cancer types, downregulation of ADAMs might also promote cancer development. Thus, ADAM23 gene silencing in breast cancer by promoter hypermethylation may result in abnormal cell–cell interactions favoring cell migration. Signaling Function Beside the involvement of ADAM molecules in the EGFR transactivation, only few data about the signaling function of ADAM molecules are known. It is intriguing that interactions between integrins and/or ECM- and ADAM-binding domains may induce outside–in signaling. ADAM inside–out signaling pathways might regulate shedding and/or adhesion function of the molecules. However, many ADAM cytoplasmatic domains contain binding motives for the Src homology region 3 (SH3 domain) of various intracellular proteins. Tyrosine residues could be substrates for tyrosine kinases or could act as ligands for phosphotyrosine-binding domains, when phosphorylated. A number of binding partners

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have been identified for the cytoplasmatic domains of various ADAM molecules. Interaction of the cytoplasmatic domain of ADAM9 and ADAM15 with endophilin and SH3PX1 is reported. ADAM12 and ADAM15 are associated with ▶ Src protein–tyrosine kinases. However, the shedding of the L1 adhesion molecules in breast cancer cells might involve a Scr protein–tyrosine kinase. Furthermore, mitotic arrest-deficient-2 (MAD2) was found as binding partner of ADAM17/TACE and ADAM15; MAD2b is linked to ADAM9. To date, the physiological role of these interactions as well as the implication in malignancies is speculative. Other Functions Within the ADAM molecules, ADAM11 might play a special role in malignancies. ADAM11 represents a candidate tumor suppressor gene for human breast cancer. This is based on its location within a minimal region of chromosome 17q21 previously defined by tumor deletion mapping. Taken together, there are rapidly increasing data supporting a critical implication of ADAM molecules in malignancies. But there are still more questions than answers on the function of ADAMs in human cancer and cancer development.

Cross-References

ADAM17 increases the malignant potential in human pancreatic ductal adenocarcinoma. Cancer Res 66(18):9045–9053 Seals DF, Courtneidge SA (2003) The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 17:7–30

ADAM17 Aleksandra Franovic and Stephen Lee Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

Synonyms CD156b antigen; TACE; Tumor necrosis factoralpha converting enzyme

Definition ADAM17 is a zinc-dependent metalloprotease belonging to the ADAM (A disintegrin and metalloproteinase) family of type I transmembrane proteins. ADAM17 is involved in the ectodomain shedding of a wide variety of membrane-bound ligands and cytokines that are implicated in diverse biological processes including growth and ▶ inflammation.

▶ Extracellular Matrix Remodeling

References Gschwind A, Hart S, Fischer OM et al (2004) TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. EMBO J 22:2411–2421 Iba K, Albrechtsen R, Gilpin BJ et al (1999) Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion. Am J Pathol 54:1489–1501 Karan D, Lin FC, Bryan M et al (2003) Expression of ADAMs (a disintegrin and metalloproteases) and TIMP3 (tissue inhibitor of metalloproteinase-3) in human prostatic adenocarcinomas. Int J Oncol 23:1365–1371 Ringel J, Jesnowski R, Moniaux N et al (2006) Aberrant expression of a disintegrin and metalloproteinase 17/tumor necrosis factor-alpha converting enzyme

Characteristics Structure The 50 kb ADAM17 gene, which is located at chromosome 2p25, consists of 19 exons, and encodes an 824 amino acid protein. ADAM17 is synthesized as an inactive precursor protein consisting of five domains: the pro-, metalloprotease, cysteine-rich, transmembrane, and cytoplasmic domains. Prior to ADAM17 maturation, a conserved cysteine residue within the pro-domain interacts with the active site zinc atom maintaining the enzyme biologically inert. The active site of the metalloprotease domain contains

ADAM17

a histidine consensus sequence (HExxHxxGxxH) that coordinates zinc atoms and water required for the enzymatic processing of ADAM17 substrates. Removal of the pro-domain occurs through a ▶ furin cleavage site (RVKR), by an unidentified furin or proprotein convertase, enabling the active site zinc to interact with the required histidine residues and to generate the active protease. While the structural and functional aspects of the pro- and metalloprotease domains have been studied extensively and are well defined, the precise functions of the remaining ADAM17 domains are still somewhat obscure. The cysteine-rich domain consists of two subdomains: the disintegrin and EGF-like domains. A role in cellular ▶ adhesion has been proposed for the disintegrin domain. In support of this hypothesis, ADAM17 has been shown to interact with at least one integrin (a5b1) and modulate cell migration as a result of this interaction. It has also been demonstrated that the cysteine-rich domain is indispensable for the ectodomain shedding of select ADAM17 substrates and, thus, might function in substrate recognition through the recruitment of accessory proteins or direct contact with the substrates themselves. The transmembrane domain tethers mature ADAM17 in the cell membrane where it exerts most of its physiological functions. Finally, the cytoplasmic domain comprises several Src homology 2 (SH2) and 3 (SH3) domain binding sites as well as phosphorylation sites, and is likely involved in regulatory signal transduction pathways. Expression and Regulation ADAM17 mRNA is ubiquitously expressed in most adult tissues, albeit at lower levels than those observed in fetal tissues at various stages of development. The ADAM17 zymogen is synthesized in the rough endoplasmic reticulum and is processed in the late Golgi compartment to produce the mature protease lacking the inhibitory pro-domain. This maturation step seems to entail a constitutive process as the majority of cellular ADAM17 exists in its mature form. The greater part of ADAM17 protein is localized in the perinuclear area while the remaining fraction resides at the cell surface, as expected. Notably,

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it appears that the membrane-bound ADAM17 population is exclusively in the processed form. This surface pool of ADAM17 is relatively stable with a half-life of ~8 h. The mechanism by which ADAM17 function is regulated is not entirely clear; however, two methods by which the protease can be activated have been described. The first method involves the activation of ADAM17 by growth factors, such as the ▶ fibroblast growth factor (FGF) and the ▶ platelet-derived growth factor (PDGF). ADAM17-mediated ligand shedding can also be induced by non-physiological stimuli such as phorbol esters (phorbol myristate acetate). Treatment of cells with phorbol esters, such as PMA, results in increased ligand shedding without affecting the quantity or localization of endogenous ADAM17 in the cell. There is conflicting evidence with respect to the mechanism by which this stimulation occurs. One study demonstrated that PMA exerts its effects by activating the extracellular signal-regulated kinase (ERK) signaling pathway, which results in the phosphorylation of ADAM17 at Thr735 in its cytoplasmic tail, while another group showed that the cytoplasmic tail of ADAM17 is not required for PMA-induced ligand shedding. Although there is no evidence that phorbol esters regulate ADAM17 activity in vivo, the ERK signaling pathway has also been implicated in growth factor stimulated ADAM17 activation. For this reason, the ERK signaling pathway will likely be the focus of future studies aimed at delineating the mechanisms involved in the positive regulation of ADAM17 activity. In addition to stimulating ADAM17-mediated ligand cleavage, the treatment of cells with PMA also triggers the establishment of a negative feedback mechanism. Following an increase in ADAM17 activity and ligand shedding, the protease itself is internalized and degraded in response to prolonged treatment with PMA. This negative regulatory mechanism is probably in place to prevent over-stimulation of ligandactivated signaling pathways. In attempt to identify potential regulators of ADAM17 activity, two ADAM17 binding partners were uncovered by at least two-hybrid screens: synapse associated

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ADAM17

protein 97 (SAP97) and protein tyrosine phosphatase PTPH1. Overexpression of either molecule results in decreased ligand shedding implicating them in the negative regulation of ADAM17 activity. Whether either of these two proteins regulates ADAM17 activity in vivo remains to be seen. The only known endogenous inhibitor of ADAM17 is the tissue metalloprotease inhibitor, TIMP3. The mechanism by which TIMP3 expression results in reduced ADAM17 activity is unknown.

These substrates include the TNF receptors (TNF-RI and TNF-RII), the chemokine fractalkine, and the leukocyte adhesion molecule L-selectin to name a few. While many ADAM17 substrates have been identified to date, there is no obvious sequence or structural homology between their cleavage sites. How ADAM17 achieves substrate specificity is a key question that remains to be answered. Nonetheless, it is evident that ADAM17 substrates play an important role in a broad range of fundamental cellular processes.

Biological Function ADAM17 was initially identified as the secretase responsible for the cleavage of tumor necrosis factor-alpha (TNFa), a pro-inflammatory cytokine. The generation of transgenic mice expressing ADAM17 lacking the zinc-binding sequence in its metalloprotease domain (ADAM17DZn/DZn) allowed for the identification of a multitude of additional ADAM17 substrates. The vast majority of the ADAM17DZn/DZn mice die at birth as a result of severe deficiencies in skin, muscle, lung, and neuronal system development that cannot be entirely attributed to loss of TNFa shedding. This indicates the existence of other biologically relevant ADAM17 substrates. Interestingly, the few animals that do survive display a phenotype that is comparable to that of transforming growth factor alpha (TGFa) or ▶ epidermal growth factor receptor (EGFR) knockout mice. This includes the failure of eyelids to fuse as well as defects in skin and hair follicle development. Upon further investigation it was confirmed that TGFa, an EGFR ligand, is in fact an ADAM17 substrate. Moreover, ADAM17 appears to be the major convertase of several EGFR ligands which are involved in a variety of cellular processes including cellular proliferation, survival, migration, and differentiation. The bulk of ADAM17 substrates, including the EGFR ligands, are involved in cell development and differentiation. Other examples include the neurogenic signaling molecule Notch, the neurotrophin receptor TrkA, and the EGFR-family receptor HER4. The remaining substrates can be classified as those involved in cellular immunity and regulation of immunogenic responses, like TNFa.

Clinical Relevance Due to its involvement in TNFa processing, ADAM17 is considered to be a central mediator in human inflammatory diseases such as rheumatoid arthritis. Direct inhibition of TNFa or ADAM17 in arthritis-affected cartilage has been shown to reduce inflammation. For these reasons ADAM17-based therapies, such as zinc-chelating sulfonamide hydroxamates, are in use for the treatment of such diseases. In addition to its role in inflammatory diseases, ADAM17 is becoming increasingly implicated in the development and progression of cancer as a result of its role in the processing of EGFR ligands. The upregulation of EGFR expression and signaling is a common feature in human cancer. Unfortunately, EGFR inhibitors have rendered disappointing results in ▶ clinical trials and there is an apparent resistance of several cancer cell lines to these agents. Importantly, ADAM17 is also overexpressed in several neoplastic tissues including breast carcinomas, colon carcinomas, pancreatic ductal adenocarcinomas, and ovarian carcinomas. There is also a positive correlation between ADAM17 expression and the aggressiveness of the malignancy. Thus ADAM17 is most highly expressed in advanced tumors, suggesting that ADAM17 and its substrates play a role in tumor progression. In accordance with these observations, there is a growing amount of evidence supporting the use of anti-ADAM17 drugs in the treatment of cancer. Several studies have shown that inhibition of ADAM17 activity using a variety of approaches is sufficient to inhibit EGFR ligand release and to prevent the proliferation, migration, and survival

ADAM17

of squamous cell, kidney cancer, ▶ bladder cancer, and ▶ breast cancer cell lines in vitro. It was demonstrated that ▶ siRNA-mediated silencing of ADAM17 inhibits the release of soluble TGFa in highly malignant renal carcinoma cells, thereby abolishing their ability to form tumors in nude mice. This was the first in vivo evidence that ADAM17-mediated ligand cleavage is a pivotal step in the establishment of the TGFa/EGFR autocrine (▶ autocrine signaling) growth stimulatory loop and thus in tumorigenesis. Another study revealed that targeting ADAM17, using a small molecule inhibitor, prevents heregulin cleavage and hence HER3 activation in non–small cell lung cancer cells. Not only did this inhibition abolish tumor growth in vivo but it also enhanced the sensitivity of the cancer cells to gefitinib, an anti-EGFR based therapy. This result suggests that the concomitant inhibition of ADAM17 and EGFR should improve patient responsiveness to such agents and increase survival. Thus targeting ADAM17 is a promising new alternative to traditional EGFR-based therapies in the treatment of human cancer. Summary ADAM17 was originally characterized for its role in TNFa processing and the regulation of inflammatory responses. It has since been demonstrated that ADAM17 is also a physiological convertase of a wide variety of signaling molecules implicated in the development and progression of cancer. The importance of ADAM17 in these oncogenic pathways is highlighted by the finding that silencing of ADAM17 is sufficient to abolish tumor formation in vivo. These results validate ADAM17 as a rational therapeutic target and endorse the use of ADAM17 inhibitors in the treatment of human cancer.

Cross-References ▶ ADAM Molecules ▶ Adhesion ▶ Autocrine Signaling ▶ Bladder Cancer

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▶ Breast Cancer ▶ Epidermal Growth Factor Receptor ▶ Extracellular Signal-Regulated Kinases 1 and 2 ▶ Fibroblast Growth Factors ▶ Furin ▶ Inflammation ▶ Platelet-Derived Growth Factor ▶ Renal Cancer Clinical Oncology ▶ Renal Cancer Genetic Syndromes ▶ SH2/SH3 Domains ▶ SiRNA ▶ Transforming Growth Factor-Beta

References Blobel CP (2005) ADAMS: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 6:32–43 Franovic A, Robert I, Smith K et al (2006) Multiple acquired renal carcinoma tumor capabilities abolished upon silencing of ADAM17. Cancer Res 66:8083–8090 Lee DC, Sunnarborg SW, Hinkle CL et al (2003) TACE/ ADAM17 processing of EGFR ligands indicates a role as a physiological convertase. Ann N Y Acad Sci 995:22–38 Seals DF, Courtneidge SA (2003) The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 17:7–30 Zhou BS, Peyton M, He B et al (2006) Targeting ADAMmediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell 10:39–50

See Also (2012) EGFR In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1828 (2012) ERK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1307–1308. doi:10.1007/978-3-642-16483-5_1987 (2012) Extracellular Signal-Regulated Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1365. doi:10.1007/978-3-64216483-5_2070 (2012) HER3. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1680. doi:10.1007/978-3-642-16483-5_2678 (2012) Heregulin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1684. doi:10.1007/978-3-642-16483-5_2685 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084

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62 (2012) Metalloproteases. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2259. doi:10.1007/978-3-642-16483-5_3666 (2012) Notch Signaling. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2559. doi:10.1007/978-3-642-16483-5_4131 (2012) Phorbol 12-Myristate 13-Acetate. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2865. doi:10.1007/978-3-642-164835_4523 (2012) PMA. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2930–2931. doi:10.1007/978-3-642-16483-5_4641 (2012) Renal Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3225– 3226. doi:10.1007/978-3-642-16483-5_6575 (2012) Small-Molecule Inhibitors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3455. doi:10.1007/978-3-642-16483-5_5375

Adaptive Immunity

a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to cancer cells, and the ability to mount these tailored responses is maintained in the body by “memory cells.” Cells of the adaptive immune system are B and T lymphocytes. Adaptive humoral responses are mediated by tumorspecific antibodies.

Cross-References ▶ DNA Vaccination ▶ Immunoediting ▶ Immunoprevention ▶ Inflammation

Adaptive Immunity Definition Adaptive immune responses occur when the host comes into contact with immunogenic molecules or organisms. These stimulate the expansion of the antigen-specific lymphocytes, antibody-secreting B cells and T cells of the cytotoxic and helper phenotypes, which recognize cells expressing foreign antigens. B cells and T cells are the effector cells of the adaptive immune response. They bear antigen-specific receptors of great diversity that are generated by random rearrangement of gene segments and other mechanisms. This results in a vast array of antigen-specific receptors clonally distributed on T and B cells, which clonally expand on contact with antigen. As the immunogen is cleared, these clonal populations shrink but leave behind long-lived populations of memory cells that are easily recalled on subsequent exposure to the same immunogen. Unlike the innate immune response, adaptive responses are not immediate, requiring 3–5 days for clonal expansion and differentiation of effector lymphocytes. The adaptive immune system allows for a strong immune response as well as immunological memory, where a tumor antigen is “remembered.” The adaptive immune response is antigen-specific and requires the recognition of tumor antigens during

Adaptor Proteins Alessio Giubellino Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

Definition Adaptor proteins are cell signaling molecules linking intracellular proteins, including cell surface receptors to cytosolic effectors.

Characteristics In their pure form, adaptor proteins are devoid of any intrinsic enzymatic activity and serve as intracellular platforms for the amplification and coordinated assembly of multimeric protein complexes. Adaptor proteins provide a diverse array of functions, including: The entry “Adaptor Proteins” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

Adducts to DNA

1. Co-localize signaling proteins in a specific area of the cell 2. Bring together enzymes and substrates to facilitate specific reactions 3. Coordinate diverse signals in a timely fashion A common feature of adaptor proteins is the organization in modular structures; a limited number of highly evolutionary conserved protein sequences (“domains” or “modules”) are combined to produce a diverse array of protein structures with specific cellular functions and diverse connecting capabilities. In an oversimplified model, upon stimulation by extracellular ligands (e.g., growth factors), cell surface receptors become activated. The activation is responsible for the transient posttranslational modification (e.g., phosphorylation) of specific residues inside a defined amino acid sequence domain, which is recognized as specific docking sites by intracellular adaptor proteins acting as signaling transducers. The presence of such modular sequence was originally characterized through structural analysis of the protein kinase Src, which uncovered the presence, besides the catalytic domain (named SH1 domain), of other sequences with peculiar and distinct structures; those sequences were named consecutively Src-homology 2 (SH2) and Src-homology 3 (SH3) domains because of the proximity to the catalytic domain. Computational analysis has allowed the identification of similar domains in other proteins. In addition, several other protein binding modules were discovered, each recognizing specific binding motifs on partner proteins. An up-to-date list and description of known modular domains, building blocks for adaptor proteins, can be found at the lab website of Dr. Tony Pawson (http://pawsonlab.mshri.on.ca/) who has pioneered the discovery and study of such modules.

References Pawson T (2007) Dynamic control of signaling by modular adaptor proteins. Curr Opin Cell Biol 19(2):112–116, Review PubMed PMID: 17317137

63 Pawson T, Nash P (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300(5618):445–452, Review PubMed PMID:12702867 Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326(5957):1220–1224, Review PubMed PMID: 19965465; PubMed Central PMCID: PMC3041271

Adducts to DNA Helmut Bartsch Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany

Synonyms DNA-bound carcinogens

Definition DNA adducts reflect the amount of a ▶ xenobiotic that covalently reacts with nucleic acid bases at the target site (biologically effective dose) or in surrogate tissues. DNA adducts are mechanistically more relevant to ▶ carcinogenesis than the internal dose of a carcinogen, since they take into account interindividual differences in metabolism and of DNA repair capacity (Fig. 1). Several hundred DNA adducts, many with miscoding properties, are known to be produced by some 20 classes of carcinogens and through endogenous oxidative processes. DNA adducts are used in human ▶ biomonitoring as dosimeters of early biological effects and predictors of cancer risk. These ▶ biomarkers also provide tools for studying disease pathogenesis, etiology, and for verifying preventive measures in human cancer.

Characteristics Rationale for Using DNA Adducts as Biomarkers for Exposure and Adverse Effects Evidence for the biological significance of DNA adducts in carcinogenesis is supported by the following:

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Adducts to DNA

Ambient exposure

Internal dose

Biologically effective dose

Endogenous processes Genotoxic exposure

DNA Metabolism adducts

Early biological effect

Atered structure function

Mutations transduction disruption of signaling cell proliferation changed DNA methylation patterns

Repair

Clinical disease

Cancer

Metastasis

Apoptosis

Adducts to DNA, Fig. 1 Paradigm for the multistage process of ▶ carcinogenesis with DNA adducts as initiating lesions. They are used mostly as biomarkers for the biologically effective dose both of exogenous carcinogens and of DNA-reactive agents produced by endogenous

processes, such as chronic oxidative stress. Over the past 40 years, emphasis has been placed on the development of accurate and sensitive methods for the detection and quantitation of DNA adducts

• Over 80% of identified or suspected human carcinogens react often after metabolic activation with nucleic acids and proteins to form macromolecular adducts • Carcinogen-DNA adducts represent the initiating events leading to mutations in ▶ oncogenes and ▶ tumor suppressor genes and to ▶ carcinogenesis • The carcinogenic potency of a large number of carcinogens is proportional to the extent they bind to rodent liver DNA • Humans with inherited or acquired defects in ▶ DNA repair have an elevated risk of developing cancer

kinetic parameters are taken into account. These include the steady state adduct concentration; the amount of the miscoding adduct compared to others of lesser biological relevance; the adduct half-life after carcinogen exposure has stopped; and the organ, cell, and gene selectivity of the adduct (Fig. 2).

Biological effect markers are defined as indicators of irreversible genetic damage that result from genotoxic interactions at the target site. As DNA adducts do not often cause completely irreversible lesions, because the DNA undergoes repair (which may not be complete), they are not in the strict sense biological effect markers. However, as carcinogen dosage is linked to cancer outcome, and permanent mutations can be caused by DNA adducts, they are associated with cancer risk. This has been shown for many carcinogens and their DNA adducts, when critical toxico-

Advantages and Disadvantages of DNA Adducts Compared to Other Biomarkers For human biomonitoring, both DNA and protein adducts can be used for exposure assessment as long as the response in target organs versus surrogate tissue is shown to be proportional. The latter has to be determined individually for each carcinogen. The advantage of certain protein adduct measurements is that they often reflect cumulative past exposure (of several months), while the majority of DNA adducts is rapidly repaired or lost after exposure has ceased. However, a small portion of DNA adducts either with slow repair and/or subpopulations of nondividing cells can survive for several months or even years. Since somatic genetic or cytogenetic effect markers are neither chemical- nor exposurespecific, only macromolecular adducts allow identification of the structure and thus the

Adducts to DNA

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Increasing proximity to critical lesions

Target DNA sequence cell(s) specificity Cancer relevant gene(s)

White blood cells, urine

Non-target tissue

Exfoliated cells, alveolar macrophages

Target organ

Surrogate DNA adducts

Exposure to exogenous and endogenous chemicals

Adducts to DNA, Fig. 2 Measurement of carcinogenDNA adducts in target tissue and cells or in surrogates. The predictive value of DNA adducts for disease risk increases

with the proximity of measurements to critical lesions. Accordingly, from right to left, the specificity of this biomarker increases for predicting disease outcome

determination of the genotoxic exposure sources. Also, cytogenetic markers are more easily affected by lifestyle and environmental components (confounders) that often act as uncontrolled or uncontrollable variables in biomonitoring and molecular epidemiology studies. In addition, at equal levels of carcinogen exposure, DNA adduct levels are a measure for the host’s capability of carcinogen metabolism and adduct repair and can be used to determine the overall effect of genetic polymorphisms on DNA damage and cancer susceptibility by a given carcinogen.

causes hereditary nonpolyposis colorectal cancer (HNPCC). Genetic defects in these DNA repair functions or inhibition of repair proteins may have dramatic consequences when DNA adducts, DNA mismatches, and DNA loops are not repaired prior to cell replication and when damaged cells are not eliminated by apoptosis. Thus, characterization of germ-line and somatic mutations in DNA-repair genes can identify high-risk subjects who especially in the case of biallelic mutations suffer from functional defects of proteins that repair DNA adducts leading to genetic instability and cancer.

Cellular Defense: Repair of DNA Adducts DNA repair (▶ repair of DNA) systems such as base and ▶ nucleotide excision repair, O6-alkylguanine-DNA alkyltransferase, and ▶ mismatch repair operate in human cells to remove adducted and oxidatively damaged DNA bases. Deficiency in nucleotide excision repair genes cause ▶ xeroderma pigmentosum (XP) and a high-rate occurrence of skin cancers, as well as a high susceptibility to UV light and ▶ polycyclic aromatic hydrocarbon-induced carcinogenesis. A defective mismatch repair system

Adduct Measurements in Disease Epidemiology Cross-sectional and longitudinal studies in cancer epidemiology assess the relationship between carcinogen exposures and biomarker (adduct) levels. Adduct measurement exposed in humans allow the detection, quantification, and structural elucidation of specific DNA damage. Findings from such studies include the detection of background exposures manifested in “unexposed” populations and a significant interindividual variation in

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adduct levels in persons with comparable exposure. The latter is in part due to genetic variation in carcinogen metabolism and DNA-repair processes. Positive correlations between the extent of occupational and environmental exposures, adduct levels, and adverse effects, e.g., mutations in oncogenes and tumor suppressor genes have been observed. For example, large-scale studies on geographical variations of ▶ hepatocellular carcinoma and exposure to ▶ aflatoxins have used aflatoxin-bound albumin adducts, urinary aflatoxin B1-N7-guanine adducts, and mutational hotspots in the ▶ TP53 gene as biomarkers. They revealed more than an additive interaction between the hepatocarcinogen and hepatitis B virus infection. ▶ Case–control studies in disease epidemiology allow the evaluation of the role of biomarkers as cancer risk factors and the exploration of underlying mechanisms, but such studies cannot establish causality between biomarker response and cancer causation. This is especially the case when the latency period (between exposure and cancer) is long. Here, adduct measurements are of greater relevance for cancer risk estimation when exposure has been continuous. An optimal study design that can establish causality is a nested case–control study that uses questionnaire data and biological sample collection prior to disease manifestation. Once diagnosis of cancer has been made, cases are matched to appropriate controls and their stored samples analyzed. The predictive value in terms of specificity and sensitivity of a DNA adduct biomarker in biological samples can thus be determined. Association of DNA Adducts with Cancer Risk Not all types of DNA adducts are associated with the same cancer risk. Using alkylating agents, aflatoxins, and aromatic amines (that induced 50% tumor incidence) DNA adduct levels were compared in animal experiments. A 40- to 100-fold difference in the ability of DNA adducts to induce the same tumor incidence in target tissues was detected. Thus, it is difficult to predict the tumor induction potential of unknown DNA adducts. In the past, assays for DNA adduct determination provided mostly information on the total

Adducts to DNA

amount of adducts in bulk genomic DNA. However, new methods are capable of pinpointing adduct profiles in critical target genes (Fig. 2). Because of the multistage and complex nature of human carcinogenesis, carcinogen-DNA adducts per se cannot precisely and quantitatively predict an individual’s cancer risk. At present, risk estimation is limited to a group level. Background DNA Adduct Levels: Sources, Variations, and Cancer Risk Prediction The major analytical challenge has been to detect levels of DNA adducts at a concentration of 0.1–1 adducts per 108 unmodified DNA bases using only low microgram amounts of DNA, and with high specificity and accuracy. Several methods are available, including 32P-postlabeling assays often in combination with immunopurification and liquid chromatography coupled to electrospray ionization-mass spectrometry. By using ultrasensitive detection methods, background DNA adduct levels have been found in organs of unexposed humans and untreated animals. These are due to physiological lipid peroxidation (LPO) processes, whereby end products, such as 4-hydroxynonenal and malondialdehyde when formed in excess in the body, can react with DNA to yield background levels of a variety of exocyclic DNA adducts. These types of adducts generally increase with age but are significantly increased in human subjects affected by cancer risk factors that induce chronic oxidative stress. These include chronic inflammatory processes and infections, nutritional imbalances, and metal storage disorders. In addition, oxidized DNA bases and LPO-derived DNA adducts occur more frequently in cells with impaired antioxidant defense. Exogenous carcinogens can also induce oxidative stress causing agent-specific DNA adducts and secondary oxidative DNA base damage. The biological relevance of both oxidative and LPO-derived DNA damage is supported by the fact that these adducts are miscoding lesions which are recognized by specific DNA-repair enzymes. There is a growing evidence that both types of DNA lesions, either derived from exogenous and endogenous agents, play a role in the initiation and progression of the multistage

Adducts to DNA

carcinogenesis process, as well as other chronic degenerative diseases. Current research addresses some open questions: • What is the significance of endogenously formed DNA adducts in human cancer, particularly associated with chronic inflammatory conditions and also in relation to spontaneous tumors? • Has the proportion of cancers that result from environmental agents been overestimated compared to those arising from endogenous DNA damaging processes? • Can one protect humans against endogenously derived DNA damage and prevent chronic degenerative diseases by administration of chemopreventive (antioxidative) agents, using DNA adduct measurements to verify their efficacy? • Will LPO-derived DNA adducts serve as potential prognostic markers for assessing progression of chronic inflammatory cancer-prone diseases? Contributions of DNA Adduct Measurements to Disease Etiology and Pathogenesis New insights are gained since • Adduct analysis permits identification of hitherto unknown exogenous and endogenous DNA-reactive agents and of carcinogenic components in complex exposures, thus increasing the power to establish causal relationships in molecular epidemiology. • Highly exposed individuals can be more readily identified, and exposure to carcinogenic risk factors can be minimized or even avoided. • Subgroups in the population (so called pharmacogenetic variants) that are, due to genetic polymorphism of xenobiotic-metabolizing and DNA-repair enzymes, more susceptible to carcinogens are identifiable by a combination of genotyping and DNA adduct measurements. • Repeated applications of dosimetry methods for macromolecular adducts can evaluate the effectiveness of primary and secondary interventions, either by reduction of carcinogen exposure or through (chemo-)preventive strategies. • Incorporation of DNA adduct measurements (and of other critical endpoints involved in

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carcinogenesis) can reduce (i) the enormous uncertainties currently associated with highto-low dose and species-to-species extrapolation and (ii) yield information on interindividual risk assessment procedures. • The role of specific carcinogen exposures may be retrospectively implicated in cancer etiology by analyzing decades after the period of exposure, mutational fingerprints in tumors that arise from exogenous and endogenous agents after their reaction with DNA. Specific mutational signatures, detected in the tumor suppressor gene TP53, were associated with distinct past carcinogen exposures (e.g., tobacco smoke, aflatoxin B1, vinyl chloride, and UV light) or inflammatory disease state (such as chronic inflammatory bowel diseases). • Adducts and derived mutations should allow to study pathogenesis and preventive approaches of chronic degenerative diseases other than cancer (e.g., atherosclerosis, Alzheimer disease).

Cross-References ▶ Biomarkers in Detection of Cancer Risk Factors and in Chemoprevention ▶ Case Control Association Study ▶ Clinical Cancer Biomarkers ▶ Hepatitis B Virus ▶ Mismatch Repair in Genetic Instability ▶ Repair of DNA ▶ Surrogate Endpoint

References Bartsch H, Nair J (2006) Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation. DNA Damage Repair Langenbecks Arch Surg 391:499–510 Gupta RC, Lutz WK (eds) (1999) Background DNA damage. Mutat Res 424:1–288 Singh R, Farmer PB (2006) Liquid chromatographyelectrospray ionization-mass spectrometry: the future of DNA adduct detection. Carcinogenesis 27:178–196 Toniolo P, Boffetta P, Shuker DEG et al (eds) (1997) Application of biomarkers in cancer epidemiology,

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68 vol 142, IARC scientific publications. IARC, Lyon, pp 143–158 Vineis P, Perera F (2000) DNA adducts as markers of exposure to carcinogenesis and risk of cancer. Int J Cancer 88:325–328

Adenine Nucleoside

years, first as a noncancerous polyp (adenoma) and later as cancer. By the age of 50, one in four people has polyps.

Cross-References

Adenine Nucleoside ▶ Adenosine and Tumor Microenvironment

Adenine-9-b-d-Ribofuranoside ▶ Adenosine and Tumor Microenvironment

▶ Adenoma ▶ Appendiceal Epithelial Neoplasms ▶ Bile Duct Neoplasms ▶ Colorectal Cancer ▶ Colorectal Cancer Premalignant Lesions ▶ Lung Cancer

See Also (2012) Polyp. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2955. doi: 10.1007/978-3-642-16483-5_6524

Adenocarcinoma Definition A form of carcinoma that originates in glandular tissue. To be classified as adenocarcinoma, the cells do not necessarily need to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. The term adenocarcinoma is derived from “adeno” meaning “pertaining to a gland” and “carcinoma” which describes a cancer that has developed in the epithelial cells, i.e., cells that line the walls of various organs. This type accounts for about 40% of ▶ lung cancer. It is usually found in the outer part of the lung. The cancer cells are arranged in the gland-like structure. Morphologically, adenocarcinomas are classified according to the growth pattern (e.g., papillary, tubular, alveolar) or according to the secreting product (e.g., mucinous, serous). Virtually all adenocarcinomas develop from ▶ adenoma. In general, the bigger the adenoma, the more likely it is to become malignant. For example, in ▶ colorectal cancer, a polyp larger than 2 cm has a 30–50% chance of being cancerous. By the time colorectal cancer is diagnosed, it has often been growing for several

Adenoma Definition Is a benign tumor that develops from epithelial cells. Adenoma in the colon is often referred to as adenomatous polyp. Although adenomas are not cancerous, they have the potential to become so. Colon cancer usually develops from adenomatous polyps. Adenomas that turn into cancer are referred to as adenocarcinoma.

Cross-References ▶ Colorectal Cancer Premalignant Lesions

See Also (2012) Benign Tumor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 381. doi:10.1007/978-3-642-16483-5_579. (2012) Polyp. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2955. doi: 10.1007/978-3-642-16483-5_6524.

Adenosine and Tumor Microenvironment

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Definition

Adenomas

▶ APC Gene in Familial Adenomatous Polyposis

Adenosine is a small molecule that is released into the tissue at high concentrations in response to a deficiency of oxygen, which occurs characteristically in solid tumors. Adenosine has multiple effects within the tumor, including controlling cancer cell growth, locally inhibiting the immune system, and increasing blood vessel formation.

Adenomatous Polyps

Characteristics

▶ Colorectal Cancer Chemoprevention ▶ Colorectal Cancer Premalignant Lesions

Adenosine (adenine-9-b-D-ribofuranoside, Fig. 1) is a small organic molecule that plays an important part in general cellular biochemistry. Chemically, it is a purine nucleoside. Adenosine is abundant within all cells, predominantly in the form of adenine nucleotides (AMP, ADP, and ATP) which participate widely in cellular energy metabolism and act as precursor molecules in many processes. However, adenosine itself can exist in a free form both inside and outside of cells, and extracellular adenosine is responsible for the regulation of many processes throughout the body. Adenosine becomes particularly important when tissues become deprived of oxygen (a state known as ▶ hypoxia). This can happen in certain pathological situations, including cancer. It may occur suddenly when blood flow is interrupted, as it takes place in a stroke within the brain or during a heart attack. In solid tumors, however, hypoxia is a chronic condition because the blood vessels that the cancer forms to nourish itself are not well made and are unable to supply the tissue with sufficient oxygen and other nutrients. For cells to be well oxygenated, they need to be within a distance of about 150 150 mm mu;m of a properly functioning blood vessel. Tumor vessels are typically far apart, are irregular in both size and orientation, and can be so poorly regulated that the blood flow may periodically change direction. Cancer cells respond to these harsher conditions by changing their metabolism. In hypoxic cancer tissues, the balance of energy metabolism in the cells becomes altered. Specific changes in the biochemical pathways of

▶ Colorectal Cancer Premalignant Lesions

Adenomatous Polyposis Coli

Adenomucinosis ▶ Appendiceal Epithelial Neoplasms

Adenopathy Definition An enlargement or increase in size of glandular organs or tissues usually resulting from disease processes.

Adenosine ▶ Adenosine and Tumor Microenvironment

Adenosine and Tumor Microenvironment Jonathan Blay Department of Pharmacology, Dalhousie University, Halifax, NS, Canada

Synonyms Adenine nucleoside; Adenine-9-b-D-ribofuranos ide; Adenosine; Purine nucleoside

A

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Adenosine and Tumor Microenvironment

hypoxic cells dramatically change the fate of adenosine. Free adenosine is normally formed principally from adenine nucleotides by the enzyme 50 -nucleotidase inside the cell (some tissues have another pathway that also contributes)

NH2 N

N

Adenine

N

N

HOCH2 O H H H H OH

Ribose

OH

Adenosine and Tumor Microenvironment, Fig. 1 The chemical structure of adenosine. Adenosine is composed of a purine base (adenine) linked through a glycosidic bond to a sugar (ribose). Successive phosphate groups may be added at the position indicated by the arrow to give AMP (adenosine monophosphate), ADP (adenosine diphosphate), and ATP (adenosine triphosphate)

and adjacent to the exterior of the cell membrane by a series of proteins including CD39 and CD73, the latter of which also has 50 -nucleotidase activity (Fig. 2). In hypoxia, the 50 -nucleotidase pathways that lead to adenosine production from adenine nucleotides are activated, while the adenosine kinase enzyme which serves to convert adenosine to AMP is inhibited. These and other changes rapidly increase the concentrations of adenosine within and outside the cell. Since adenosine can pass freely into and out of the cell through various nucleoside transporters in the outer membrane, any excess adenosine in the cytoplasm escapes from the cell and further accumulates in the extracellular space. These sources of adenosine contribute to very high extracellular adenosine concentrations in hypoxic tissues. In the tumor tissue, the average concentration of adenosine in the extracellular space is approximately 10 mM. Such high concentrations can be found in small tumor nodules of about 2–3 mm in diameter, so are likely to be present in the extracellular fluid of early cancers even before the

ATP ADP AMP ATP

CD39 Adenosine CD73

ADP

Inosine

5’-NT AMP IMP

AK

Adenosine ADA Inosine

ADA CD26

Hypoxanthine Xanthine Uric acid

Adenosine and Tumor Microenvironment, Fig. 2 Adenosine production in and around the tumor cell. Adenosine is produced in the cell principally from AMP through the action of 50 -nucleotidase (50 -NT). This pathway is more active under hypoxic conditions, such as that exist in solid tumors. Hypoxia also inhibits adenylate kinase (AK), which catalyzes the reverse reaction to convert adenosine to AMP. Outside the cell, adenosine is

Inside

Outside

produced from ATP that is present in the extracellular fluid, by the sequential enzyme activities of CD39 and CD73. The major factor restraining the levels of adenosine that can be reached is the activity of the enzyme adenosine deaminase (ADA), which breaks adenosine down to inosine. This is present both within the cell and as an enzyme outside of the cell (ecto-enzyme) that is held in place by an anchoring protein, CD26

Adenosine and Tumor Microenvironment

angiogenic switch. Furthermore, because the level of hypoxia varies through the tumor depending upon the proximity of blood capillaries, local levels can be much higher. Finally, adenosine concentrations are highly regulated by ectoenzymes such as adenosine deaminase (ADA) at the cell surface (Fig. 2) so that the ultimate effects of adenosine depend heavily on events at the cell surface. In normal tissues, where the concentrations of adenosine are low (in the nanomolar range), the principle pathway through which adenosine is metabolized involves phosphorylation to AMP by adenosine kinase. At higher adenosine concentrations, as are present inside a tumor, the major route through which disposal of adenosine occurs is by deamination to inosine through ADA. ADA is found both within the cell and in the external milieu. The ADA that is present in the extracellular fluid does not remain free but is largely captured by a 110-kDa binding protein present at the surface of many cells, particularly those of epithelial origin. This ADA-binding protein (ADAbp) is found embedded as a dimer in the outer membrane of many cancer cells, where it functions to hold ADA. There is also evidence that some ADA can bind directly to adenosine ▶ receptors of A1 and A2B subtypes. ADA held in this way is then able to modify adenosine concentrations immediately next to the cell surface (where the adenosine receptors are located). One factor that complicates our understanding of how adenosine levels may be regulated within the cancer tissue is the fact that adenosine has the capacity to regulate its own levels. This interesting complication arises because ADAbp (also known as CD26 or DPPIV) can be downregulated at the cell surface by adenosine. That reduces the capacity of the cell to bind ADA at the cell surface and therefore the local rate of degradation of adenosine. This will extend the half-life of adenosine and increase the persistence of its action. As a result, in the high-concentration environment of a tumor, adenosine has the capacity to suppress its own breakdown and enhance its actions still further (See also ▶ CD26/DPPIV in Cancer Progression and Spread).

71 Adenosine and Tumor Microenvironment, Table 1 The different types of cellular receptors for adenosine

Receptor subtype A1

Affinity for adenosine High

Major Ga protein (s) Gi/o

A2A

High

Gs

A2B

Low

Gs, Gq/11

A3

Low

Gi/o, Gq/ 11

Signaling pathways used by receptor Adenylyl cyclase (# cAMP) Phospholipase C K+ channels Adenylyl cyclase (" cAMP) Phospholipase C Adenylyl cyclase (" cAMP) Phospholipase C Phospholipase A2 PI3K Adenylyl cyclase (# cAMP) Phospholipase C KATP channels

Although adenosine is a common molecule and has a relatively simple structure, it is able to regulate cellular behavior by interacting with specific receptors. The different types of adenosine receptors are outlined in Table 1. There are four known types, all of which are G-protein-coupled receptors with seven transmembrane segments in their structure, embedded in the outer membranes of responsive cells. Adenosine receptors may be found on any of the cell types within a tumor including the cancer cells, supporting stromal cells, endothelial cells within blood vessels, or inflammatory cells that are infiltrating the tumor. All four of the adenosine receptor subtypes have been shown to exist on cancer cells; indeed, it is possible for a single cancer cell population to express all four forms of the receptor. However, adenosine receptor subtypes A3 and A2B are the most commonly observed in cancers. The adenosine concentrations that exist in tumors are sufficient to activate all four of the adenosine receptor subtypes. There are four different types of adenosine receptor in Table 1, which differ in their affinity for

A

72 Adenosine and Tumor Microenvironment, Fig. 3 The multiple potential actions of adenosine within a tumor. This diagram summarizes the different ways in which adenosine might act to facilitate the survival and expansion of a malignant tumor. This figure is drawn based upon studies on individual tumor cell populations and other studies in vivo in which these responses have been observed

Adenosine and Tumor Microenvironment

Decreased function of cell adhesion molecules Enhanced growth of cancer cells

Inhibition of immune response against tumor cells Adenosine

Interactions with nucleoside transporters

Stimulation of angiogenesis

adenosine and the signaling pathways to which they are linked through G proteins. All of the receptor subtypes are able to act on adenylyl cyclase but may either increase or decrease the production of cAMP as shown. The receptors can also be coupled to phospholipase C (leading to calcium release and activation of protein kinase C), to phospholipase A2 (causing generation of arachidonic acid and subsequent production of eicosanoid lipid mediators), and to phosphatidyl inositol 3-kinase (PI3K, leading to increased activity of the phospholipase D pathway) or in certain cell types can cause the activation of potassium (K) channels. The interaction of adenosine with its receptors on the different cell types in a tumor leads to a myriad of different cellular responses. Although it is at times difficult to extrapolate from the experimental approach to the disease itself, these are such as to generally favor the expansion and spread of the cancer (Fig. 3). There is evidence that synthetic agents which target individual receptor subtypes may have different actions to adenosine, sometimes not clearly directed through the adenosine receptor. When adenosine itself is studied at concentrations that are known to be present within the tumor extracellular fluid, it is typically shown to increase the growth of cancer cells. At very high concentrations of adenosine, cells may be triggered to undergo ▶ apoptosis, although some tumor cells are resistant to this action of adenosine.

Increased cellular migration (chemotaxis)

In addition to effects on cancer cell growth and survival, adenosine acts on isolated cancer cell populations to increase cell motility, adhesion to the extracellular matrix, the expression of cell attachment proteins, and receptors for molecules that can direct cell movement. The patchiness of hypoxia within the tumor tissue leads to local areas of high adenosine concentrations that would be capable of influencing tumor cell behavior directionally in this way. While not yet proven, it is possible that within the context of the tumor itself, adenosine may have an influence on the distribution of cells within the tumor and perhaps their dissemination at the later stage of metastasis. Adenosine receptors are also found on endothelial cells, which are the flattened cells that line blood vessels and which are the major cellular component of the newly formed vasculature that is formed to supply the expanding cell population with nutrients. Adenosine is able to promote endothelial cell division and motility and has been shown to enhance the formation of blood vessels (▶ angiogenesis) in experimental animal models. Adenosine may therefore have an ancillary role alongside other angiogenic factors such as VEGF in regulating the formation of the tumor microvascular network. Probably the greatest potential role for adenosine in the context of cancer, however, is as a local immunosuppressant within the tumor. It has long been known that the local tissue environment in

Adenovirus

cancer is capable of suppressing the immune response and that this is one of the factors that limits the capacity of our immune system to eliminate the cancer. Experimental studies have shown that a significant proportion of the immunosuppressive activity is mediated by soluble factors that it increases in proportion to tissue bulk, and it is seen to decline substantially when the cancer tissue is removed from the animal or patient and dissociated into isolated cells. Adenosine is one of the possible factors responsible for this phenomenon of “metabolic suppression” of the antitumor immune response. The capacity for adenosine to act as an immunosuppressant is dramatically illustrated by a rare but well-known genetic disease involving a lack of ADA. In this disorder, levels of adenosine within lymphoid tissues rise and (through a combination of events involving both toxic metabolites and adenosine acting through its receptors) cause a severe immunodeficiency (well known because of the need to protect afflicted children from infection in “biobubble” tents). Adenosine is capable of interfering with the immune response at different levels and by acting on different cell types. It works through cell surface adenosine receptors (principally A2A and A3 subtypes) to suppress various functions of T lymphocytes, natural killer (NK) cells, polymorphonuclear granulocytes, and phagocytic cells such as tissue macrophages that play a key role in recognizing the targets for immunological attack. In the case of T lymphocytes and NK cells, whose infiltration and activity is of key importance to the fate of the tumor and prognosis of the patient, adenosine suppresses successive stages in the evolution and function of the cells. It inhibits the proliferation of the cells, the expression of key molecules on the cell surface that are needed to allow full activation, the extent of interaction with the cancer cell, the release of toxic molecules involved in cell killing, and the overall capacity for killing of the cellular targets. Given the extensive effects of adenosine on nearly all of the cell types present in tumors, it would be appealing to attempt to use drugs that interfere with adenosine pathways as a way of interfering with the growth of the cancer cells, blocking the formation of new blood vessels to

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nourish the tumor, or relieving the immunosuppression that is due to adenosine. The challenge here lies in the fact that this is a primitive regulatory network in evolutionary terms, and adenosine has a role in the regulation of most organ systems in the mammal. Adenosine receptors of the four subtypes are found on cells throughout the body. Drugs that would block adenosine’s action at its receptors (antagonists) or mimic its actions at a certain receptor subtype (selective agonists) run the risk of interfering with normal processes such as the control of blood flow or the transmission of nerve signals. Nevertheless, there is hope that careful targeting of certain receptors (particularly the A3 subtype) in cancer may prove to be a useful intervention.

Adenovirus Stefan Kochanek Division of Gene Therapy, University of Ulm, Ulm, Germany

Definition Adenoviruses were originally isolated as etiologic agents for upper respiratory infections. Their name is derived from the initial observation that primary cell explants from human adenoids were found to degenerate secondary to the infection by an, at the time, unknown virus. According to the current official taxonomy, there are four adenovirus genera (Mastadenovirus, Atadenovirus, Aviadenovirus, and Siadenovirus), indicating that adenovirus is widely distributed in vertebrates. More than 50 human serotypes have been identified. The individual serotypes are distinguished by different parameters such as immunological properties, tumorigenicity, and DNA sequence. Some serotypes may cause more serious infectious diseases such as epidemic keratoconjunctivitis, gastroenteritis, or hemorrhagic cystitis. The adenoviral particle is composed of an outer icosahedral protein capsid with an inner linear double-stranded DNA genome of

A

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approximately 36 kilobases (kb) in size. There are 11 structural proteins, seven to form the capsid, among them hexon, penton base, and fiber being the major constituents of the adenoviral capsid, and four that are packaged in the core. Internalization of the viral particle during infection requires the interaction of the fiber and the penton base with surface proteins (receptors) of the cell. Several virally encoded proteins are associated with the viral DNA. Adenovirus is being used as a gene carrier for ▶ gene therapy. Most adenoviral vectors (see below) are derived from the serotypes 2 and 5 (Ad2, Ad5) which are frequent causes for mild colds. During childhood most individuals will become immunized against different adenoviral serotypes by natural infection. Ad2 and Ad5 are not oncogenic in humans Adenoviruses have a good safety record based on vaccination studies that have been performed in military recruits two to three decades ago. As detailed below, during natural infection of permissive cells, the adenoviral DNA is transcribed, replicated, and packaged into capsids within the nuclei of infected cells. Similar to other DNA viruses, two main phases can be distinguished during infection: • An early phase that is characterized by the expression of the early virus genes E1, E2, E3, and E4 • A late phase after onset of viral replication in which the viral structural proteins are produced

Characteristics Infection and Viral Transcription A productive infectious cycle takes approximately 2–3 days, and under optimal conditions more than 50,000 particles are produced in every infected cell. In the case of most human adenoviral serotypes, the infection begins with the attachment of the virus particle to the cell surface via interaction of the tip of the capsid fiber protein with the membrane protein CAR (coxsackie–adenovirus receptor). As it is apparent from the name, CAR is also used by some coxsackie viruses as receptor for entry. Naturally, CAR plays an important role

Adenovirus

in the interaction of neighboring cells. The adenoviral particle is internalized by receptormediated endocytosis into clathrin-coated pits requiring a secondary interaction of the penton base with an av-integrin. Following endocytosis the viral particle is sequentially disassembled, initially losing the fiber proteins, later most of the other viral structural proteins. Finally, the viral DNA is released as a DNA–protein complex through nuclear pores into the nucleus of the host cell. Shortly thereafter, transcriptional activation of the early genes E1A and E1B initiates a complex transcriptional program designed to first replicate the viral DNA and later to generate new infectious viral particles (Fig. 1). The activation of early and late transcription units follows a relatively well-understood transcriptional pattern. The gene products of the E1A and E1B genes are involved in the activation of both viral and cellular genes. Under certain conditions, in particular if infection of a cell does not result in a productive but rather abortive infection (abortive infection, the infectious cycle is blocked at an early stage following infection of the host cell) together with the rare event of integration of the viral DNA into the chromosome, cellular transformation may be a consequence. The E2A and E2B gene products are involved in the replication of the viral genome and include the viral DNA polymerase (Ad-Pol), the terminal protein (TP), and the DNA-binding protein (DBP). The E3 and E4 gene products have diverse functions leading to transcriptional activation of other promoters, preferential export of viral RNAs out of the nucleus of infected cells, and suppression of host defenses. With the beginning of replication of the viral genome approximately 6 h after infection, late-phase transcription units are activated. Most of the late-phase proteins are capsid proteins or proteins that are involved in the organization and packaging of the viral genome inside the viral capsid. The most active promoter at this stage is the major late promoter (MLP) that directs the transcription of a large primary RNA transcript that covers more than two thirds of the viral genome. From this transcript five families (L1–L5) of structural proteins are generated by differential splicing and polyadenylation. During the course of an

Adenovirus

75 Nucleotides

10 000

20 000

MLP

30 000

A

MLTU

E1 AE1 B L1

L2

L3

E3

L4

L5 Ad5 genome

E2 B

E2 A

Ad-pol,TP

DBP

E4

Therapeutic gene

E1-deleted vector (first-generation vector)

Therapeutic gene ΔE2

ΔE4

Therapeutic gene Non-coding stuffer DNA

Non-coding stuffer DNA

E1 + E4/E2-deleted vector (Second-generation vector)

High-capacity vector (‘gutless’ vector)

Adenovirus, Fig. 1 Organization of the adenovirus genome and the different adenoviral vector types employed for gene transfer. Promoters are indicated by arrowheads and transcribed genes by arrows. The genes that are transcribed early during infection are the E1A, E1B, E2, E3, and E4 genes. The main gene products, generated late during infection, are transcribed from the major late promoter (MLP), which directs a very long RNA message (MLTU, major late transcription unit). Different RNA species (L1–L5) that code for structural proteins are

generated by alternative splicing and differential polyadenylation (for clarity not all adenoviral genes and gene products are indicated). First-generation adenoviral vectors are characterized by deletion of the E1 genes and second-generation adenoviral vectors by the additional deletion of the E2 and/or E4 genes. High-capacity adenoviral vectors have most of the viral genome removed and retain only the noncoding viral ends. In high-capacity adenoviral vectors, stuffer DNA is included in the vector genome for stability reasons

infection, the metabolism of infected cells is redirected to support a predominant production and assembly of viral proteins.

hamsters was the first direct demonstration of a human virus causing malignant cellular transformation. This observation greatly stimulated the interest in using viruses as experimental systems in the study of the pathogenesis of cancer. While there is no epidemiological evidence for an involvement of adenoviruses in the pathogenesis of human cancers, several serotypes have been shown to cause tumors in rodents. Some serotypes, such as Ad12 or Ad18, are highly oncogenic in animals; others, for example, Ad4 or Ad5, have a low oncogenic potential. Based on several complementing observations, cellular transformation is mediated by the viral E1A and E1B genes: In most virus-transformed cells, the viral E1 genes are consistently found integrated

Adenoviral Functions and Oncogenesis Adenoviruses have played important roles as experimental tools in the discoveries of several fundamental principles in molecular biology, including RNA splicing and oncogenic transformation of cells. In fact, the 1993 Nobel Prize for Physiology or Medicine was awarded to Dr. Phillip Allen Sharp and Dr. Richard John Roberts for the discovery of RNA splicing and was based on their work with adenovirus RNA transcription. The induction of malignant tumors by injection of adenovirus type 12 in newborn

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into the cellular genome where they are expressed. Transfection of cells with the E1A and E1B genes is necessary and sufficient for cell transformation, and viruses with mutations in the E1 genes are defective for transformation. Several RNAs are transcribed from the E1A genes, the main species in Ad5 being the 12S and the 13S RNAs coding for E1A proteins of 243 and 289 amino acids. To a large extent, the E1A proteins exert their transforming activity by interaction with cellular proteins that are involved in cell cycle regulation such as the tumor suppressor pRB. While E1A alone is capable of immortalizing cells, cooperation with E1B functions is required to achieve a full transformation phenotype. Two main proteins are produced from the E1B gene by alternative splicing: the 21 kD E1B protein that has been shown to inhibit apoptosis and the 55 kD E1B protein that interacts with the tumor suppressor protein p53. The expression of additional viral functions may contribute to E1-mediated tumorigenesis. For example, a 19 kD protein expressed from the E3 region can decrease MHC class I levels in transformed cells, and certain functions expressed from the E4 region can cooperate with the transforming activity of the E1B 55 kD protein. Gene Therapy: First- and Second-Generation Adenoviral Vectors First-generation adenoviral vectors do not replicate in human cells under normal conditions because the E1A and E1B genes are deleted from the vector genome (Fig. 1). These vectors are produced in complementing cell lines that express the E1A and E1B genes. First-generation vectors have been used for gene transfer in cultured cells, animals, and even clinical trials in humans to express a large number of genes in different cell types and tissues. So far the results of experiments performed in animals and clinical studies in humans have been relatively disappointing. Several significant disadvantages of first-generation adenoviral vectors have been acknowledged: • Because first-generation vectors still contain a nearly complete set of viral genes, toxicity and

Adenovirus

antiviral immune responses are frequently observed resulting in the clearance of transduced cells. Consequently, gene expression is only transient. Contributing factors for shortterm gene expression include immune responses directed to the transgenic proteins expressed from the vector, if the organism is not tolerant to that protein. • The upper DNA packaging limit for adenoviruses is about 38 kb. Because most viral genes are retained on the vector, only about 7–8 kb of nonviral DNA can be incorporated into such vectors. However, in many conditions the therapeutic cDNAs are either large, additional elements have to be included to achieve regulated gene expression, or multiple genes need to be expressed to obtain a therapeutic effect. Thus, it is clear that the size constraints in first-generation adenoviral vectors may be a limiting factor for many potential applications. In order to further decrease expression of late viral proteins, adenoviral vectors with inactivation of the E2 and/or E4 functions in addition to the deletion of the E1 region have been generated. These vectors are produced in cell lines that complement both E1 and E2 and/or E4 functions. Currently it is controversial whether these secondgeneration adenoviral vectors have any significant advantages over first-generation vectors and lead to a longer duration of gene expression. “Gutless” Adenoviral Vectors In an attempt to address several of the problems observed with first-generation adenoviral vectors, a novel adenoviral vector has been developed that will be useful for the functional analysis of genes in vivo and clinical studies. This vector has been variably called the “high-capacity (HC),” “gutless,” “gutted,” or “helper-dependent (HD)” adenoviral vector. Because all viral genes are deleted from this vector, the capacity for the uptake of foreign DNA is more than 30 kb. The current production system involves the use of an adenoviral helper virus and takes advantage of the Cre–loxP recombination system. In this production scheme, a first-generation adenoviral vector

ADEPT

carries two loxP recognition sequences that flank the adenoviral packaging signal. The vector is produced in E1-complementing cells that express the Cre-recombinase of bacteriophage P1. After infection of these cells by both the helper virus and vector, the packaging signal of the helper virus is excised. Therefore, the vector and only little helper virus are packaged. From several in vivo experiments performed in different animal species, it is apparent that these new vectors have clear advantages compared to earlier versions of adenoviral vectors and are considerably improved in safety and expression profiles. Their increased capacity for foreign DNA allows gene transfer of several expression cassettes, large promoters, and some genes in their natural genomic context, a significant advantage over first- and secondgeneration adenoviral vectors. Replication-Competent Adenoviral Vectors for Cancer Gene Therapy While the abovementioned adenoviral vectors have been widely used in preclinical and, with the exception of “gutless” adenoviral vectors, also in clinical studies to express a wide variety of transgenes including cytokines, p53, and thymidine kinase (TK), it would be desirable to achieve gene transfer into all or most neoplastic cells within a tumor. This is clearly not possible with current vector technology. A new concept has been proposed that is based on the use of an adenovirus that is both replication competent and tumor restricted in its growth. This virus is based on an Ad5 mutant virus that has an inactivating deletion within the E1B gene and does not express the E1B 55 kD protein. Initially, it was thought that replication of the virus was dependent on the p53 status of the host cell and that the virus was able to grow only in cells deficient for function p53 expression. However, results indicate that the growth of this virus is independent of the p53 status cells and may depend on other cell cycle-related factors. Although clinical studies so far have not been or only partially been successful, such a virus has been approved in 2005 in China for cancer therapy and is currently used in combination with chemotherapy and/or radiotherapy.

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In addition, replication-competent adenoviral vectors are being developed, in which expression of essential viral genes, in particular of E1A, is under control of a tumor-specific promoter. These vectors have been named CRADs (conditionally replicating adenoviruses). Adenoviral Vectors for Genetic Vaccination One of the most promising applications of adenoviral vectors is in the area of genetic vaccination. For many common diseases including AIDS or malaria, there are currently no vaccines available. Since adenoviral vectors have been found to induce strong cellular and humoral (antibody) immune responses against expressed genes, many preclinical studies have been performed with the aim of vaccine development. In these studies adenoviral vectors have been found to belong to the strongest inducers of antigenspecific immune responses against different antigens. Therefore, clinical studies have been initiated, in which adenoviral vectors, either alone or in combination with proteins or other vectors, are evaluated for their potential as a vaccine against different infectious diseases.

References Berk AJ (2007) Adenoviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM (eds) Fields virology, 3rd edn. Lippincott-Raven, Philadelphia/New York, pp 2355–2394 Doerfler W, Böhm P (eds) (1995) The molecular repertoire of adenoviruses. Current topics in microbiology and immunology, 199/I–III. Springer, Berlin Imperiale MJ, Kochanek S (2004) Adenovirus vectors: biology, design, and production. Curr Top Microbiol Imunol 273:335–357 Wold WSM, Horwitz MS (2007) Adenoviruses. In: Fields virology, 5th edn. Lippincott-Raven, Philadelphia/New York, pp 2395–2436

ADEPT ▶ Antibody-Directed Enzyme Prodrug Therapy

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ADF ▶ Thioredoxin System

Adherens Junctions Jun Miyoshi1 and Yoshimi Takai2 1 Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan 2 Faculty of Medicine, Osaka University Graduate School of Medicine, Suita, Japan

Synonyms Intermediate junction; Zonula adherens

Definition Adherens junctions are specialized cell–cell attachments composed of transmembrane proteins and cytoplasmic proteins that anchor to the actin cytoskeleton (Fig. 1). Anchoring proteins are clustered with several actin-binding proteins in the cytoplasm adjacent to the junctional membranes. Adherens junctions form punctate or streak-like attachments in nonepithelial tissues, whereas they encircle the apical portion of adjacent epithelial cells below ▶ tight junctions. Adherens junctions have prototypic roles in stabilizing the epithelium, establishing apical–basal polarity of epithelial cells, and facilitating cell–cell communication that regulates cell proliferation and movement. Since most human cancers are of epithelial origin, disruption of adherens junctions is one of the hallmarks of cancer cells exhibiting malignant transformation.

Characteristics Adherens junctions are sites of mechanical attachment regulated by dynamic changes in the actin

ADF

cytoskeleton, and they also serve as sites of cell–cell communication. Adherens junctions are abundant in many tissues that are subjected to mechanical stress. In epithelial cells, adherens junctions coalesce into the mature zonula adherens. In cooperation with the zonula occludens (tight junctions), the zonula adherens defines apical–basal polarity by physically separating the membrane into apical and basolateral membrane domains. In addition, adherens junctions mediate nuclear ▶ signal transduction induced by cell contact. For example, molecules clustered at adherens junctions could mediate contact-dependent inhibition of cell proliferation and movement: the arrest of the cell cycle in G1 phase that occurs when cell density increases to confluence in culture. Thus, the coupling of cell contact and signaling at adherens junctions reflects structural and functional regulations involved in establishing multicellular organisms. Cadherins and nectins are two major ▶ cell adhesion molecules in the extracellular space. Cadherins are a superfamily composed of classical cadherins, which are the main components of adherens junctions, and nonclassical cadherins, which include desmosomal cadherins and protocadherins. The classical cadherins share a motif of five cadherin repeats in the extracellular domain, and they are divided into several subtypes including epithelial (E) cadherin, placental (P) cadherin, neural (N) cadherin, and vascular endothelial (VE) cadherin. On the other hand, nectins are immunoglobulin-like adhesion molecules composed of four members. Adherens junctions facilitate cell–cell adhesion through homophilic binding between cadherin molecules, as well as homophilic and heterophilic bindings between nectin molecules on adjacent cells. It remains controversial whether or not the extracellular domain of E-cadherin first binds to form cis-dimers on the surface of the same cells, and then promotes cell-cell contacts by forming trans-dimers in a Ca2+-dependent manner. On the other hand, each member of nectins forms cis-dimers, and then promotes homophilic or heterophilic trans-dimer formation in a Ca2+-independent manner. Heterophilic trans-interactions have been detected between

Adherens Junctions Apical surface

Tight junction

A JAM

ZO

Claudin

ZO

Occludin

ZO

F-Actin

Nectin

Afadin

Rap1

Afadin

Afadin

F-Actin

Afadin FAK

Adherens junction

Integrin ανβ3

Rap1 IQGAP1

Src Cdc42

β-Catenin

p120 ctn

β-Catenin α-Catenin

E-Cadherin

p120 ctn

α-Catenin

nectin-2 and nectin-3, between nectin-1 and nectin-3, and between nectin-1 and nectin-4. Importantly, heterophilic trans-dimers form stronger cell–cell attachment than homophilic trans-dimers, which actually determines the type of cell–cell adhesion. Namely, cadherins exclusively promote adhesion between homotypic cells, whereas nectins have a dual role in promoting adhesion between homotypic cells and between heterotypic cells. Heterophilic engagement of nectins may thus play key roles in cell recognition and sorting in vivo.

p120 ctn

Rac

p120 ctn

Adherens Junctions, Fig. 1 Epithelial cells joined by the apical adhesion complex. Adherens junctions are located below tight junctions near the apical end of the lateral cell interface in epithelial cells. Nectin and E-cadherinbased cell adhesions are connected via several cytoplasmic proteins into belts of actin filaments that underlie adherens junctions. Nectins are localized to adherens junctions via afadin, and they are associated with integrin avb3 in the extracellular space. Afadin binds to the tail of nectin cis-dimers as well as F-actin directly, interacting with Rap1. b-catenin binds to the tail of E-cadherin cis-dimers directly, and then a-catenin binds to b-catenin. The catenins can mediate interactions to F-actin through binding to several actin-binding proteins such as ZO proteins, afadin, vinculin, a-actinin, VASP, formin-1, and Arp2/3 complex. c-Src, Rac, Cdc42, and FAK play roles in regulating dynamic changes of the actin cytoskelton, facilitated by E-cadherin and nectin clustering

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Vinculin VASP

α-Catenin α-Actinin β-Catenin

Arp2 Arp3

β-Catenin

Formin1

α-Catenin Src

IQGAP1

Rac

Basal interface Extracellular matrix

The intracellular domain of cadherins is associated with a cytoplasmic complex consisting of a-catenin and b-catenin, and forms structural links to the actin cytoskeleton. a-catenin does not act as a stable link to filamentous actin (F-actin) but possibly acts as a molecular switch that regulates actin dynamics at adherens junctions. The catenins could also mediate interactions with F-actin via binding to proteins such as ZO protein-1, afadin, vinculin, and a-actinin. The intracellular domain of nectins directly binds to afadin that links nectins to the actin cytoskeleton.

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Localization of nectins to adherens junctions depends on the presence of afadin. Thus, the catenins and afadin cooperatively contribute to form adherens junctions that are strong yet easily remodeled. Nectin-based cell–cell adhesions establish adherens junctions, both independently and cooperating with cadherin-based cell–cell adhesions. In Madin–Darby canine kidney (MDCK) cells in culture, nectins first form cell–cell adhesion and then recruit cadherins to the nectin-based cell–cell adhesion sites to establish adherens junctions. Nectins further promote formation of tight junctions in MDCK cells by recruiting JAM (junctional adhesion molecule)-A, claudin-1, and occludin. On the other hand, nectins and integrin avb3 are physically associated through their extracellular domains to cooperatively regulate cell movement, proliferation, adhesion, and polarization. Thus, nectins play roles in establishing apical junctional complex, as well as in communication between cell–cell and cell–matrix junctions. Trans-interacting E-cadherin induces activation of Rac small ▶ G-protein, which stabilizes nontrans-interacting E-cadherin on the cell surface by inhibiting endocytosis through the reorganization of the actin cytoskeleton. p120 catenin (p120ctn) also plays a role for inhibiting endocytosis of E-cadherin. In contrast, E-cadherin undergoes endocytosis when adherens junctions are disrupted by the action of an extracellular signal, such as hepatocyte growth factor/▶ scatter factor. Activated c-Src enhances endocytosis of E-cadherin by inducing the tyrosine phosphorylation and ubiquitylation of the E-cadherin complex. On the other hand, trans-interaction of nectins activates Cdc42 and Rac, which promotes the formation of adherens junctions mediated by the IQGAP1-dependent actin cytoskeleton. In addition, afadin and activated Rap1 complex interacts with p120ctn to strengthen the binding between p120ctn and E-cadherin. Furthermore, the cell polarity proteins Par-3, Par-6, and aPKC that form a ternary complex could be implicated in the assembly of adherens junctions. They regulate the association of afadin with nectins in MDCK cells. These cell polarity proteins and afadin could play

Adherens Junctions

cooperative roles in the formation of adherens junctions and tight junctions although the mechanism is largely unknown. Thus, E-cadherin and nectin trans-interactions induce elaborate interactions between peripheral proteins to establish mature adherens junctions. b-catenin is able to translocate to the nucleus, where it binds to lymphoid enhancer factor–T-cell factor (LEF/TCF) that regulates gene transcription. b-catenin is involved in several signaling pathways including the wingless-type mammary virus integration-site family (Wnt) signaling pathway. When Wnt proteins bind their receptors, they inactivate the serine/threonine kinase GSK3b that phosphorylates b-catenin and targets it for destruction in the proteosome. Mutations involving the serine/threonine residues of b-catenin that are phosphorylated by GSK3b can stabilize the b-catenin protein or increase its nuclear localization. Furthermore, tyrosine phosphorylation of b-catenin also disrupts the association between E-cadherin and b-catenin, allowing b-catenin to transduce signals to the nucleus. Necl-5, a member of nectin-like cell adhesion molecules (Necls), originally identified as a poliovirus receptor, could mediate growth arrest that has been known as contact inhibition of cell proliferation and movement. Necl-5 is overexpressed in human colon carcinoma, as well as in NIH3T3 cells transformed by ▶ RAS activation. Necl-5 colocalizes with integrin avb3 and growth factor receptors at leading edges of migrating cells and regulates growth factor induced cell migration. When Necl-5 interacts in trans with nectin-3 at cell–cell contacts in NIH3T3 cells, Necl-5 undergoes downregulation from the cell surface, resulting in reduction of cell proliferation and movement. Thus, nectins and Necls have roles in mechanical cell–cell adhesion as well as cell–cell communication. Implications in Cancer Adherens junctions control epithelial cell polarity while other adhesion apparatus tends to inhibit cell migration, which is crucial for the differentiation and morphogenesis of many tissues. Loss of adherens junctions, as well as aberrant signaling involving the Wnt pathway, could contribute to

Adherens Junctions

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p120 ctn

E-Cadherin

p120 ctn

A α-Catenin

Wnt

β-Catenin

F-Actin

β-Catenin α-Catenin GSK-3β β-Catenin Endocytosis disassembly

Dissociation

p120 ctn

P β-Catenin Degradation

α-Catenin Rho

Stress fibers

Rac

Cdc42

Lamellipodia

Filopodia

TCF β-Catenin

Adherens Junctions, Fig. 2 Signaling induced by loss of E-cadherin. Disruption of adherens junctions is caused by mutation or transcriptional repression of E-cadherin and growth-factor signaling. Dissociation of homophilic binding of E-cadherin promotes the endocytosis of E-cadherin and the disassembly of the catenins. p120ctn further promotes cell motility by activating Rac and Cdc42 to form lamellipodia and filopodia, and inhibits Rho activity that leads to stress-fiber formation. b-Catenin dissociated from

the E-cadherin and catenin complex accumulated in the cytoplasm. Part of b-catenin translocates to the nucleus and binds to TCF to activate transcription of key genes required for survival of detached cells, while the other part of b-catenin is modified by phosphorylation and ubiquitination, leading to proteosome degradation. The Wnt pathway promotes b-catenin signaling by repressing the phosphorylation of b-catenin mediated by GSK-3b

carcinogenesis and ▶ metastasis by causing cell depolarization, loss of contact-dependent inhibition of proliferation, and increased ▶ motility and invasiveness (Fig. 2). Cancer cells that show migratory properties undergo ▶ epithelial to mesenchymal transition (EMT), with the induction of transcriptional repressor proteins, such as ▶ snail transcriptional factor, slug, and twist, that downregulate E-cadherin gene expression. EMT is a basic mechanism that mediates disruption of epithelial polarity and disintegration of cancer cell nests. Reduced E-cadherin levels in cancer cells are accomplished by genetic events such as somatic mutation and reduced gene expression mediated

by repressor proteins or by methylation of the promoter region of the E-cadherin gene. The genetic defects of E-cadherin have been found in human lobular breast carcinomas and scirrhoustype gastric cancers, both of which have highly metastatic potentials. Mutations of b-catenin also promote migration and ▶ invasion of cancer cells by the loss of interaction of adherens junctions with the actin cytoskelton. Distributions of E-cadherin and b-catenin tend to change depending on sites of tumor remodeling. In epithelial structures in the centre of cancer, E-cadherin and b-catenin are mostly present in adherens junctions. However, solitary cells at the invasive front of cancer plates shows

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no signal for E-cadherin but often produce signals for nuclear b-catenin. Thus, decreased E-cadherin expression promotes the release of solitary cancer cells at the invasive front and increases the survival of cancer cells by stimulating b-catenin signaling. Strategy for restoring adherens junctions, as well as cell–cell and cell–matrix communication, may prevent cancer-cell invasiveness. Therapeutic targets might be molecules involved in pathways affecting the adhesive properties of E-cadherin and the assembly of the adherensjunction complex: c-Src and other tyrosine kinases, tyrosine phosphatases such as PTP-LAR, Rho, Rac, and Rap small G-proteins, transcriptional repressor proteins, and ▶ merlin and the ▶ ERM proteins. For example, c-Src regulates both disruption of adherens junctions and focal-adhesion turnover that are required for cancer cell motility. Twist is highly expressed in human cancers with reduced E-cadherin mRNA expression levels. In contrast, podoplanin promotes cancer cell invasion in the absence of EMT, suggesting cancer cells can also migrate as a mass, not necessarily as a single cell. Restoring E-cadherin-mediated cell adhesion could be means of preventing EMT in cancer and metastasis although EMT is not essentially required for cancer-cell invasion.

Adhesion

▶ Snail Transcription Factors ▶ Tight Junction

References Christofori G (2006) New signals from the invasive front. Nature 441:444–450 Kobielak A, Fuchs E (2004) Alpha-catenin: at the junction of intercellular adhesion and actin dynamics. Nat Rev Mol Cell Biol 5:614–625 Takai Y, Nakanishi H (2003) Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci 116:17–27 Takeichi M (1993) Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 5:806–811 Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142

See Also (2012) Rap1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3168. doi:10.1007/978-3-642-16483-5_4947 (2012) Wnt. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3953. doi:10.1007/978-3-642-16483-5_6255 (2012) ZO-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3977. doi:10.1007/978-3-642-16483-5_6301

Adhesion Cross-References ▶ Cell Adhesion Molecules ▶ E-Cadherin ▶ Epithelial-to-Mesenchymal Transition ▶ ERM Proteins ▶ Gastric Cancer ▶ G Proteins ▶ Invasion ▶ IQGAP1 Protein ▶ Merlin ▶ Metastasis ▶ Motility ▶ RAS Activation ▶ Scatter Factor ▶ Signal Transduction

Dario Rusciano Friedrich Miescher Institute, Basel, Switzerland

Definition Cell adhesion is a dynamic process that results from specific interactions between cell surface molecules and their appropriate ligands. Adhesion can be found between adjacent cells (cell-cell adhesion) as well as between cells and the extracellular matrix (ECM) (cell-matrix adhesion). Besides keeping a multicellular organism together, cell adhesion is also a source of specific signals to adherent cells; their phenotype can thus be regulated by their adhesive interactions. In fact,

Adhesion

83

A

Adhesion, Fig. 1 Cell adhesion in normal (a, b) and cancer (c, d) cells. Normal mesenchymal cells show regular actin stress fibers (a, stained with phalloidin) and focal contacts (b, stained with anti-vinculin antibodies). In contrast, cancer cells (a highly motile melanoma cell is shown)

often present with a completely disorganized actin cytoskeleton (c) and few focal contacts (d). Vinculin is typically arranged in patches at the periphery of the cell (d) (Confocal micrograph courtesy of Dr. Jörg Hagmann, FMI, Basel)

most of the cell adhesion receptors were found to be involved in ▶ signal transduction. By interacting with growth factor receptors they are able to modulate their signaling efficiency. Therefore, gene expression, cytoskeletal dynamics, and growth regulation all depend, at least partially, on cell adhesive interactions (Fig. 1).

• Integrins represent a family of cell surface glycoproteins that depend on divalent cations and are important in cell-ECM and cell-cell adhesion. The noncovalent association of an alpha and a beta subunit results in heterodimers that span the plasma membrane, enabling contacts with elements of the ▶ Cytoskeleton and signal transducing intermediates. • The immunoglobulin superfamily of adhesion receptors is mainly involved in cell-cell adhesion. Named after a 90–100 amino acid domain that is also present in Ig molecules, these kind of receptors can be expressed either as plasma membrane-spanning molecules. However, some of them are alternatively spliced and are anchored to the cell membrane by covalent linkage to phosphatidylinositol.

Characteristics Cell Adhesion Receptors Cell adhesion molecules were grouped into distinct classes according to structural and/or functional homologies. The following receptors have been directly implicated in the malignant phenotype of tumor cells.

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Adhesion

Adhesion, Table 1 Adhesion receptors Family Integrins

IgG superfamily

Cadherins

Selectins Connexins

Cell surface proteoglycans CD44

Main members Characterized by the different a- and b-subunits ICAM-1, V-CAM, N-CAM, CD2 (LFA2), LFA3, CD4, CD8, MHC (class I and II) E, P, L

E, P, N 26 (tumor suppressor) 32 (liver) 43 (glial cells) Syndecan, glypican CD44s, CD44v

Type of adhesion Cell-ECM cell-cell Cell-ECM cell-cell

Cell-cell (adherens junction) Cell-cell Cell-cell (gap junctions) Cell-ECM cell-cell Cell-ECM cell-cell

• Selectins represent a class of structurally related monomeric cell surface glycoproteins that bind specific carbohydrate ligands via their lectin-like domains. Since the ligands are expressed in a specific way by vascular endothelial cells, selectins are important in lymphocyte trafficking and homing of malignant tumor cells. • Cell surface proteoglycans consist of glycosaminoglycans (GAG) attached to core proteins through an O-glycosidic linkage. They can mediate cell-cell and cell-ECM adhesion. • ▶ CD44 comprises a large family of proteins generated from one gene by alternative splicing. Variants of CD44 (CD44v) differ from the standard form (CD44s) by their implementation of ten variant exons in various combinations. Some variants have been causally related to the metastatic spread of some tumor cells. Among the ligands for CD44 are hyaluronic acid (HA), fibronectin and collagen, and chondroitin sulfate-modified proteins. • Cadherins are surface glycoproteins involved in cell-cell interactions. They are involved in the formation of adherens-type functions between cells. Through their cytoplasmic tail they interact with catenins, which are

important for the signal transducing ability of cadherins. • ▶ Connexins are gap junction-forming proteins that oligomerize into specialized intercellular channels, connecting apposing plasma membranes. They allow the exchange of low molecular weight metabolites such as second messengers that are important in signal transduction (Table 1). Adhesion and Cancer The selective adhesion of one cell to another or to the surrounding ECM is of paramount importance during embryonic development as well as for the maintenance of normal adult tissue structure and function. Severe perturbations of these interactions can be, at the same time, cause and consequence of malignant transformation and also play a fundamental role during malignant progression and metastatic dissemination (Fig. 1). • Adhesion to the ECM through integrin receptors is important for anchorage dependent cell growth and cell survival. Normal cells that are detached from the ECM are locked in the G1 phase of the cell cycle (by loss of activity of the cyclinE/cdk2 complex) and undergo apoptosis (anoikis). Transformed cells, in which integrin signaling is altered, acquire the ability to grow in suspension and do not succumb to anoikis. • Adhesion to neighboring cells, mediated by cell-cell adhesion molecules (e.g., N-CAM and C-CAM) and by gap-junctions, inhibits growth of normal cells (what is commonly known as “contact growth inhibition”). Loss of these contacts due to the disrupted function of the relative adhesion molecules may result in uncontrolled proliferation. • The differentiated state of mature cells (their “identity”) is also maintained through specific adhesion to the ECM and adjacent cells: a loss of identity is thus a likely consequence if specific contacts are lost, finally resulting in the ambiguous phenotype of many tumor cells (Fig. 2). Certain genes that code for cell adhesion molecules may therefore be considered as ▶ tumor

Adhesion

85

Loss of growth control, resistance to anoikis, motility, invasion Apoptosis

ECM

Cancer

Proliferation survival motility

E CM

Growth inhibition, survival polarization, differentiation

E CM

Adhesion, Fig. 2 Cell adhesion and maintenance of a normal differentiated phenotype: Detachment of a normal cell from the extracellular matrix (ECM) would normally lead to apoptosis. Normal cells that keep contact with the ECM are protected from apoptosis and may migrate and grow. Normal cells tend to be organized as sheets onto the ECM, which contributes to their polarization and

differentiation. Extensive intercellular contacts among cells adhered onto the ECM lead to contact-mediated growth inhibition. Tumor cells do not undergo apoptosis when detached from the ECM and may grow, migrate, and invade into the matrix, to enter the circulation and give rise to distant metastases

suppressor genes or even ▶ metastasis suppressor genes since their loss or a functional mutation can strongly contribute to the acquisition of the malignant phenotype.

contribute to the release of such mutant cells from the primary tumor mass. Indeed, it was found that tumor cells separate more easily from solid tumors than normal cells from corresponding tissues.

Adhesion in Metastasis Adhesive interactions play a very critical role in the process of metastatic tumor dissemination, and the abnormal adhesiveness that is generally displayed by tumor cells appears to contribute to their metastatic behavior. Both positive and negative regulation of cell adhesion are required in the metastatic process, since metastatic cells must break away from the primary tumor, travel in the circulation where they can interact with blood cells and then adhere to cellular and extracellular matrix elements at specific secondary sites.

• Cadherin expression has been shown to influence intercellular cohesion in direct correlation with invasive behavior. An increased cadherin expression in tumor lines generally causes a tighter association of tumor cells. In vitro experiments have shown that cells which do not express cadherins or in which cadherins are functionally inhibited are more invasive than cells with normal cadherin activity. In cases where E-cadherin was involved, re-introduction of a wild type copy of the gene could revert the invasive phenotype. The loss of cadherin activity, however, is not sufficient to make cells invasive. ▶ Invasion also requires other cellular activities, such as ▶ motility and protease production. In vivo, tumors expressing low levels of cadherins tend to be less differentiated and to exhibit higher invasive potential, although they are not necessarily more metastatic. In human cancer, a reduction in cadherin activity correlates with the infiltrative ability of tumor cells, a

Adhesion Within The Tumor Mass The majority of normal adult cells are restricted by compartment boundaries that are usually conserved during the early stages of development of a tumor. Therefore, the detachment of malignant cells from the primary tumor is an essential step for the initiation of the metastatic cascade. During tumor progression, changes on the cell surface that lead to a weakening of the cellular constraints

A

86

correlation that in many tumors is also retained in distant metastasis. • A different type of cellular constraint is provided by gap junction communication. Gap junctions play an essential role in the integrated regulation of growth, differentiation, and function of tissues and organs. The disruption of gap junction communication can cause irreversible damage to the integrity of the tissue and finally contribute to tumor promotion and malignant progression by favoring local cell isolation. There is experimental evidence that a loss of intercellular junction communication affects the metastatic potential of cell lines. Normal cells use gap junctions to control the growth of tumor cells. Once gap junctional communication is lost, the signaling mechanism responsible for the exertion of such growth control is also lost. Both quantitative and qualitative changes in gap junction protein (connexins) expression were found to be associated with tumor progression during multistage skin carcinogenesis in the mouse model system as well as with tumorigenesis in a rat bladder tumor cell line. Malignant Tumor Cells in the Blood Stream: Adhesion to Blood Cells and Platelets Blood-borne tumor cells undergo various homotypic and heterotypic interactions, the effect of which will also influence their metastatic behavior. Some of these interactions may be detrimental to circulating tumor cells such as tumor cell recognition by natural killer (NK) cells, or by tumor infiltrating lymphocytes (TIL). Others may provide, to a certain extent, a protective effect and/or contribute to metastatic spreading, such as interactions with platelets or, in certain cases, with leucocytes. • De novo expression of the cell adhesion molecule ICAM-1 by melanomas might lead to heterotypic adhesion between melanoma cells and leukocytes bearing the relative receptor (LFA-1). Such interaction might thus enhance tumor cell adhesion to migratory and invasive leukocytes, thereby contributing to further dissemination of malignant tumor

Adhesion

cells. In this regard, it has been suggested that site specificity of cancer metastasis might be, at least partially, a consequence of the formation of “multicellular metastatic units” (MSU) consisting of tumor cells, platelets, and leukocytes. A subset of leukocytes within the “MSU” would be responsible for sitespecific endothelium recognition, adhesion, and stable attachment, thus serving as “carrier cells” targeting the metastatic “spheroids” to specific sites of secondary tumor foci formation. • Several lines of evidence have provided strong support for the concept that tumor cell-platelet interaction significantly contributes to hematogenous metastasis. Two categories of molecules can trigger tumor cell induced platelet aggregation (TCIPA) and activation: soluble mediators and adhesion molecules. The latter are likely to be responsible for the initial contact between tumor cells and platelet cells, and might later stabilize the interaction. P-selectin and aIIbb3 integrin on the platelet surface may bind Lex carbohydrate determinants and fibrin on the surface of tumor cells, thus triggering platelet activation. Sialylation appears to be a general requirement for TCIPA, and sialoglycoconjugates present on both tumor cells and platelets have been involved in tumor cellplatelet interactions. Mechanistically, platelets may contribute to metastasis by stabilizing tumor cell arrest in the vasculature, shielding tumor cells from physical damage, providing additional adhesion mechanisms to endothelial cells and subendothelial matrix, and serving as a potential source of growth factors. If tumor cell interaction with host platelets occurs while tumor cells are circulating, an organ-specific colonization ability of blood-borne tumor cells may be influenced. In fact, the resulting embolus will be more easily arrested in the vasculature of the first organ downstream from the primary tumor site. If this organ represents a favorable milieu for tumor growth, then interaction with platelets will enhance tumor metastasis at that site; if this is not the case, it may prevent tumor cells from reaching their preferred organ and thus cause a reduction of the

Adhesion

metastatic potential. It seems, however, that in most cases platelets are involved only after tumor cells have arrested, and platelet activation may then stabilize the initial tumor cell arrest in the microvasculature. Adhesion in the Target Organ Circulating tumor cells, either as single cells or most likely as homotypic and/or heterotypic aggregates that have escaped killing by the host immune system and lysis by mechanical shear forces associated with passage in the blood stream, need now to arrest in the microvasculature and extravasate into the organ parenchyma. In fact, the survival time of tumor cells entering the circulation is very short, usually less than 60 min. Therefore those cells that can rapidly arrest and are able to get out the blood stream might have a selective advantage in giving rise to metastatic colonies. Specific adhesion in the target organ has been proposed as a critical determinant of organ specific metastasis, and experimental data indicates that malignant tumor cells preferentially adhere to organ-specific adhesion molecules. Tumor cells, for instance, adhered more efficiently to disaggregated cells or to histologic sections prepared from their preferred site of metastasis than from other organs. These type of assays, however, do not accurately mimic the physiological situation in vivo, where the first contact of circulating tumor cells happens with the luminal surface of the vascular endothelium, and, after endothelial retraction, with the subendothelial basement membrane. The basement membrane (BM) is a thin mat of extracellular matrix that separates epithelial sheets and many types of cells, such as muscle cells and fat cells, from connective tissue. The characteristic components of BMs are laminin, collagen type IV, and heparan sulfate proteoglycan. Adhesion to Endothelial Cells (EC) The arrest of tumor cells in the capillary bed of secondary organs and their subsequent extravasation occur through interactions with the local microvascular endothelium and the subendothelial matrix.

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• Biochemical heterogeneity of EC is related to both the heterogeneous microenvironment within tissues and the size of the vessel. Heterogeneity is seen in the differential expression of plasma membrane glycoproteins, cytoskeletal proteins, and surface receptors in microvascular endothelium of different organs. Such heterogeneity of endothelium underscores the importance of using organ-specific capillary endothelium in studying the role of organspecific tumor cell adhesion in metastasis. • The specificity of the adhesive interactions that depends on the heterogeneity of microvascular EC and tumor cells may favor, in a selective way, the initial adhesive events in preferred metastatic sites. As a consequence it may also facilitate metastatic dissemination to those organs, in a way that is similar to extravasation of lymphocytes from high endothelial venules of lymphoid tissues. In fact, lymphocyte “homing” represents the paradigm for organ-specific cell adhesion, and it has been shown to follow specific interactions between surface “homing” receptors on lymphocytes with vascular “addressins” expressed on the high endothelial venule surface. In a similar way, tumor cells express various combinations of cell surface molecules that may serve as ligands for EC surface receptors, which are typically induced upon stimulation by mediators of inflammation. A local inflammatory response might thus facilitate circulating tumor cells adhesion and arrest. The relevance of this type of interaction in directing tumor metastasis has been demonstrated in vivo using strains of transgenic mice that constitutively express cell surface E-selectin either in all tissues or in the liver alone. Metastatic tumor cells that do not express the ligand colonized mostly the lung. However, following the induction of ligand expression, tumor cell colonization was redirected to the liver with tremendous efficiency. Adhesion to Extracellular Matrix Components Mammalian organisms are composed by a series of tissue compartments separated from one another by two types of extracellular matrix

A

88

(ECM): basement membranes and interstitial stroma. ECM consists of three general classes of macromolecules, including collagens, proteoglycans, and noncollagenous glycoproteins (such as fibronectin, laminin, entactin, and tenascin among others), which are expressed in a tissue-specific fashion. Malignant cells arrested in the microcirculation sometimes do not migrate further into the organ parenchyma but grow locally in an expansive fashion until they rupture the vessel wall. In most cases, however, the contact between tumor cells and the endothelium results in EC retraction with exposure of the underlying basement membrane, followed by invasion of tumor cells in the tissue. The presence of specific adhesion receptors on the membrane of metastatic cells, and the peculiar composition of the extracellular matrix at a given site, will influence tumor cell retention, motility and invasion, and growth at target organs. • Electron microscopy observation on the formation of pulmonary metastasis has shown that tumor cells often adhere to regions of exposed basal lamina. The exposed subendothelial matrix is usually a better adhesive substrate for tumor cells than the endothelial cell surface. • In order to move through the ECM, tumor cells must make firm contacts with matrix molecules, be able to break these adhesive contacts as they move on and respond to chemotactic molecules that direct their movement. Interactions with the ECM may fulfill all these scopes, through the signaling effect of several cytokines (growth factors, motility factors, enzymes, and enzyme inhibitors) that are stored bound to ECM molecules, and released upon interaction with tumor cells. Moreover, ECM macromolecules themselves may also function as motility attractants, and have been shown to stimulate both chemotaxis and haptotaxis. Haptotactic migration over insoluble matrix components may occur predominantly during the initial stages of metastatic invasion, while at later stages partially degraded matrix proteins, derived from proteolytic processing of the

Adhesion

matrix, could be the major determinant of directed motility. • Finally, it has to be considered that some ECM components may actually impede cell adhesion and thus might influence directional tumor cell motility by promoting the localized detachment of the trailing edge of migrating cells. ECM-associated chondroitin sulfate proteoglycans, such as decorin, or the glycoprotein tenascin have been suggested to modulate tumor cell adhesion and motility in this way. Adhesion and Drug Resistance The malignant phenotype of tumor cells depends, at least partially, on the weakening of cell-matrix and cell-cell interactions that occurs during tumor progression. However, late stage tumors maintain some level of intercellular adhesion, or even tend to reactivate certain adhesion mechanisms, indicating that modulation of cell adhesion is a dynamic process. Given the beneficial effect of cell adhesion on apoptosis resistance, an increased level of adhesion may facilitate survival of tumor emboli, and there is evidence that it can help tumor cells to evade the cytotoxic effects of anticancer therapy.

Cross-References ▶ CD44 ▶ Connexins ▶ Cytoskeleton ▶ Invasion ▶ Metastasis Suppressor Gene ▶ Motility ▶ Signal Transduction ▶ Tumor Suppressor Genes

References Boudreau N, Bissell MJ (1998) Extracellular matrix signaling: integration of form and function in normal and malignant cells. Curr Opin Cell Biol 10:640–646 Hedrick L, Cho KR, Vogelstein B (1993) Cell adhesion molecules as tumor suppressors. Trends Cell Biol 3:36–39

Adiponectin Ruohslahti E, Öbrink B (1996) Common principles in cell adhesion. Exp Cell Res 227:1–11 Rusciano D, Welch DR, Burger MM (2000) Cancer metastasis: experimental approaches. In: Laboratory Techniques in Biochemistry and Molecular Biology, vol 29. Elsevier Science B.V, Amsterdam Yamasaki H, Omori Y, Zaidan-Dagli ML et al (1999) Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis. Cancer Detect Prev 23:273–279

See Also (2012) Basement membrane. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 349. doi:10.1007/978-3-642-16483-5_537 (2012) Cadherins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 581– 582. doi:10.1007/978-3-642-16483-5_770 (2012) Chemotaxis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 793. doi:10.1007/978-3-642-16483-5_1081 (2012) Extracellular matrix. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Glycoprotein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2451 (2012) Glycosaminoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2453 (2012) Haptotaxis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1631. doi:10.1007/978-3-642-16483-5_2565 (2012) Heparan sulfate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1647. doi:10.1007/978-3-642-16483-5_2637 (2012) Lectin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1999. doi:10.1007/978-3-642-16483-5_3303 (2012) Proteoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3100. doi:10.1007/978-3-642-16483-5_4816 (2012) Selectins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3355. doi:10.1007/978-3-642-16483-5_5218 (2012) Sialoglycoconjugates. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3402. doi:10.1007/978-3-642-16483-5_5292 (2012) Tumor progression. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6046

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ADI ▶ Arginine-Depleting Deiminase

Enzyme

Arginine

Adipocyte C1q and Collagen Domain Containing ▶ Adiponectin

Adipocyte Complement-Related Protein of 30 kDa ▶ Adiponectin

Adipocytic Tumors ▶ Adipose Tumors

Adiponectin Jie Chen, Janice B. B. Lam and Yu Wang Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China

Synonyms ACDC; ACRP30; Adipocyte C1q and collagen domain containing; Adipocyte complementrelated protein of 30 kDa; AdipoQ; Adipose most abundant gene transcript 1; aPM1; GBP28; Gelatin-binding protein 28

Definition

Adhesion Molecules ▶ Cell Adhesion Molecules

Adiponectin is a major adipokine secreted exclusively from adipocytes. This adipokine possesses

A

Signal Sequence

Hypervariable Region

-O-GG

-SH

a

-O-GG -O-GG -O-GG

Adiponectin -O-GG

90

Collagenous Domain

Globular Domain

b

Monomer

Trimer (LMW)

Hexamer (MMW)

HMW

Adiponectin, Fig. 1 Schematic representation of the primary structure (a) and the oligomeric complexes of adiponectin (b). Adiponectin contains a NH2-terminal signal sequence peptide and a hypervariable region, followed by a conserved collagenous domain and a COOH-terminal globular domain. A cysteine residue within the hypervariable region is involved in the disulfide bond formation.

Several lysine residues located within the collagenous domain are hydroxylated and glycosylated. (b) Adiponectin exists as three oligomeric species, including the trimer (LMW), hexamer (MMW), and HMW. Disulfide bond formation and glycosylation are involved in its oligomeric formation. GG, glucosyla(1–2)galactosyl group; S −S, disulfide bonds

insulin-sensitizing, antidiabetic, antiangiogenic, antiatherogenic, antiinflammatory and antitumorigenic properties.

adiponectin complex is a trimer or low molecular weight (LMW) oligomer, which is formed via hydrophobic interactions within its globular domain. Two trimers self-associate to form a disulfide-linked hexamer or middle molecular weight (MMW) oligomer, which further assembles into a bouquet-like high molecular weight (HMW) multimeric complex that consists of 12–18 monomers (Fig. 1b). Posttranslational modifications, including disulfide bond formation at a conserved cysteine residue and glycosylations occurred on several hydroxylated lysine residues within the collagenous domain, are involved in the assembly and stabilization of the oligomeric structures. Different oligomeric complexes of adiponectin activate distinct signaling pathways and possess different biological functions. Two putative adiponectin receptors, termed AdipoR1 and AdipoR2, have been identified. AdipoR1 is highly expressed in skeletal muscle whereas AdipoR2 is most abundantly expressed in liver. Both receptors are integral membrane proteins containing seven transmembrane spanning domains. AdipoR1/R2 may mediate the effect of adiponectin on activation of

Characteristics Adiponectin was originally identified as an adipose-specific gene dysregulated in ▶ obesity. Human adiponectin gene is located on chromosome 3q27 and encodes a 244 amino acids polypeptide comprising of an NH2-terminal secretory signal sequence, followed by a hypervariable region, a collagenous domain, and a COOH-terminal globular domain (Fig. 1a). Circulating concentrations of adiponectin range from 3 to 30 mg/ ml and account for about 0.05% of total human blood proteins. Despite the fact that it is produced in adipose tissue, serum concentrations of adiponectin are paradoxically reduced in obese individuals and obesity-related pathological conditions. Endogenous adiponectin is predominantly present as several characteristic oligomeric complexes. The basic building block of the

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Adiponectin, Fig. 2 Adiponectin acts as a negative regulator in the tumor-stromal microenvironment

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AMP-activated protein kinase (AMPK), a fuelsensing enzyme that plays a central regulatory role in cellular energy metabolism. T-cadherin, which is highly expressed in endothelium and smooth muscle, has been identified as an adiponectin coreceptor with preference for hexameric and HMW adiponectin multimers. Both adiponectin analogues and adiponectin receptor agonists represent the potential therapeutic targets for obesity-linked diseases.

non-cancer (stromal) cells in the tumor microenvironment, including adipocytes. Experimental evidence suggest that adiponectin acts as a major stromal factor to suppress carcinogenesis via the regulation of energy metabolism, cell growth and survival, as well as angiogenesis in the tumor microenvironment (Fig. 2). For example, adiponectin inhibits tumor neovascularization, through suppression of endothelial cell proliferation, migration, and tubular formation.

Adiponectin and Carcinogenesis In humans, adiponectin deficiency is closely associated with increased cancer risks. Numerous clinical studies have confirmed an inverse association between the blood concentrations of adiponectin and the risks of obesity-related cancers, including ▶ lung, ▶ prostate, ▶ breast, ▶ endometrial, ▶ gastric, liver and ▶ colorectal cancers. In addition to cancer cells, there are multiple types of

Prostate Cancer

Obesity is associated with prostate cancer progression, increased tumor aggressiveness, and poor prognosis. Low levels of adiponectin are an independent risk factor for prostate cancer and associated with the histologic grade and stage of the disease. Genetic variations of adiponectin affect its circulating levels, the tumor grade, clinical stage and aggressiveness in prostate cancer

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patients. Higher adiponectin concentrations predispose men to a lower risk of developing and dying from prostate cancer. Thus, adiponectin represents a molecular link countering the adverse effects of obesity on prostate cancer, particularly in earlier stages of the disease. Adiponectin, in particular the HMW form, has been shown to inhibit leptin- and/or ▶ insulin-like growth factor-1 (IGF-1)-stimulated DU145 androgen independent prostate cancer cell growth and dihydrotestosterone-stimulated growth of androgen-dependent LNCaP-FGC cells at subphysiological concentrations. It suppresses oxidative stress in human prostate cancer cell lines. In addition, adiponectin enhances the inhibitory effects of the cytotoxic chemotherapy agent, doxorubicin, on prostate cancer cell growth. These data suggest that adiponectin plays an important role in the pathogenesis of prostate cancer, and may be used as a drug target for therapeutic interventions. Breast Cancer

Excess adiposity over the pre- and postmenopausal years is an independent risk factor for the development of breast cancer, and is associated with late-stage disease and poor prognosis. Clinical studies have shown that low plasma adiponectin levels are significantly associated with an increased risk for breast cancer in both pre- and postmenopausal women, particularly in a low estrogen environment. Moreover, tumors from women with low plasma adiponectin levels are more likely to show a biologically aggressive phenotype. Higher serum adiponectin levels, especially the HMW form, are associated with a decreased breast cancer risk. The association is more pronounced in oestrogen- and progesteronenegative cases. Genetic polymorphisms of adiponectin gene are significantly associated with breast cancer. In line with these clinical findings, experimental evidence supports the role of adiponectin as an inhibitory factor for breast cancer development. Adiponectin at physiological concentrations suppresses the proliferation and induces ▶ apoptosis in the ▶ estrogen receptor (ER)-negative human breast carcinoma MDA-MB-231 cells and the ER-positive human

Adiponectin

MCF7 breast cancer cells. It also inhibits insulinand growth factors-stimulated cell growth in another ER-positive T47D human breast cancer cells. Furthermore, adiponectin replenishment therapy suppresses mammary tumorigenesis of MDA-MB-231 cells in nude mice. Endometrial Cancer

Adiponectin is decreased in obesity, insulin resistance, type 2 diabetes, and polycystic ovary syndrome, all of which are well-established risk factors for endometrial cancer. A number of case-control studies and meta-analyses have demonstrated an inverse correlation between plasma levels of adiponectin and the risk of endometrial cancer, independent of other obesity-related risk factors. Moreover, genetic polymorphisms in the adiponectin gene are associated with endometrial cancer risk. In addition, the oligomeric status of and the ratio of leptin (another hormone secreted by adipose tissue and elevated in obese individuals) to adiponectin show predictive values for endometrial cancer. Treatment with adiponectin reduces the viability of endometrial stromal cells, and inhibits leptin-induced proliferation and invasion of several types of endometrial cancer cells. Further studies are needed to investigate whether adiponectin deficiency plays a causative role in the pathogenesis of endometrial cancer. Lung Cancer

Adiponectin levels are significantly lower in lung cancer patients with advanced disease in comparison with those with limited disease. Increased circulating adiponectin levels are associated with reduced risk for lung cancer. However, serum adiponectin levels at diagnosis are not predictive for survival and progression of the disease. The expression of AdipoR1/R2 is increased in tumor tissues of both non-small (NSCLC) and small cell (SCLC) lung cancer. The genetic variations in the adiponectin gene are associated with increased susceptibility of NSCLC. A direct effect of adiponectin on the proliferation and inflammation status of lung epithelial A549 cells supports a functional role of adiponectin in lung cancer development.

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Kidney Cancer

Esophageal Cancer

The link between obesity and renal cell carcinoma (RCC) is well-established. Lower plasma adiponectin levels are associated with larger tumor size and metastasis of RCC. In patients with end-stage renal disease, low adiponectin levels are an independent predictor of developing malignancy. Adiponectin treatment inhibits the invasive and migratory capacities of RCC cells. Reducing the expression of AdipoR1 increases the growth, dissemination and angiogenesis of RCC. Thus, the deficiency of adiponectin represents a link between obesity and RCC.

The incidence of esophageal adenocarcinoma (EAC) has increased by approximately 600% in the past 40 years. Obesity is an independent risk factor for the development of EAC, independent of gastro-esophageal reflux. Decreased adiponectin levels contribute to the influence of obesity on EAC. Leptin and adiponectin exert mutually antagonistic actions on cells of Barrett esophagus, which appear to influence the progression of malignant behaviour.

Pancreatic Cancer

Genetic variants of adiponectin gene show significant associations with pancreatic cancer. Prediagnostic plasma levels of adiponectin are inversely associated with risk of pancreatic cancer, independent of other markers of obesity. In patients with pancreatic cancer, low adiponectin levels are associated with the development of pancreatic cancer. Treatment with adiponectin inhibits proliferation and induces apoptosis of pancreatic cancer cells. However, a number of case-control studies suggest that adiponectin levels are significantly higher in patients with pancreatic cancer. Moreover, the tumor tissues of pancreatic cancer patients show positive or strong expression of AdipoR1/R2. Liver Cancer

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Obesity and related metabolic abnormalities increase the risk of HCC. However, serum levels of adiponectin in HCC patients are significantly higher than those in healthy controls and associated with worsened overall survival, due to a reduced excretion of this adipokine via the biliary route. Different roles of adiponectin in virusinduced and metabolic-related liver diseases have been proposed, although the underlying mechanism remains unknown. Experimental studies indicate that lack of adiponectin enhances hepatic tumor formation, and treatment with adiponectin induces apoptosis and leptin-stimulated proliferation of HCC cells.

Gastric and Colorectal Cancer

Lower plasma levels of adiponectin have been observed in patients with gastric cancer, especially in those with upper gastric cancer. The negative correlation is more significant in undifferentiated forms than in differentiated forms of gastric cancers. Plasma adiponectin levels tend to decrease as the tumor size, depth of invasion, and tumor stage increases. These data raise the possibility that adiponectin might play a potential role in the progression of gastric cancer, especially in the upper stomach. Obesity is implicated in the pathogenesis of colorectal cancer. Negative, positive or null associations between adiponectin and the risk of developing colorectal cancer have been reported, although polymorphisms of adiponectin gene are associated with colorectal pathogenesis. Experimental studies suggest that adiponectin elicits growth-promoting and proinflammatory actions in HT-29 colonic epithelial cancer cells, but prevents interleukin 1b-regulated malignant potential in colon cancer cell lines. Adiponectin reduces chronic inflammation-induced colon cancer at early stage of carcinogenesis. However, increased adiponectin levels do not confer protection against the development of colon tumors. Leukemia and Myeloma

Adiponectin secretion by bone marrow adipocytes represent a promising drug target in haematological malignancies. Adiponectin is decreased in bone marrow from patients with leukemia at diagnosis. In serum, decreased levels of adiponectin are associated with acute myeloblastic leukemia (AML) and acute lymphoblastic

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Adiponectin

Adiponectin, Fig. 3 Molecular signalling pathways involved in adiponectinmediated antitumorigenic activities

leukemia (ALL). However, it is worthy to note that adiponectin concentrations can be modulated by various inflammatory cytokines and interferon therapy in these conditions. Whether low adiponectin level is a causal factor of leukemia, or a secondary response to ▶ inflammation, needs to be further clarified. Adiponectin levels are also reported to be inversely associated with ▶ chronic lymphocytic leukemia and myeloproliferative diseases. Obesity and lower serum adiponectin levels increase the risk of developing multiple myeloma. Adiponectin inhibits cell proliferation and induces apoptosis in myelomonocytic cell lines. Thus, adiponectin deficiency may play an important role in obesity-related myelomagenesis. Mechanisms In addition to the direct inhibitory effects on cancer cell growth, invasion and migration, as an insulin-sensitizing hormone, adiponectin elicits antitumorigenic activities indirectly by alleviating hyperglycemia and insulin resistance, the two established risk factors for many obesity-related cancers. Furthermore, adiponectin possesses antiinflammatory functions by inhibiting the production or actions of a number of inflammatory factors involved in promoting tumorigenesis. Moreover, adiponectin acts as a decoy for a number of proangiogenic growth factors, including basic fibroblast growth factor (bFGF), plateletderived growth factor BB (PDGF-BB), and

heparin-binding epidermal growth factor (HBEGF), and prevents these growth factors from activating their respective receptors to promote tumor development (Fig. 3). Several key signalling cascades mediate the suppressive effects of adiponectin on the survival and growth of various cancer cells. AMPK

AMPK stimulates fatty acid oxidation and glucose uptake, inhibits cholesterol and triglyceride synthesis, and modulates cell growth and death. The phosphorylation-dependent activation of AMPK mediates the insulin-sensitizing effects of adiponectin in liver and muscle. It is also involved in the regulatory activities of adiponectin on endothelial cell functions and cardiac remodeling. The upstream kinase LKB1 that activates AMPK is a tumor suppressor. AMPK activation inhibits ▶ mammalian target of rapamycin (mTOR) and its downstream effector kinases. Through inactivation of mTOR, AMPK negatively regulates protein and de novo fatty acid synthesis, two essential elements for rapid cancer cell growth. In addition, AMPK controls phosphorylation and activation of the P53 tumor suppressor and expression of the cell cycle inhibitor ▶ p21. Phosphorylation of AMPK further activates protein phosphatase 2A, which can negatively regulate Akt in response to adiponectin stimulation. These molecular events might represent the potential mechanisms through which adiponectin

Adiponectin

regulate carcinogenesis. Indeed, it has been reported that adiponectin at subphysiological concentrations induces AMPK phosphorylation and reduces the cell growth in human breast, colon, liver, endometrial and prostate cancer cells. Glycogen Synthase Kinase (GSK) 3b/b-Catenin Signaling Pathway

Hyperactivation of the canonical Wnt/b-Catenin pathway is one of the most frequent signal abnormalities in many types of cancers. The central event in this pathway is the stabilization and nuclear translocation of b-catenin, where it binds to the transcription factor TCF/LEF and consequently activates a cluster of genes that ultimately establish the oncogenic phenotype. b-Catenin is phosphorylated by GSK3b and then modified by polyubiquitination for ▶ proteasome-mediated degradation. In MDA-MB-231 cells, prolonged treatment with adiponectin markedly reduces serum-induced phosphorylation of GSK3b, decreases intracellular accumulation and nuclear translocation of b-catenin, and suppresses ▶ Cyclin D expression. Tumor cells derived from an adiponectin-deficient stromal microenvironment exhibit a hyperactivated phosphatidylinositol-3-kinase (PI3K)/Akt/b-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN) activities. Adiponectin promotes the thioredoxin/thioredoxin reductase balance, disruption of which in the tumor microenvironment causes PTEN inactivation. In addition, adiponectin enhances the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. These information suggest that the cross-talk between adiponectin and the Wnt signaling pathway represents a key mechanism underlying the development of obesity-related cancers. Other Pathways

Both c-Jun N-terminal kinase (▶ JNK) and signal transducer and activator of transcription 3 (STAT3) are important regulators of cell proliferation, apoptosis, and differentiation in various physiological and pathophysiological conditions. Constitutive activation of STAT3 is crucial in

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malignant transformation and cancer progression. It has been reported that adiponectin stimulates the phosphorylation of JNK in prostate cancer DU145, PC-3, and LNCaP-FGC cells, as well as in hepatocellular carcinoma HepG2 cells. On the other hand, adiponectin inhibits constitutive activation of STAT3 in DU145 and HepG2 cells, suggesting that activation of JNK and inhibition of STAT3 may contribute to the suppressive effect of adiponectin on carcinogenesis. Adiponectin inhibits leptin-induced oncogenic signalling in oesophageal cancer cells by activation of PTP1B. Adiponectin also increases suppressor of cytokine signaling (SOCS3). In addition, the inactivation of p42/p44 MAP kinase has been implicated in the antiproliferative effects of adiponectin in human beast carcinoma MCF-7 and T47D cells. In prostate cancer cells, NF-kB signalling pathway is involved in adiponectin-mediated integrin upregulation and cellular migration. Adiponectin-based Therapeutics Adiponectin and its analogues represent a novel class of anticancer agents for the treatment of obesity-related malignant tumors. The peptide ADP-355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSerPhe-Ala-DSer-NH2) mimics the actions of adiponectin to dose-dependently inhibit cancer cell proliferation and suppress breast cancer xenograft growth. A small molecular compound, AdipoRon, binds with high affinity to AdipoR1/R2 and elicits protective effects on obesity-induced metabolic dysfunctions. However, considering the increased or ubiquitous expression of AdipoR1/R2 in tumor tissues, the application of AdipoRon in cancer treatment needs to be scrutinized. Other agents that increase the endogenous adiponectin levels include PPARg ligands, antidiabetic drug methormin and apolipoprotein AI mimetic peptide L-4 F. Further studies are warranted to investigate their potential applications in obesity-related cancers.

Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Acute Myeloid Leukemia

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▶ Angiogenesis ▶ Apoptosis ▶ Breast Cancer ▶ Chronic Lymphocytic Leukemia ▶ Cyclin D ▶ Endometrial Cancer ▶ Estrogen Receptor ▶ Gastric Cancer ▶ Inflammation ▶ Insulin-Like Growth Factors ▶ JNK Subfamily ▶ Lung Cancer ▶ Mammalian Target of Rapamycin ▶ Obesity and Cancer Risk ▶ p21 ▶ Prostate Cancer Clinical Oncology ▶ Proteasome

References Holland WL and Scherer PE (2013) Ronning after the adiponectin receptors. Science 342:1460–1461 Li H, Stampfer MJ, Mucci L, Rifai N, Qiu W, Kurth T, Ma J (2010) A 25-year prospective study of plasma adiponectin and leptin concentrations and prostate cancer risk and survival. Clin Chem 56:34–43 Macis D, Guerrieri-Gonzaga A, Gandini S (2014) Circulating adiponectin and breast cancer risk: a systematic review and meta-analysis. Int J Epidemiol 43:1226–1236 Ntikoudi E, Kiagia M, Boura P, Syrigos KN (2014) Hormones of adipose tissue and their biologic role in lung cancer. Cancer Treat Rev 40:22–30 Otvos L Jr, Haspinger E, La Russa F, et al. (2011) Design and development of a peptide-based adiponectin receptor agonist for cancer treatment. BMC Biotechnol 11:90 Peterson SJ, Drummond G, Kim DH, Li M, Kruger AL, Ikehara S, Abraham NG (2008) L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice. J Lipid Res 49:1658–1659 Saxena NK, Fu PP, Nagalingam A, Wang J, Handy J, Cohen C, Tighiouart M, Sharma D, Anania FA (2010) Adiponectin modulates c-jun N-terminal kinase and mammalian target of rapamycin and inhibits hepatocellular carcinoma. Gastroenterology 139: 1762–1773 Wang Y, Lam JB, Lam KS et al. (2006) Adiponectin modulates the glycogen synthase kinase-3β/β-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res 66:11462–11470

AdipoQ Wang Y, Lam KS, Yau MH, Xu A (2008) Post-translational modifications of adiponectin: mechanisms and functional implications. Biochem J. 409:623–633

See Also (2012) Adipokine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 67. doi: 10.1007/978-3-642-16483-5_102 (2012) AMPK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 160. doi: 10.1007/978-3-642-16483-5_244 (2012) Colorectal cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 916. doi: 10.1007/978-3-642-16483-5_1265 (2012) Hyperglycemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1781. doi: 10.1007/978-3-642-16483-5_2907 (2012) Insulin resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1877. doi: 10.1007/978-3-642-16483-5_3078 (2012) Jun N-terminal kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1929. doi: 10.1007/978-3-64216483-5_3187 (2012) Leptin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2001. doi: 10.1007/978-3-642-16483-5_6734 (2012) Liver cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2063. doi: 10.1007/978-3-642-16483-5_3393 (2012) MTOR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2384. doi: 10.1007/978-3-642-16483-5_3867 (2012) Obesity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2595. doi: 10.1007/978-3-642-16483-5_4185 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi: 10.1007/978-3-642-16483-5_4331 (2012) Polyubiquitination. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2957. doi: 10.1007/978-3-642-164835_4678 (2012) Tumor suppressor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3803. doi: 10.1007/978-3-642-16483-5_6056 (2012) Wnt/beta-catenin pathway. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3957. doi: 10.1007/978-3-64216483-5_6256

AdipoQ ▶ Adiponectin

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Adipose Most Abundant Gene Transcript 1 ▶ Adiponectin

Adipose Tissue-Specific Secretory Factor (ADSF) ▶ Resistin

Adipose Tumors Florence Pedeutour1 and Antoine Italiano2 1 Laboratory of Solid Tumors Genetics, Faculty of Medicine, Nice University Hospital, Nice, France 2 Early Phase Trials and Sarcoma Units, Institut Bergonie, Bordeaux, France

Synonyms Adipocytic tumors; tumors; Liposarcomas

Lipomas;

Lipomatous

Definition Adipose tumors (AT) are mesenchymal neoplasms that form the largest group of human tumors. They include benign tumors, such as the very common lipomas, as well as rare malignant tumors with various degrees of clinical aggressiveness. Histologically, AT consist of adipocytic cells showing different levels of differentiation, from mature adipocytes in benign lipomas up to undifferentiated lipoblastic cells in high-grade liposarcomas. The 2002 World Health Organization classification distinguishes seven entities of benign AT: lipoma, lipoblastoma/ lipoblastomatosis, angiolipoma, myolipoma of soft tissue, chondroid lipoma, spindle cell/pleomorphic lipoma, and hibernoma. Malignant AT, also called liposarcomas, include three types:

well-differentiated liposarcoma/dedifferentiated liposarcoma, ▶ myxoid/round cell liposarcoma, and pleomorphic liposarcoma. Except for the ordinary superficial lipomas, differential diagnosis between benign and malignant AT and between AT and other kinds of tumors is sometimes difficult. Studies based on tumor karyotypes have identified chromosomal abnormalities specific to benign and malignant AT and advances in molecular cytogenetics improved AT diagnosis. It is now possible to directly detect the genic rearrangements resulting from chromosomal alterations on interphase nuclei such as those in formalin-fixed and paraffinembedded tumor tissue sections using fluorescence in situ hybridization (FISH) (▶ interphase cytogenetics) or polymerase chain reaction (PCR).

Characteristics Benign Adipose Tumors The most common benign AT is the so-called superficial conventional lipomas. The other types of benign AT are rare and may be the cause of diagnostic difficulties because of their clinical or histological resemblance to malignant soft tissue tumors. In most cases benign AT do not require any treatment. Surgical removal may be necessary in case of functional or cosmetic impairment. Conventional lipomas are the most common soft-tissue neoplasm in adults. They occur mainly in the fifth to seventh decades of life and are generally located superficially in subcutaneous fat. They can also be situated deeply in muscles or on the surface of bones or rarely in visceral and other organ sites. Lipomas usually present as a small (90% long-term virological and clinical response when treating viral infections. Enhancing the Function of Adoptively Transferred Cells Lymphodepletion

To increase treatment efficacy of adoptively transferred cells, investigators are lymphodepleting patients prior to adoptive cell transfer or genetic modifying cells to enhance effector function. Lymphodepletion, removal of the host’s lymphocytes, prior to adoptive transfer should allow the infused cells to expand using the body’s own homeostatic cytokines like IL-7 and IL-15. The most common regimens for lymphodepletion utilize cyclophosphamide and fludarabine prior to adoptive cellular transfer; however, groups have used a variety of options including monoclonal antibodies targeting CD45, single agent chemotherapy, or chemotherapy and total body irritation. Published reports have shown improved outcomes in patients, especially in metastatic melanoma, where lymphodepletion was used prior to T cell infusion. TCR and Chimeric Antigen Receptors

Additionally, infused T or NK cells may be genetically modified with artificial receptors targeting tumor antigens or with molecules that may confer resistance to tumor evasion strategies. In addition to TCRs, effector lymphocytes can be genetically modified to express chimeric antigen receptors (CARs). CARs can combine the specificity and antitumor effects of monoclonal antibodies with the direct cytotoxicity and long-term persistence of T cells. The most successful use of CAR T cell (▶ Chimeric Antigen Receptor on T Cells) therapy has targeted the CD-19 antigen for the treatment of CD-19 positive leukemia and lymphoma and GD-2 for the treatment of neuroblastoma. Investigators are now focusing on increasing the activity of the infused CAR T cells, improving antitumor targeting, and reducing sensitivity of the modified cells to the inhibitory microenvironment of the tumor. Further, as the affinity of CARs

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and/or TCRs increases with genetic modification, the likelihood of on- and off-target toxicity secondary to low-level antigenic expression on normal tissues increases. Thus, further evaluation of antigenic expression on tumor cells and normal tissues and improved methods of preclinical toxicity assessments are critically important.

Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Acute Myeloid Leukemia ▶ Adenovirus ▶ Chimeric Antigen Receptor on T Cells ▶ Colorectal Cancer ▶ Epstein-Barr Virus ▶ Hepatocellular Carcinoma ▶ Hodgkin Disease ▶ Hodgkin Lymphoma, Clinical Oncology ▶ Human Herpesvirus 6 ▶ Multiple Myeloma ▶ Nasopharyngeal Carcinoma ▶ Natural Killer Cell Activation

Adoptive T-Cell Transfer Papadopoulou A, Gerdemann U, Katari UL et al (2014) Activity of broad-spectrum T cells as treatment for AdV, EBV, CMV, BKV, and HHV6 infections after HSCT. Sci Transl Med 6(242):242ra83 Rosenberg SA, Restifo NP (2015) Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348(6230):62–68 Zhang J, Zhu L, Zhang Q et al (2014) Effects of cytokineinduced killer cell treatment in colorectal cancer patients: a retrospective study. Biomed Pharmacother 68(6):715–720

See Also (2012) Cytotoxic T lymphocytes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1058. doi:10.1007/978-3-642-16483-5_1501 (2012) Glioblastoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1554. doi:10.1007/978-3-642-16483-5_2421 (2012) Lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2124. doi:10.1007/978-3-642-16483-5_3463 (2012) Non-Hodgkin lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-164835_4110 (2012) Renal-Cell carcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3252. doi:10.1007/978-3-642-164835_5023

References Bollard CM, Rooney CM, Heslop HE (2012) T-cell therapy in the treatment of post-transplant lymphoproliferative disease. Nat Rev Clin Oncol 9(9):510–519 Brentjens RJ, Davila ML, Riviere I et al (2013) CD19targeted T Cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5(177):177ra38 Dillman RO, Duma CM, Ellis RA et al (2009) Intralesional lymphokine-activated killer cells as adjuvant therapy for primary glioblastoma. J Immunother 32(9):914–919 Grupp SA, Kalos M, Barrett D et al (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368(16):1509–1518 Heczey A, Louis CU (2013) Advances in chimeric antigen receptor immunotherapy for neuroblastoma. Discov Med 16(90):287–294 Lee JH, Lee JH, Lim YS et al (2015) Adjuvant immunotherapy with autologous cytokine-induced killer cells for hepatocellular carcinoma. Gastroenterology 148(7):1383–1391 Levine BL, Humeau LM, Boyer J et al (2006) Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci USA 103(46):17372–17377

Adoptive T-Cell Transfer Mingjun Wang Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA

Synonyms Infusion of T cells; Transfer of T cells; Transfusion of T cells

Definition Adoptive T-cell transfer is an approach to treat various types of diseases, particularly malignant tumors by intravenous injection of autologous T cells modified with or without a gene encoding

Adoptive T-Cell Transfer

a specific antigen receptor. T cells are usually isolated from the tumor tissues or peripheral blood mononuclear cells (PBMCs) of cancer patients, stimulated with tumor antigens or modified with a gene encoding a specific antigen receptor, then expanded in vitro to a large quantity before infusion back into the patient for treatment.

Characteristics Adoptive T-cell transfer is an approach of cancer immunotherapy, which has emerged as a novel and promising approach for treatment of cancer patients with advanced or refractory diseases, since traditional cancer treatments, including surgery, chemotherapy, and radiation therapy, have demonstrated very limited efficacy for patients with late-stage diseases. In addition, compared to considerable side effects caused by traditional therapies, adoptive T-cell transfer-based immunotherapy holds several key advantages: (1) high specificity, (2) little or no side effects, although adverse effects may occur in adoptive cell transfer using genetically modified T cells, and (3) good safety profile. To date, adoptive T-cell transfer among various types of cancer immunotherapy has been demonstrated to be the most effective immunotherapy method for cancer treatment and has achieved very promising results in cancer clinical trials, thus leading to its being named as the Science “Breakthrough of the Year” in 2013. Importantly, a number of pharmaceutical industries are starting to invest heavily to rapidly facilitate the development of such an adoptive T-cell transfer-based approach to treat various types of cancer. T cells used for adoptive T-cell transfer are usually isolated from the tumor tissues or peripheral blood mononuclear cells (PBMCs) of cancer patients, stimulated with tumor antigens or modified with a gene encoding a specific antigen receptor in vitro, and then expanded to a large quantity before infusion back into the patient for treatment. Adoptive T-cell transfer, which has demonstrated dramatic potency in cancer treatment and has shown encouraging therapeutic effects in clinical trials, includes tumor infiltrating

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T cells (TILs), cancer antigen-induced T cells, T cell receptor (TCR)-transduced T cells, and chimeric antigen receptor (CAR)-transduced T cells (Yee 2014; Ruella and Kalos 2014; Hinrichs and Rosenberg 2014; Cheadle et al. 2014; Restifo et al. 2012). TILs TILs are isolated from the tumor tissues of cancer patients, expanded in vitro using a high concentration of interleukin (IL)-2, and then infused back into the patient. TIL-based adoptive T cell transfer for treatment of cancer patients was pioneered by Dr. Rosenberg at NIH in 1988 and was first demonstrated in melanoma with a low objective response at that time. Current objective response rate of 49–72% can be achieved when lymphodepleting preparative regimen is performed prior to TIL infusion. Despite the clinical benefits of TIL-based therapy, there are limitations to its successful implementation: (1) TIL-based immunotherapy is an individualized treatment that requires surgical removal of tumor tissues and a highly skilled medical staff to isolate and cultivate TILs; (2) TIL-based immunotherapy is currently only effective against melanoma even though TILs can be also isolated from other solid tumors including colorectal cancer, breast cancer, lung cancer, and ovarian cancer. Nevertheless, successful TIL-based immunotherapy has promoted the rapid development of adoptive T-cell transfer using antigen-induced T cells and genetically modified T cells for treatment of other types of cancer. Cancer Antigen-Induced T Cells Cancer antigen-specific T cells are present in PBMCs of cancer patients and can be cultured or enriched from PBMCs or TILs following in vitro stimulation using autologous antigen-presenting cell pulsed with peptides derived from cancer antigens. Cancer antigen-induced T cells targeting MART1/MelanA, gp100 and NY-ESO-1 have been used for adoptive T-cell transfer to treat metastatic melanoma with little or no side effects. Adoptive T-cell transfer using cancer antigeninduced T cells has several advantages: (1) easy to collect PBMCs from patients for preparation of

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cancer-reactive T cells by in vitro cancer antigen stimulation; (2) a number of cancer antigens and their derived HLA-restricted epitopes (www. cancerimmunity.org/peptide) are available to facilitate the development of antigen-specific T cells; and (3) peptides are synthesized cheaply and can be easily and safely delivered to any medical center for stimulation of cancer-reactive cells. With identification of more and more cancer antigens, it will be favorable to use such antigens to generate cancer antigen-induced specific T cells in vitro for the treatment of various types of cancer. Antigen-Specific TCR-Transduced T Cells T cells genetically engineered with antigenspecific TCRs in vitro can specifically recognize and kill cancer cells. TCR-transduced T cells targeting several tumor antigens including MART1, CEA, gp100, NY-ESO-1, and MAGEA3 have been tested in clinical trials. Although, the results based on adoptive T-cell transfer using TCR-transduced T cells in clinical trials have shown great promise in treating various types of cancers including metastatic melanoma, metastatic colorectal cancer, metastatic synovial cell sarcoma, and epithelial malignancies, severe adverse events (SAEs) due to cross-reactivity with cancer antigens expressed at low levels in vital organs have become a barrier to wide application of TCR-transduced T cells in clinical trials. To date, NY-ESO-1 has been only shown as one of ideal tumor targets for TCR-transduced T cells, since NY-ESO-1 is expressed in various types of cancer cells, but not in normal somatic tissues or cells except normal testes, ovary, and placenta. Thus, theoretically TCR-transduced T cells targeting NY-ESO-1 will only eradicate or attack cancer cells, but not normal cells. Therefore, autoimmune toxicities in this setting will not occur. Indeed, TCR–transduced T cells targeting NY-ESO-1 have achieved objective tumor responses in patients with metastatic synovial cell sarcoma and melanoma without the induction of autoimmune toxicities. In the future, it is of great importance to identify ideal cancer antigen targets with tumor-restricted expression (e.g., NY-ESO-1) to minimize the risk of developing

Adoptive T-Cell Transfer

SAEs due to cross-activity or “on-target/offtumor” effects. CAR-Transduced T Cells The concept of CAR was firstly introduced in 1989. The CAR structure is composed of an extracellular single-chain variable fragment (scFv) of an antibody, a transmembrane domain, and intracellular signaling domains derived from molecules involved in T cell signaling. When T cells are transduced with a lentiviral or retrovial vector encoding a CAR targeting a surface antigen, the ectodomain scFv of CAR can specifically recognize and bind to the surface antigen expressed on cancer cells and deliver activating signals to T cells through CD3x, which in turn trigger T cell effector functions to eliminate the cancer cells. CAR-transduced T cell therapy can target a variety of cell surface molecules including proteins with varying glycosylation and nonprotein structures such as gangliosides and carbohydrate antigens; in addition, CAR-transduced T cell function is unaffected by tumor escape mechanisms related to HLA downregulation and altered processing. CAR is generally classified into three generations according to the number of signaling domains including CD28, 4-BB, OX-40, etc. Firstgeneration CAR-transduced T cells targeting neuroblastoma, lymphoma, renal cancer, and ovarian cancer have achieved only limited clinical activity due to the lack of T cell expansion and long-term persistence in vivo. Currently, most clinical trials are using second-generation CAR-transduced T cells; adoptive T-cell transfer with secondgeneration GD2-specific CAR-transduced T cells has showed clinical benefits that are associated with the long-term low-level presence of CAR-expressing T cells. However, the most promising results from CAR-transduced T cell therapy have been with CD19-based targeting of B cell malignancies. It has been shown that T cells transduced with second generation anti-CD19 CARs containing either CD28 or 4-1BB costimulatory endodomain are effective to treat advanced or refractory lymphoma, chronic lymphocytic leukemia, and acute lymphocytic leukemia. The complete remission rate is now about

Adrenocortical Cancer

60–90% after infusion of CAR-transduced T cells targeting CD19 into patients with refractory or recurrent acute lymphocytic leukemia. Although CAR-transduced T cells targeting CD19 have achieved encouraging results, this treatment can also cause side effects. B cell aplasia due to infusion of anti-CD19 CAR-transduced T cells is an expected “on-target/off-tumor” effect, which may be treated by injection of immunoglobulins. The other associated toxicity is described as a cytokine release syndrome including high-grade fevers, hypotension, hypoxia as well as neurologic disturbances, which may need supportive treatment. Despite the great success to date with antiCD19 CAR-transduced T cells in the treatment of patients with B cell malignancies, clinical trials targeting solid cancers have achieved limited efficacy and observed “on-target, off-tumor responses” with serious consequences. The failure of CAR-transduced T cells to treat solid cancers may be due to several reasons including lack of ideal cancer antigens, short-term persistence of CAR-transduced T cells, and inefficient trafficking of sufficient numbers of CAR-transduced T cells to tumor sites. Furthermore, an immunosuppressive tumor environment also inhibits the functions of CAR-transduced T cells at tumor sites. Strategies to overcome these barriers should be taken into consideration to construct CAR-transduced T cells capable of treating solid cancers as well as hematopoietic malignancies in future clinical trials.

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▶ Chronic Lymphocytic Leukemia ▶ Immunotherapy ▶ NY-ESO-1

References Cheadle EJ, Gornall H, Baldan V, Hanson V, Hawkins RE, Gilham DE (2014) CAR T cells: driving the road from the laboratory to the clinic. Immunol Rev 257(1):91–106 Hinrichs CS, Rosenberg SA (2014) Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev 257(1):56–71 Restifo NP, Dudley ME, Rosenberg SA (2012) Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 12(4):269–281 Ruella M, Kalos M (2014) Adoptive immunotherapy for cancer. Immunol Rev 257(1):14–38 Yee C (2014) The use of endogenous T cells for adoptive transfer. Immunol Rev 257(1):250–263

Adrenocortical Cancer Rossella Libè and Jérôme Bertherat Endocrinology, Metabolism and Cancer Department, INSERM U567, Institut Cochin, Paris, France

Synonyms Carcinoma of the adrenal cortex; Malignant adrenocortical tumor

Conclusions Adoptive T-cell transfer-based immunotherapy has achieved encouraging results in clinical trials. Future research is required to develop safe and efficient adoptive T-cell transfer-based immunotherapies with broad efficacy against various types of cancer.

Definition

Cross-References

Characteristics

▶ Autoimmunity and Cancer ▶ B-cell Tumors ▶ Cancer ▶ Chimeric Antigen Receptor on T Cells

Epidemiology of Adrenocortical Cancer ACC is a rare disease with an estimated incidence between 1 and 2 per million and per year in adults in North America and Europe.

Adrenocortical cancer (ACC) is a malignant tumor from the adrenal cortex. It is a rare tumor with a poor prognosis. The consequences of ACC are due to tumor growth and metastasis and also due to steroid oversecretion.

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Pathophysiology of Adrenocortical Cancer Analysis of the pattern of X-chromosome inactivation in heterozygous female tissue has shown that ACC consists of monoclonal populations of cells. A large number of molecular techniques, such as comparative genomic hybridization (CGH) and microsatellite analysis, have identified alterations affecting various chromosomes and loci in ACC. Most of the changes observed concern losses on chromosomes 2, 11q and 17p, and gains on chromosomes 4 and 5. Studies using microsatellite markers have demonstrated a high percentage of loss of heterozygosity (LOH) or allelic imbalance at 11q13 (90%), 17p13 (85%), and 2p16 (92%) in ACC. IGF-II (Insulin-Like Growth Factor II)

The insulin-like growth factors system is involved in the development of the adrenal cortex and its role has been largely documented in adrenocortical tumors. The IGF-II gene located at 11p15 encodes an important fetal growth factor, is maternally imprinted, and is therefore expressed only from the paternal allele. Genetic or epigenetic changes in the imprinted 11p15 region, resulting in increases in IGF-II expression, and mutations of the p57kip2 gene have been implicated in Beckwith-Wiedemann syndrome. This overgrowth disorder is characterized by macrosomia, macroglossia, organomegaly and developmental abnormalities (in particular abdominal wall defects with exomphalos), embryonal tumors – such as Wilms’ tumor – and ACC, neuroblastoma, and hepatoblastoma. Many studies have demonstrated that IGF-II is strongly overexpressed in malignant adrenocortical tumors, with such overexpression observed in ~90% of ACC. Transcriptome analysis of adrenocortical tumors has demonstrated that IGF-II is the gene most overexpressed in ACC by comparison with benign adrenocortical adenomas or normal adrenal glands. The mechanisms underlying IGFII overexpression are paternal isodisomy (loss of the maternal allele and duplication of the paternal allele) or, less frequently, loss of imprinting.

Adrenocortical Cancer

variety of cancers. Adrenocortical tumors have been observed in some case reports of patients with familial APC. Furthermore, familial APC patients, with germline mutations of the APC gene that lead to an activation of the Wnt signaling pathway, may develop ACTs. The Wnt signaling pathway is normally activated during embryonic development. b-Catenin is a key component of this signaling pathway. Interestingly, gene profiling studies in various types of adrenocortical tumors have shown the frequent activation of Wnt signaling target genes. In both benign and malignant ACT, b-catenin accumulation can be observed. These alterations seem very frequent in ACC, consistent with an abnormal activation of the Wnt signaling pathway. This is explained in a subset of adrenocortical tumors by somatic mutations of the b-catenin gene altering the Glycogen synthase kinase 3-b (GSK3-b) phosphorylation site. TP53

The tumor suppressor gene TP53 is located at 17p13 and involved in the control of cell proliferation. Germline mutations in TP53 are identified in 70% of families with Li-Fraumeni Syndrome (LFS). This syndrome displays dominant inheritance and confers susceptibility to breast carcinoma, soft tissue sarcoma, brain tumors, osteosarcoma, leukemia, and ACC. Germline mutations in TP53 have been observed in 50–80% of children with apparently sporadic ACC in North America and Europe. The incidence of pediatric ACC is about ten times higher in Southern Brazil than in the rest of the world, and a specific germline mutation has been identified in exon 10 of the TP53 gene (R337H) in almost all cases. In sporadic ACC in adults, somatic mutations of TP53 are found in only 25–35% of cases. LOH at 17p13 has been consistently demonstrated in ACC but not in adrenocortical adenomas. LOH at 17p13 was reported to occur in 85% of ACC. Diagnosis and Treatment of Adrenocortical Cancer

b-Catenin Activation in Adrenocortical Cancer

Genetic alterations of the Wnt signaling pathway were initially identified in familial adenomatous polyposis coli (APC) and have been extended to a

Clinical and Hormonal Investigations

Symptoms leading to the diagnosis of ACC can be due to hormone hypersecretion and/or tumor mass

Adrenocortical Cancer

and metastasis. The majority of ACC are usually secreting tumors when careful hormonal investigations are performed. By contrast with benign adrenocortical tumors (that usually secrete a single class of steroid), ACC can secrete various types of steroids (glucocorticoids, androgens, and mineralocorticoids). Cosecretion of androgens and cortisol is the most frequent and highly suggestive of a malignant adrenocortical tumor. Cortisol oversecretion (classified as “ACTHindependent Cushing’s syndrome” in case of ACC) can induce centripetal obesity, protein wasting with skin thinning and striae, muscle atrophy (myopathy), diabetes, hypertension, psychiatric disturbances, gonadal dysfunction, and osteoporosis. Imaging of Adrenocortical Cancer

Imaging is an essential diagnostic step for ACC. It is important for both, not only for the diagnosis of malignancy of an adrenal mass but also for the extension work-up. Adrenal computed tomography scan (CT-scan) is a very informative imaging procedure for adrenocortical tumors. In ACC, it shows a unilateral mass, which is most often large (above 5–6 cm, typically 10 cm and above), lowering the kidney. MRI can also be used in the diagnosis of liver nodules and venous invasions. Studies have demonstrated that ACCs almost invariably have a high uptake of 18-fluorodesoxyglucose ((18)-FDG). Thus (18)FDG PET scan appears to distinguish between benign and malignant adrenal tumors. This simple, nontraumatic imaging procedure also participates in the extension work-up. Pathology and Molecular Analysis

As often with endocrine tumors, the diagnosis of malignancy of adrenocortical lesions is not always easy for the pathologist. Combinations of various histological parameters allowing the calculation of a “score” for a given tumor have been developed. The most widely used is the Weiss score made of nine different items. It is assumed that a score above three is most likely associated with a malignant tumor. Since the Weiss score has limitations and is dependent on the experience of the pathologist, there is an effort to develop

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molecular markers of malignancy. IGF-II overexpression and allelic losses at 17p13 have been suggested as useful markers. Immunohistochemistry of Cyclin E or Ki-67 that are higher in malignant adrenocortical tumors has also been suggested in the literature as potential useful tools. Prognosis of Adrenocortical Cancer

The overall prognosis of ACC is poor with a 5-year survival rate below 35% in most series. Among the various clinical parameters that have been shown to impact on ACC prognosis, tumor staging has been demonstrated as one of the most important. The MacFarlane staging is the most commonly used and relies on surgical finding and extension work-up. Four different stages are differentiated with this score. Stage 1 and 2 tumors are localized to the adrenal cortex and present a maximum diameter below or above 5 cm, respectively. Locally invasive tumors or tumors with regional lymph node metastases are classified as Stage 3, whereas Stage 4 consists of tumors invading adjacent organs or presenting with distant metastases. The prognosis of Stage 1 and 2 tumors is better than that of Stage 3 or 4 tumors. A better survival is usually reported in younger patients. Some pathological features as a high mitotic rate or atypical mitotic figures have been shown to be associated with a poor prognosis. In the future, it is expected that molecular tools will help a better prediction of the prognosis of ACC. Gene profiling approach can already differentiate malignant from benign tumors. Treatment of Adrenocortical Cancer

Surgery of the adrenal tumor is the major treatment of Stage 1 to 3 ACC. It can also be discussed in Stage 4 patients. Only complete tumor removal can lead to long-term remission. Radiofrequency thermal ablation of liver and lung metastasis below 4–5 cm of maximal diameter can be an alternative to surgical removal. Chemoembolization has also been used for liver metastasis. Surgery of bone metastasis can be indicated to reduce fracture risk, or, in case of spinal localization, neurological symptoms. Radiation therapy is usually considered as not very effective to control tumor growth. However, it has been suggested

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that tumor bed radiation therapy could help prevent local recurrence after surgical removal. When complete tumor removal is not possible, or in case of recurrence, medical treatment with o, p’-DDD (ortho, para’, dichloro-, diphenyl-, dichloroethane, or Mitotane) is recommended. It has both an anticortisolic action and a cytotoxic effect on the adrenocortical cells. Objective tumor regression could be observed in 25–35% of the patients. A mitotane blood level of at least 14 mg/l seems to improve the tumor response rate. However, the side effects of mitotane (mainly digestive and neurologics) often limit the ability to reach this suggested optimal level. Since o,p’-DDD can induce adrenal insufficiency, substitutive glucocorticoid, and mineralocorticoid therapy should be associated. Several cytotoxic chemotherapy regimens have been used in ACC. They are usually considered in patients with tumor progression under mitotane therapy reaching the plasma blood level of 14 mg/l or presenting severe side effects limiting its use. Various drugs have been used and the experience is still limited. It is currently accepted that the combined treatment with cisplatine, etoposide, doxorubicin (EDP regimen) associated with o,p’-DDD, and streptozotocin also given with o,p’-DDD are the better regimens. However, there is obviously an important need for prospective controlled studies and for new therapies in patients with advanced ACC.

Adrenomedullin

Adrenomedullin Enrique Zudaire and Franck Cuttitta NCI Angiogenesis Core Facility, National Cancer Institute, National Institutes of Health, Advanced Technology Center, Gaithersburg, MD, USA

Definition Adrenomedullin (AM) is a member of the ▶ calcitonin superfamily of peptides. It is produced in virtually every organ by many different cell types, and it is secreted into the plasma where it occurs at picomolar concentrations. Over the past several years, AM has increasingly received the attention of the scientific community by virtue of its implication in many normal and disease states.

Characteristics

References

Adrenomedullin is a small peptide (52 amino acids) first isolated from a pheochromocytoma in 1993. It was initially described as a hypotensive peptide although after more than a decade of research and about 2,000 articles published, AM is now recognized as a pluripotent peptide hormone implicated in many normal and pathological processes ranging from vascular tone and diabetes to ▶ angiogenesis and embryogenesis/ ▶ carcinogenesis.

Allolio B, Hahner S, Weismann D et al (2004) Management of adrenocortical carcinoma. Clin Endocrinol (Oxf) 60(3):273–287 Bertherat J, Groussin L, Bertagna X (2006) Mechanisms of disease: adrenocortical tumors – molecular advances and clinical perspectives. Nat Clin Pract Endocrinol Metab 2(11):632–641 Giordano TJ, Thomas DG, Kuick R et al (2003) Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 162(2):521–531 Libe R, Bertherat J (2005) Molecular genetics of adrenocortical tumours, from familial to sporadic diseases. Eur J Endocrinol 153(4):477–487 Sidhu S, Sywak M, Robinson B et al (2004) Adrenocortical cancer: recent clinical and molecular advances. Curr Opin Oncol 16(1):13–18

Adrenomedullin: Peptide and Gene Structure Adrenomedullin is generated as part of a larger precursor molecule named preproadrenomedullin (preproAM) (Fig. 1). PreproAM is 185-aminoacid long and contains an N-terminal 21-aminoacid signal peptide which is cleaved during the transport of the molecule across the cell membrane to produce the 164-amino-acid prohormone proAM. Further processing of proAM by endopetidases generates four peptides termed proadrenomedullin N-terminal 20 peptide (PAMP), mid-regional proadrenomedullin (proAM 45–92), adrenomedullin (AM), and adrenotensin (proAM 153–185). From these,

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Adrenomedullin, Fig. 1 Genomic organization of the AM gene

Ex 1

5’

Ex 2 Ex 3

Ex 4

3’

A Form A Form B UAA mRNA

Preprohormones

Gly

Gly ? Proteins present in plasma

Signal peptide

PAMP

PAMP, proAM 45–92, and AM are present in plasma, and PAMP, AM, and adrenotensin are biologically active peptides. Both PAMP and AM peptides are produced as glycine (Gly)extended inactive peptides which coexist in plasma with the active form generated upon enzymatic amidation. AM shares homology with several vasoactive peptide members of the calcitonin superfamily including calcitonin, calcitonin generelated peptide (CGRP), amylin, and intermedin. Members of this family share the presence of an intramolecular disulfide bond which generates a six-member ring structure and an amidated carboxy-terminal, both of which are required for biological activity. In humans, the single locus of the adrenomedullin gene is located in the short arm of chromosome 11. The complete gene (2,319 bp) contains four exons and three introns which are alternatively spliced during the transcription process to generate two different transcripts (Fig. 1).

?

Mid-regional pro-adrenomedullin

Adrenotensin

Adrenome dullin

Amide group

HRE

The shortest mRNA form includes exons 1–4 and therefore codes for a complete preprohormone which results in stoichiometric amounts of the four peptides referred above. The longest transcript incorporates the third intron that contains an early termination codon, resulting in a truncated preprohormone which only expresses PAMP. AM is an ancient gene that, based on our current knowledge, first appeared in the starfish with a potential dual function of neurotransmission and host defense. It shows a remarkable degree of conservation in genomic organization and peptide structure from fish to humans which supports its critical role in species survival. Signal Transduction As most soluble peptides, AM transduces its signal upon interaction with a receptor located in the cellular surface. The discovery of the AM receptor in 1998 represented a novel paradigm in the field

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of G-protein-coupled receptor (GPCR) signaling. A functional receptor for AM requires physical interaction in the cellular membrane of the seventransmembrane domain calcitonin receptor-like receptor (CRLR) and either the receptor-activitymodifying protein (RAMP)2 or RAMP3. CRLR has two alternative pharmacological profiles that are conferred by association to the accessory proteins RAMP1 (producing the CGRP receptor) and RAMP2/RAMP3 (producing the AM receptor). Therefore, the expression pattern of functional AM receptors is determined by the presence of these two components. In healthy individuals, RAMP2/RAMP3 is equally expressed among most tissues, excluding the lung, female reproductive system, and adipocytes which show higher levels of expression. CRLR expression, although lower, parallels that of RAMP2 which suggests that the majority of CRLR signaling units in the body are complexed with RAMP2 to produce adrenomedullin receptors. Modest but robust changes in the expression of the complex CRLRRAMP2 have been reported in certain physiological and disease states such as pregnancy, sepsis, and ▶ cancer. The same physiological conditions are related to high levels of AM expression. Other stimuli which result in coordinated regulation of AM, CRLR, and RAMP2 include hypoxia, endocrine hormones, and inflammatory cytokines. Upon binding to its receptor, AM induces cAMP elevation through an adenylyl cyclasePKA-mediated pathway. While multiple reports including the seminal paper by Kitamura have consistently demonstrated cAMP-mediated effects of AM, other more scarce ones have shown cAMP-independent actions such as vasodilation via elevation of Ca2+ and K+-ATP and activation of endothelial nitric oxide synthase. AM also activates Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells which mediate its angiogenic potential. AM Serves as a Common Language Between the Different Cellular Components of the Tumor Microenvironment Many disease states have been reported to modulate the expression of AM including cancer. As we mentioned before, AM was originally isolated

Adrenomedullin

from an adrenal gland tumor. A wealth of subsequent studies have found that AM and its receptor are overexpressed in many human cancers and tumor cell lines establishing an autocrine loop mechanism that tumor cells exploit to maintain an autonomous proliferative state. AM is intimately intertwined at several levels in the multistep process of tumor development. At the initial stage of tumor growth, rapid accumulation of malignant cells results in the establishment of an avascular nutrient-depleted hypoxic environment. Low oxygen tension within and surrounding the tumor body triggers a number of survival mechanisms which allow neoplastic cells to overcome this inhospitable microenvironment. Many of these encompass the upregulation of AM’s expression. In fact one, if not the most important, driving force for AM upregulation in tumor cells is hypoxia. Cellular responses to hypoxia are mediated through a well-known hypoxia inducible factor (HIF)-dependent mechanism. HIF is a heterodimeric transcription factor which is stabilized under hypoxic conditions and binds to specific DNA sequences denoted as hypoxia response elements (HRE) which are present in the promoter regulatory region of the AM gene (Fig. 1). Hypoxia also upregulates the expression of the AM receptor gene in many tumor types hence establishing a rational explanation behind the aforementioned autocrine growth mechanism underlying carcinogenesis (Fig. 2). As tumor-derived AM is released into the microenvironment, it establishes a peptide gradient which ultimately disseminates to reach a teeming collection of cell types known to be able to respond to this peptide and to be involved in further development of the tumor, including the cancer cell itself. AM not only stimulates tumor cell proliferation via its mitogenic activity but also by involving an antiapoptotic state. Although the advantageous effects of AM for the tumor cell are apparent, its actions are not restricted to this compartment within the tumor. On the contrary, AM acts as an integrative molecule allowing the crosstalk between all different compartments within the tumor microenvironment. As an example, AM is a migratory factor for different inflammatory cells, including ▶ mast cells.

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O2 gradient

A VEGF

AM

bFGF

MCP-1

Directional ECM degradation migration

Tumor growth

Preexisting vasculature

Neovasculature AM gradient

Adrenomedullin, Fig. 2 Model of the AM/tumor cell/ inflammatory cells’ relationship in human carcinogenesis. The microenvironment around the tumor is hypoxic and stimulates expression of AM by the tumor cells. Tumorderived AM is released into the microenvironment setting up a concentration gradient of peptide that contributes to angiogenesis and attracts distal MCs to infiltrate the tumor site. Neovasculature makes possible tumor metastasis, and it is used as a point of entrance for inflammatory cells (i.e.,

MC). As MCs migrate up the peptide gradient, higher AM concentrations are reached stimulating MC-derived angiogenic factor (AM, VEGF, bFGF, MCP-1) expression and ultimately release at the tumor site. AM mediates a paracrine tumor survival effect (direct mitogen, angiogenic factor, and anti-apoptosis) and functions as a paracrine recruitment factor drawing additional MCs to the area, thus perpetuating the inflammatory process and enhancing tumor promotion

Mast cells migrate toward the tumor mass following the preestablished tumor-derived AM gradient. Hence, as mast cells approach the tumor, they are exposed to increasingly higher concentrations of AM. Only when certain concentration of AM is reached in the proximity of the tumor, mast cells degranulate liberating to their immediate milieu numerous inflammatory factors (including AM) which not only enhance the tumor progression but also perpetuate the inflammatory process. AM is also implicated as a potential immune system suppressor, inhibiting macrophage function and acting as a negative regulator of the complement cascade, protective properties which help cancer cells circumvent immune surveillance. One of the most significant features distinctive of hypoxic tumors is their ability to induce

angiogenesis. Tumor-induced angiogenesis is a pathological condition that results in ectopic neovascularization. Of most therapeutic interest is the finding that AM is an essential factor that regulates normal and pathological vascularization. AM was first described as a potent hypotensive peptide although its connection to the normal and pathological biology of the vascular system is much deeper than initially thought. AM is an essential factor for the normal development of vasculature as revealed in mice lacking the AM gene which is embryonically lethal due to abnormal vascularization. AM also induces pathological neovascularization via CRLR-RAMP2 present in the endothelial cells. Angiogenesis is a multistep process which commences with the growth of endothelial cells which is enhanced by

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tumor-derived AM. AM also prevents hypoxiatriggered apoptosis in endothelial cells enhancing the neovascularization process. Additionally, AM participates in the remodeling of the extracellular matrix and tridimensional rearrangement of endothelial cells in the tissue which results in the establishment of the new intratumoral vasculature by stimulating migration and tube formation of endothelial cells. AM increases the permeability of the endothelial cells in the newly established vasculature which supplies the tumor with the necessary nutrients for expansion; additionally it creates an access route for inflammatory cells which are attracted to the tumor site and migrate in following gradients of chemoattractant and migratory factors produced by the tumor, such as AM. The same route can simultaneously be utilized by tumor cells as the entrance point to the vascular system facilitating the metastasis process. The invasive capability of tumor cells is thus enhanced by AM.

Adriamycin Kitamura K, Kangawa K, Kawamoto M et al (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560 Zudaire E, Martinez A, Cuttitta F (2003) Adrenomedullin and cancer. Regul Pept 112:175–183

Adriamycin Tsutomu Takahashi1 and Akira Naganuma2 1 Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan 2 Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Synonyms 14-Hydroxydaunorubicin; Doxorubicin

Conclusion Conclusions gleaned from the studies carried over the past 14 years portrait AM as a molecular connector with competence to entangle and allow communication between the different cellular components of the tumor machinery which conspire under the tumor cell direction to promote cancer. It is not only the direct effect that AM has on tumor cells but also its ability to interact with all these cellular elements which makes this peptide an attractive therapeutic target for cancer. The collective research effort is shifting from trying to discern whether AM is a causative agent of cancer to better understanding its central role as a multifaceted exchange currency among the multiple cellular players involved in tumor development. Strategies utilizing blocking agents aimed at disruption of this loop might be proven successful to impede tumor growth.

References Cuttitta F, Portal-Nuñez S, Falco C et al (2006) Adrenomedullin: an esoteric juggernaut of human cancers. In: Kastin AJ (ed) Handbook of biologically active peptides. Elsevier

Definition Adriamycin is an antineoplastic ▶ anthracycline antibiotic isolated from cultures of Streptomyces peucetius var. caesius. It is widely used in the treatment of various different types of cancers. Proposed mechanisms for its antitumor activity include intercalation into DNA, inhibition of ▶ topoisomerase II, and promotion of free-radical formation. However, the clinical utility of this drug is seriously limited by the development of cardiomyopathy and ▶ myelosuppression.

Characteristics Chemical Properties Adriamycin is an orange-red compound, soluble in water and aqueous alcohols, moderately soluble in anhydrous methanol, and insoluble in nonpolar organic solvents. It consists of an aglycone (adriamycinone), a tetracyclic ring with adjacent quinone–hydroquinone groups in rings C-B, coupled with an amino sugar (daunosamine). It

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Adriamycin, Fig. 1 Structures of adriamycin and its analogues

is generated by C-14 hydroxylation of its immediate precursor, daunorubicin (see Fig. 1). Semisystematic derivatives of adriamycin include epirubicin, an axial-to-equatorial epimer of the hydroxyl group at C-40 in daunosamine; pirarubicin, 40 -O-tetrahydropyranyl-adriamycin; etc. Clinical Aspects Therapeutic Applications

Adriamycin has a broad antitumor spectrum. It is used to treat hematopoietic malignancies such as leukemias, lymphomas (non-Hodgkin disease, ▶ Hodgkin disease) and ▶ multiple myeloma, and different solid tumors (breast, thyroid, gastric, ovarian, bronchogenic, head and neck, prostate, cervical, pancreatic, uterine, and hepatic carcinomas, as well as transitional cell bladder carcinomas, ▶ Wilms’ tumor, ▶ neuroblastoma, and soft tissue and bone sarcomas). Adriamycin is applied as a component of combination chemotherapy, rather than a monotherapy. Adriamycin-based combination chemotherapy regimens include ABVD (adriamycin, bleomycin, vinblastine,

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dacarbazine) for non-Hodgkin disease, CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) for ▶ Hodgkin disease, and MVAC (methotrexate, vinblastine, adriamycin, cisplatin) for urothelial carcinoma. Pharmacokinetics

Adriamycin is rapidly cleared from the plasma, quickly taken up, and only slowly eliminated from organs such as the spleen, lungs, kidneys, liver, and heart. It does not cross the blood–brain barrier. Adriamycin is converted to an active metabolite, adriamycinol, through a two-electron reduction of the side chain C-13 carbonyl moiety by NADPH-dependent cytoplasmic aldo/keto reductase or carbonyl reductase. It is converted to inactive metabolites in the liver and other tissues and predominantly excreted in the bile. Clinical Toxicities

The usual toxic side effects of adriamycin, including stomatitis, nausea, vomiting, alopecia, gastrointestinal disturbance, and dermatological manifestations, are generally reversible. The dose-limiting side effects of the anthracyclines

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including adriamycin are myelosuppression and cardiotoxicity. Myelosuppression with leukopenia, neutropenia, and occasionally thrombocytopenia is dose related and potentially life-threatening. Cardiotoxicity is characteristic of the anthracycline antibiotics, of which adriamycin is the most toxic. Adriamycin-induced cardiotoxicity can be acute, chronic, or delayed. The acute effect is not dose related and is characterized by sinus arrhythmias and/or abnormal electrocardiographic (ECG) changes (nonspecific ST-T wave change, prolongation of QT interval). Acute toxicity of this type is transient and rarely a serious problem. Chronic cardiotoxicity is a much more serious problem, being related to cumulative dose. It is irreversible and leads to dilative cardiomyopathy and congestive heart failure (CHF), usually unresponsive to cardiotonic steroids (digitalis) and b-blockers. The risk of developing CHF increases markedly at total cumulative doses in excess of 500 mg/m2. Moreover, the effects of this chronic cardiotoxicity may manifest precipitously without antecedent ECG changes. The risk of life-threatening cardiac dysfunction can be decreased by regular monitoring of endomyocardial (EM) biopsy histopathological changes and left ventricular ejection fraction (LVEF) as measured by the multigated radionuclide angiography (MUGA) method and/or echocardiography (ECHO). Finally, adriamycin can also cause delayed cardiotoxicity, possibly, related to the dose. This occurs after an asymptomatic interval, mostly in people who were treated as children. Several approaches have been proposed to overcome adriamycin cardiotoxicity and that of the anthracycline antibiotics generally. Administration by slow continuous intravenous infusion (over 48–96 h) rather than the standard bolus injection decreases the likelihood of chronic cardiotoxicity. Dexrazoxane (ICRF-187), an iron chelator that prevents the formation of complexes between adriamycin and iron and subsequent production of ▶ reactive oxygen species (ROS), is sometimes used as a cardioprotectant. However, it may decrease antitumor activity. Liposomal encapsulation is designed to increase safety and efficacy by decreasing cardiac and gastrointestinal

Adriamycin

toxicity through decreased exposure of these tissues to the drug while effectively delivering it to the tumor. Polyethylene glycol-coated (pegylated) liposomal adriamycin (Doxil (USA), Caelyx (UK)) is currently used for treating AIDSrelated ▶ Kaposi sarcoma, refractory ovarian cancer, and some other solid tumors. In order to improve therapeutic efficacy and decrease side effects by promoting drug accumulation inside tumors, the water-soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, magnetic targeted carriers, and immunoliposome conjugates with the specificity of whole monoclonal antibodies (e.g., antibodies against CD19 or MUC1) or FAB’ fragments have been developed as carriers of adriamycin. In further efforts to decrease the risk of developing cardiotoxicity, several derivatives of adriamycin or daunorubicin, such as epirubicin, pirarubicin, idarubicin, and aclarubicin, have been developed. Although these agents may be less cardiotoxic than adriamycin itself, they do have a decreased antitumor activity. Pharmacological Mechanisms Mechanisms of Action

Several mechanisms appear to contribute to the cytotoxic effects of adriamycin, including inhibition of DNA replication and repair; inhibition of RNA and protein synthesis via intercalation of the aglycone portion of the molecule between adjacent DNA base pairs, especially G–C base pairs; promotion of the cleavage of DNA by formation of adriamycin–topoisomerase II-DNA ternary complexes; inhibition of topoisomerase I; and direct binding to the cell membrane. Formation of free radicals is another major mechanism of cytotoxicity. One-electron reduction of the quinone moiety in the C ring of adriamycin by some flavin-containing enzymes (mitochondrial NADH dehydrogenase, microsomal NADPHcytochrome P450 reductase, and xanthine oxidase) generates adriamycin–semiquinone radicals. These rapidly react with oxygen to form superoxide anions, which then generate hydrogen peroxide and hydroxyl radicals in the presence of redox-active metals such as iron (III) and copper

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(II). The final result is DNA damage and lipid peroxidation. The semiquinone radical can be transformed into an aglycone C7-centered radical that also mediates cellular damage by DNA alkylation and lipid peroxidation. Adriamycin can bind to metal ions such as iron, copper, and manganese, by forming adriamycin–metal complexes, which may lead to generation of ROS and damage to cell membranes. Mechanisms of Resistance

Development of resistance to the drug is a major obstacle in chemotherapy with adriamycin. Drug efflux pumps are important for defending cells against anticancer drugs. The acquisition of adriamycin resistance involves promotion of excretion of the drug by overexpressing the ATP-binding cassette (ABC) transporters ▶ P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated proteins (MRPs)/ABCC (MRP1, MRP2, MRP6, etc.), and breast cancer resistance protein (BCRP)/ABCG2. P-gp transports hydrophobic compounds including adriamycin, while MRP1 and BCRP can extrude predominantly these glutathione conjugates. In addition, RalA-binding protein 1 (RALBP1)/ Ral-interacting protein of 76 kDa (RLIP76) is a nonclassical ABC transporter involved in drug excretion. RALBP1 catalyzes ATP-dependent efflux of xenobiotics including adriamycin as well as its glutathione conjugates. In fact, the level of expression of these efflux pumps correlates with the clinical efficacy of adriamycin. ▶ Glutathione S-transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Adriamycin and its metabolites (adriamycinol) are conjugated with glutathione by GSTs and transported by MRPs, BCRP, etc. Increased expression of GSTs, especially GSTp, also confers adriamycin resistance by promoting detoxification. Lung resistance protein (LRP), the 110 kDa major vault protein (MVP), is a main component of vaults, which are multisubunit structures that may be involved in nucleocytoplasmic transport, and is involved in resistance to anticancer drugs including adriamycin. LRP may affect the intracellular distribution of adriamycin, but the

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detailed mechanisms remain unknown. Furthermore, a relationship between adriamycin resistance and qualitative and quantitative changes in the expression of topoisomerase II, a major target for adriamycin, has been reported. Mechanisms for Development of Cardiotoxicity

The molecular mechanisms leading to adriamycin-induced cardiotoxicity may include lipid peroxidation by generation of ROS; abnormalities in intracellular calcium homeostasis through inhibition of sarcomeric reticulum Ca2+ATPase (SERCA2), Na+-K+-ATPase, and Na+Ca2+ exchanger of sarcolemma; inhibition of mitochondrial creatine kinase; and interaction with cardiolipin, which is a phospholipid of the inner mitochondrial membrane in the heart. Adriamycin also promotes apoptosis by activation of p38 mitogen-activated kinases (MAPK) in cardiac muscle cells. Moreover, adriamycin downregulates the expression of genes for sarcomeric proteins (such as a-actin, myosin, troponin I, and myofibrillar creatine kinase) and for proteins involved in calcium homeostasis in the sarcomeric reticulum, such as SERCA2, cardiac muscle ryanodine receptor (RYR2), calsequestrin, and phospholamban, by suppression of transcription factors (e.g., MEF2C, HAND2, and GATA4) and/or activation of a the transcriptional repressor Egr-1. Adriamycinol (doxorubicinol), a secondary alcohol metabolite, may also be involved in the development of adriamycin-induced cardiotoxicity, via enhancing the inhibitory effects of SERCA2, Na+-K+-ATPase, and Na+-Ca2+ exchanger of sarcolemma. Adriamycinol also inhibits the iron-regulatory protein/ironresponsive element (IRP/IRE) system, which plays a crucial role in iron homeostasis, and may lead to cardiotoxicity.

References Awasthi S, Sharma R, Singhal SS et al (2002) RLIP76, a novel transporter catalyzing ATP-dependent efflux of xenobiotics. Drug Metab Dispos 30: 1300–1310

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128 Hortobagyi GN (1997) Anthracyclines in the treatment of cancer. An overview. Drugs 54(Suppl 4):1–7 Minotti G, Menna P, Salvatorelli E et al (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–229 Nielsen D, Maare C, Skovsgaard T (1996) Cellular resistance to anthracyclines. Gen Pharmacol 27:251–255 Poizat C, Sartorelli V, Chung G et al (2000) Proteasomemediated degradation of the coactivator p300 impairs cardiac transcription. Mol Cell Biol 20:8643–8654

ADT ▶ Androgen Ablation Therapy

Adult Stem Cells Rikke Christensen1 and Nedime Serakinci2 1 Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark 2 Medical Genetics, Near East University, Nicosia, Northern Cyprus

Synonyms Postnatal stem cells; Somatic stem cells; Tissue stem cells

Definition An undifferentiated cell found in a differentiated tissue that can renew itself and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.

Characteristics Adult stem cells are defined as undifferentiated tissue-specific stem cells with extensive selfrenewal capacity, which can proliferate to generate mature cells of the tissue of origin. The primary roles of adult stem cells are to maintain

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and/or regenerate the cells of damaged tissues. Stem cells may remain quiescent for long periods of time until they are activated by a need for more cells. Adult stem cells were first described in organs and tissues characterized by high cell turnover, such as blood, gut, testis, and skin, but have to date also been isolated from many other organs and tissues including brain, bone marrow, liver, heart, lung, retina, ovarian epithelium, teeth, mammary cells, and skeletal muscle. Adult stem cells mainly possess two key properties: (1) self-renewal, which is the ability to allow the cells to go through many cell divisions while remaining in an undifferentiated state, and (2) multipotency or multidifferentiative potential, which is the ability to generate progeny of several distinct cell types of the tissue or organ such as glial cells, neurons, etc. Stem cells differ from somatic cells with their different potentials and their proliferation ability. There are three kinds of stem cells – embryonic, germinal, and adult stem cells – that are classified according to their developmental potential ranging from totipotency to unipotency. The fertilized oocyte and the blastomere up to the eight-cell stage are considered as totipotent (totipotent stem cells) as they can differentiate to generate a complete organism. ▶ Embryonic stem cells, the cells derived from the inner cell mass of the blastocyst, are pluripotent (pluripotent stem cells) and have the ability to differentiate into cells and tissues from all three germ layers: the endoderm, the ectoderm, and the mesoderm. Germinal stem cells are also pluripotent and are derived from so-called primordial germ cells and give rise to the gametes (sperm and eggs) in adults. In contrast, adult stem cells are generally believed to be multipotent (multipotent stem cells) or unipotent (unipotent stem cells) which means that they can only give rise to progeny restricted to the tissue of origin. Hematopoietic stem cells (HSC), bulge stem cells in the hair follicle, and mesenchymal stem cells (MSC) are examples of multipotent stem cells, which can differentiate into multiple cell types of a single tissue, whereas epidermal stem cells, myosatellite cells of muscle, and endothelial progenitor cells are examples of unipotent stem cells, which only give rise to one mature cell

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type. Some studies have shown that many adult tissues may contain cells with pluripotent capacity capable of generating differentiated cells from an unrelated tissue. This process is termed ▶ stem cell plasticity. In most tissues/organs, renewal is compensated by tissue-specific stem cells. The stem cells normally divide very rarely, but stimuli caused by damaged or injured tissue or a need to generate progeny to maintain the tissue can induce proliferation and produce daughter cells that can differentiate into the specific cell lineages of the respective tissue type. Stem cell division typically leads to the formation of committed progenitor cells with more limited self-renewal capacity as, e.g., transit amplifying cells in the epidermis or lymphoid or myeloid progenitors in the bone marrow. Tissue progenitor or transit amplifying cells provide an expanded population of a proliferating tissue that differentiate into more mature and determined cells that eventually no longer proliferate and die. To maintain the balance in the adult tissues/organs, the number of progenitor/stem cells that proliferates must be equal to the number of cells that determinedly differentiates and dies. If the number of proliferating cells is higher than the number of cells that maturates and dies, it will give the primary feature of a cancer. Studies have shown that many of the pathways that regulate normal stem cell proliferation are dysregulated and cause neoplastic proliferation in cancer cells. Therefore, cancer may be considered a disease of dysregulated cellular self-renewal capacity. Adult stem cells reside in a special microenvironment termed the stem cell niche. Stem cell niches are composed of a group of cells that provide a physical anchorage site and extrinsic factors that control stem cell proliferation and differentiation and enable them to maintain tissue homeostasis. Deregulation of the niche signals has been proposed to lead to cancer. A decrease in proliferation-inhibiting signals, or an increase in proliferation-promoting signals, may lead to excessive stem cell production and thereby development of cancer stem cells (see later). Investigation of the interaction between stem cells and their niche may reveal possible targets for cancer treatments. For example, the blocking of proliferation

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signals, enhancing of antiproliferative signals, or induction of differentiation from the stem cell niche may be used to target the cancer stem cells. It has furthermore been suggested that targeting the stem cell niche may prevent cancer metastasis. Some cancers metastasize to sites that cannot be explained by circulation distribution, lymphatic drainage, or anatomic proximity. These sites may, however, provide favorable niches that support the survival of the cancer stem cells. Much effort is put into the identification of stem cell markers to be able to isolate the stem cells of interest. Isolation of stem cells makes it possible to enhance the knowledge of stem cell identity and to use them therapeutically. Adult Stem Cell Differentiation As it has been mentioned above and shown by many scientists, adult stem cells occur in many different tissues, and they normally differentiate to give rise to mature cell types that show characteristic morphology and specialized structures as well as functions of a particular tissue. Several researchers have reported that, in addition to their normal differentiation pathway, certain adult stem cell types can transdifferentiate into other cell types than the “predicted lineage” cell types (e.g., blood-forming cells that differentiate into cardiac muscle cells, etc.). Although transdifferentiation have been shown in some vertebrate species, it is still under debate within the stem cells scientific community if this is actually occurring in humans. In addition to transdifferentiation experiments, researchers have shown that certain types of adult cells can be “reprogrammed” into other cell types in vivo. This approach offers a way to reprogram available cells into other cell types that have been lost or damaged due to disease. Furthermore, it is now possible to reprogram adult somatic cells to become like embryonic stem cells (induced pluripotent stem cells, iPSCs) through the introduction of embryonic genes. Adult stem cells or adult cells can be reprogrammed or induced to pluripotent stem cells (iPSCs) by the introduction of four transcription factors known as the Yamanaka factors (Oct4, Sox2, cMyc, and Klf4). Thus, a source

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of cells can be generated that are specific to the donor, thereby increasing the chance of compatibility if such cells were to be used for tissue regeneration. However, like embryonic stem cells, determination of the methods by which iPSCs can be completely and reproducibly committed to appropriate cell lineages is still under investigation. Therapeutic Potential Stem cells in general, due to their high proliferative capacity and long-term survival in comparison to somatic cells, make them very ideal candidates to use for regenerative medicine and cell replacement therapy. Lately, there has been an increasing interest in the potential use of adult stem cells in cell replacement strategies and in tissue engineering, including gene therapy. This current interest rose due to the discovery of adult stem cells with pluripotential capacity and/or transdifferentiating (transdifferentiation) ability, which means that cells from one tissue can differentiate into mature and functional cells of another tissue. There are reports that HSCs under certain conditions can evolve into cells of neural lineage, liver, muscle, skin, and endothelium; skeletal muscle stem cells can evolve into blood cells and neural cells; and hair follicle stem cells can evolve into neural lineage cells. Other adult stem cells that can be induced to a different cell type include MSCs, cardiac muscle stem cells, neural stem cells, and testis-derived stem cells. These cells have the advantage that they can be used as autologous transplants and have been proposed as an attractive alternative to ▶ embryonic stem cells in genetic therapy. Adult stem cell transplantation has been used in several years for the treatment of ▶ hematological malignancies and lymphomas. The main purpose of stem cell transplantation in cancer treatment is to make it possible for patients to receive very high doses of chemotherapy and/or radiation. High-dose chemotherapy and radiation can severely damage or destroy the bone marrow while killing cancer cells. Before treatment, bone marrow or peripheral blood stem cells are harvested from the patient itself (for autologous transplantation) or from a donor (for allogen

Adult Stem Cells

transplantation), frozen down, and then transplanted after the patient has received high doses of chemotherapy, radiation therapy, or both. The transplanted healthy stem cells replace the stem cells destroyed by high-dose cancer treatment and allow the bone marrow to produce healthy cells. An alternative approach for therapeutic use of stem cells is to use them as cellular vehicles. It has been demonstrated that genetically modified MSCs can be used to target delivery of anticancer agents and ▶ suicide gene therapy vectors to tumor cells. Upon administration, MSCs can target microscopic tumors, proliferate and differentiate, and contribute to the formation of a network of cells surrounding the tumor (tumor stroma). MSCs genetically modified to express interferon beta has, for example, been shown to inhibit the growth of tumor cells by local production of interferon beta. MSCs are not the only stem cells that have been used as shuttle vectors for delivery of gene therapies into growing tumors. It has also been demonstrated that neural stem and progenitor cells migrate selectively to tumor loci in vivo in mice. These studies clearly suggest that a stem cell-directed prodrug therapy approach may have great use for eradicating tumors as well as to treat the residual cancer cells remaining after therapy. Genetic manipulation of adult stem cells may also be used to increase the functionality and proliferative capacity of these cells. HSCs are one of the most promising candidates for correction of single gene disorders as e.g., ▶ cystic fibrosis and hemoglobinopathies, due to their capability of targeting solid organs and high success rate in their isolation by using a combination of surface markers. Infants with forms of severe combined immunodeficiency syndrome have successfully been treated with genetically engineered bone marrow stem cells. The stem cells were harvested from the patients, a functional gene inserted, and the genetically modified cells reintroduced to the same patient. To increase the success of chemotherapeutic treatment, drugresistant HSCs have been produced by introduction of the multidrug resistance gene with the aim of limiting the myelosuppressive effects of standard chemotherapeutic agents on the stem cells.

Adult Stem Cells

However, even though adult stem cells have shown to carry great potential to function as therapeutic agents for targeting human diseases such as cancer, degenerative, and chronic diseases, they do have some restrictions such as having limited self-renewal capacity. This limitation can be overcome by the introduction of immortalizing genes that increases the cells proliferative capacity. ▶ Telomerase has been, in this connection, highlighted among the numerous genes that are capable of immortalizing stem or progenitor cells. However, suggested oncogenic potential of immortalized cells releases caution that before the therapeutic use of stem cells in the clinic, a thorough screen for transformation phenotype is required. Cancer Stem Cells The theory that cancer stem cells (▶ cancer stemlike cells and ▶ stem-like cancer cells) are involved in many types of cancer has gained popularity. There are many similarities between adult stem cells and cancer stem cells. Both have the ability to self-renew and differentiate into more mature diversified cells. Cancer stem cells and normal stem cells share many cell surface markers and utilize many of the same signal transduction pathways. Cancer stem cells have been identified in most types of hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma. Cancer stem cells have also been isolated from solid tumors such as breast, lung, and brain tumors. The cancer stem cells only represent approximately 1% of the tumor, making them difficult to detect and study. Studies have shown that cancer stem cells may cause tumors when transplanted into a secondary host indicating that the cancer stem cells can initiate and repopulate a tumor. A study of human leukemia shows that the normal hematopoietic stem cell and the neoplastic clone share common molecular mechanisms governing proliferation which is supportive of the normal hematopoietic stem cell being a target for transformation. Due to stem cells are able to divide over the lifespan of the individual, they seem to allow accumulation of a number of

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mutations and perhaps epigenetic changes (epigenetics) that cause neoplastic development. In addition, it has been shown that adult stem cells can be targets for neoplastic transformation by introducing the telomerase gene into a purified stem cell. The transduced cell line showed characteristic alterations of neoplastic development such as contact inhibition, anchorage independence, and in vivo tumor formation in immunocompromised mice. All these findings give a very large support to the existence of cancer stem cells, and the strong links between normal adult stem cells and cancer stem cells suggest that stem cells are targets for neoplastic transformation. Cancer stem cells may also be derived from differentiated cells. Loss of the ▶ tumor suppressor genes p16Ink4 and p19Arf combined with constitutive activation of the EGF receptor (EGFR) caused loss of differentiation in mature brain astrocytes, and the cells regained stem cell properties. The identification of cancer stem cells strongly suggests that these cells are the key targets for future therapeutic development as they fuel the replicative capacity of the cancer. Therefore, as much as understanding the nature of a cancer cell, it is very crucial to understand the neoplastic potential of the stem cells. Analysis of the differences between adult stem cells and cancer stem cells is very important to be able to specifically target the cancer stem cells while sparing the normal stem cell population. Several studies indicate that some stem cell markers are expressed differently in normal and cancer stem cells and these may be potential targets in the development of future cancer treatments.

Cross-References ▶ Cystic Fibrosis ▶ Embryonic Stem Cells ▶ Epidermal Growth Factor Receptor ▶ Epigenetic ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Side Population Cells ▶ Stem Cell Plasticity ▶ Stem-like Cancer Cells

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▶ Suicide Gene Therapy ▶ Telomerase ▶ Tissue Stem Cells ▶ Tumor Suppressor Genes

References Krampera M, Cosmi L, Angeli R et al (2006) Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 24:386–398 Lotem J, Sachs L (2006) Epigenetics and the plasticity of differentiation in normal and cancer stem cells. Oncogene 25:7663–7672 Pan CX, Zhu W, Cheng L (2006) Implications of cancer stem cells in the treatment of cancer. Future Oncol 2:723–731 Pessina A, Gribaldo L (2006) The key role of adult stem cells: therapeutic perspectives. Curr Med Res Opin 22:2287–2300 Reya T, Morrison SJ, Clarke MF et al (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111 Serakinci N, Christensen R, Fahrioglu U et al (2011) Mesenchymal stem cells as therapeutic delivery vehicles targeting tumor stroma. Cancer Biother Radiopharm 26:767–73 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676 Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45 Wyse RD, Dunbar GL, Rossignol J (2014) Use of genetically modified mesenchymal stem cells to treat neurodegenerative diseases. Int J Mol Sci 15: 1719–1745

Adult T-Cell Leukemia Heidelberg, p 2296. doi:10.1007/978-3-642-164835_3720 (2012) MSC. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2383. doi:10.1007/978-3-642-16483-5_3859 (2012) Multidrug resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2393. doi:10.1007/978-3-642-164835_3887 (2012) Multipotent stem cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2404. doi:10.1007/978-3-642-164835_3903 (2012) Pluripotent stem cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2930. doi:10.1007/978-3-642-164835_4639 (2012) Severe Combined Immunodeficiency Disease. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3395. doi:10.1007/9783-642-16483-5_5270 (2012) Somatic Cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3466. doi:10.1007/978-3-642-16483-5_5408 (2012) Totipotent Stem Cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3730. doi:10.1007/978-3-642-164835_5866 (2012) Transdifferentiation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3753. doi:10.1007/978-3-642-164835_5909 (2012) Unipotent Stem Cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3846. doi:10.1007/978-3-642-164835_6109

See Also (2012) EGFR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1828 (2012) Germinal stem cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-164835_2402 (2012) Hemoglobinopathy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1647. doi:10.1007/978-3-642-164835_2633 (2012) HSC. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1740. doi:10.1007/978-3-642-16483-5_2830 (2012) Immortalized cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1812. doi:10.1007/978-3-642-164835_2971 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/

Adult T-Cell Leukemia Synonyms ATL

Definition A leukemia of mature T lymphocytes (T cells) developing in adults, resulting from infection with the ▶ human T-lymphotropic virus (HTLV) and characterized by circulating malignant T lymphocytes, skin lesions, lymphadenopathy (enlarged lymph nodes), hepatosplenomegaly

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(enlarged liver and spleen), hypercalcemia (high blood calcium), lytic (“punched out”) bone lesions, and a tendency to infection. There are four categories of ATL, based on the aggressiveness of the disease – smoldering, chronic, lymphoma, and acute.

Aflatoxins Thomas E. Massey1 and Katherine A. Guindon2 1 Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada 2 Department of Pharmacology and Toxicology, Queen’s University, Kingston, ON, Canada

Cross-References ▶ Human T-Lymphotropic Virus

Definition

See Also (2012) T cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3599. doi: 10.1007/978-3-642-16483-5_5645

Mycotoxins are contaminants of a number of agricultural products, including peanuts, corn, and other grains in warm and moist conditions. Human exposure to aflatoxins is primarily through ingestion and results in acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy.

Adult T-Cell Leukemia-Derived Factor

Characteristics

▶ Thioredoxin System

Adult Type ▶ Ovarian Tumors During Childhood and Adolescence

Aerobic Glycolysis ▶ Warburg Effect

®

Afinitor (marketed by NOVARTIS) ▶ Everolimus

In the early 1960s, an outbreak of hepatotoxic disease in turkeys, which became known as turkey “X” disease, gained the attention of many investigators worldwide. This condition was characterized by acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy. It was deduced that the condition was caused by consumption of peanut meal contaminated with a mycotoxin, which is a toxin of fungal origin. The culprit fungi in turkey “X” disease turned out to be strains of Aspergillus flavus, A. parasiticus, and A. nomius, and thus the term aflatoxins was coined for the toxic metabolites. More specifically, A. flavus and A. parasiticus can produce aflatoxins B1, B2, G1, G2, and M1. These mycotoxins can contaminate a number of agricultural products, including peanuts, corn, and other grains in warm and moist conditions. Human exposure to aflatoxins is primarily through ingestion. In addition to outbreaks of liver failure and gastrointestinal bleeding in Southeast Asia and Africa having been attributed to aflatoxins, liver cancer incidence was observed to be elevated in regions with high endemic

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aflatoxin concentrations. The two major risk factors for human ▶ hepatocellular carcinoma, the fifth most common cancer worldwide, are hepatitis B infection and ingestion of aflatoxins. Aflatoxin B1 (AFB1), the most prevalent and carcinogenic of the aflatoxins, is classified as a group 1 carcinogen (carcinogenic to humans) by the International Agency for Research on Cancer. Although the majority of AFB1 research has focused on its hepatic effects, AFB1 also targets other organs, including the lung and the kidney. In the lung, exposure to inhaled AFB1, particularly

from contaminated grain dusts, has been linked to respiratory cancers (▶ lung cancer). Due to a significant proportion of ingested mycotoxin being excreted via the urine, the renal nephron is exposed to AFB1 and its metabolites. AFB1 accordingly alters kidney function and is a known renal carcinogen. Biotransformation AFB1 is defined as a procarcinogen, as its bioactivation is required for carcinogenicity (Fig. 1). The initial metabolism of AFB1 involves

Aflatoxins

four types of reactions: O-dealkylation, hydroxylation, epoxidation, and ketoreduction. The enzymes responsible for the metabolism include members of the ▶ cytochrome P450 family (CYPs), prostaglandin H synthase (PHS), lipoxygenase (LOX), and a cytosolic NADPHdependent reductase. In experimental animals, CYPs involved in AFB1 bioactivation include members of 1A, 2B, 2C, and 3A subfamilies. In humans, there are multiple p450 isozymes implicated, including CYP1A2, CYP2A3, CYP2B7, CYP3A3, and CYP3A4. CYP3A4 is thought to play a predominant role in the metabolism of AFB1 in human liver; although CYP1A2 has the highest affinity for AFB1 at low concentrations, it is expressed at much lower levels than CYP3A4. PHS and LOX are involved in ▶ xenobiotic bioactivation by catalyzing the oxidation of arachidonic acid to produce lipid peroxyl radicals, which are known epoxidizing agents. Cooxidation by PHS and LOX may be a significant mechanism of AFB1 bioactivation in extrahepatic tissues such as the lung, which has high PHS and LOX expression, but overall P450 activity is lower than that in the adult liver. Regardless of the enzyme catalyzing the reaction, epoxidation of AFB1 results in formation of AFB1-8,9-epoxide, which can exist in both endo and exo conformations. The exo-epoxide is the isomer implicated in the alkylation of DNA, with its reactivity being at least 1,000-fold greater than that of the endo-epoxide. Hydroxylated metabolites of AFB1 include AFM1, AFQ1, AFP1, and AFB2a. The formation of aflatoxicol from AFB1 is reversible, and therefore aflatoxicol is considered to be a “reservoir” for AFB1 rather than a bioactivation or ▶ detoxification product. The two pathways for AFB1-epoxide detoxification are glutathione conjugation and epoxide hydrolysis, with glutathione conjugation being quantitatively the most important (Fig. 1). Glutathione conjugation is catalyzed by ▶ glutathione S-transferases (GSTs), which can be highly polymorphic. Human GSTM1-1 (hGSTM1-1), which is absent in ~50% of individuals, has the highest activity toward AFB1 exo-epoxide, but the importance of this polymorphism in AFB1 carcinogenicity has not yet been clearly established.

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AFQ1, AFP1, and AFB2a are not highly mutagenic and therefore are considered to be detoxification products. They can form glucuronide or sulfate conjugates, which are excreted. AFM1, a metabolite of AFB1 identified in milk and urine, is less biologically active than AFB1, but regardless is a potent carcinogen. The AFM1-epoxide can also bind to DNA, forming AFM1-N7-guanine. Carcinogenesis AFB1 is considered to be a complete carcinogen, possessing activity as both an initiator and a promoter. Initiation occurs by ▶ DNA damage, as well as cytotoxicity, which stimulates cell division, thus promoting tumor formation. There are many characteristics of AFB1 that makes it a useful tool for investigating ▶ chemical carcinogenesis. First, the metabolites of AFB1 have been extensively investigated and their toxicity elucidated. Second, the toxicity of AFB1 is determined by a balance between bioactivation and detoxification of the AFB1-8,9-epoxide. Third, there exist multiple mechanisms of bioactivation that can be compared in terms of carcinogenic metabolites produced. Fourth, not only does the susceptibility of a species/tissue relate to DNA repair capabilities (▶ Repair of DNA), but AFB1 itself has effects on DNA repair activity. Fifth, the specific AFB1-DNA adduct formed can be used to predict the mutagenic responses. Finally, the parent compound and several metabolites fluoresce, facilitating detection. The exo-epoxide of AFB1 can alkylate proteins and nucleic acids, with the second guanine from the 50 end in guanine di- and trinucleotide sequences in DNA being the favored target. The major adduct formed by the exo-epoxide is 8,9-dihydro-8-(N7-guanyl)-9-hydroxy AFB1, also known as AFB1-N7-Gua (Fig. 2). AFB1-N7Gua can undergo three reactions: release of AFB18,9-dihydrodiol restoring guanine; depurination resulting in an apurinic site in DNA; and basecatalyzed hydrolysis to form the AFB1formamidopyrimidine adduct (AFB1-FAPY). AFB1-FAPY, representing a significant proportion of AFB1 adducts in vivo, exists in equilibrium between two rotameric forms, designated AFB1FAPY major and AFB1-FAPY minor. The

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Aflatoxins

Aflatoxins, Fig. 2 AFB1exo-8,9-epoxide and DNA damage

structure of AFB1-FAPY has not been completely defined, although the proposed structure is presented in Fig. 2. It has also been shown that metabolism of AFB1 can lead to formation of 8-hydroxy-20 -deoxyguanosine in rat, duck, and woodchuck liver and in mouse lung. G to T transversion, the most frequently observed mutation induced by AFB1, results from DNA alkylation and subsequent AFB1-N7-Gua formation and possibly by the ▶ oxidative DNA damage as well. A proportion of mutations in DNA formed by AFB1 occurs at the base 50 to the modified guanine, or even further away, due to helical distortion resulting from the AFB1 adduct. P53, a ▶ tumor suppressor gene considered the “guardian of the genome,” has controls on cell cycle, DNA repair, and ▶ apoptosis. P53 is the most frequently targeted gene in human carcinogenesis, with a mutation frequency of 50% in most major cancers. In geographical regions with a high dietary exposure to AFB1, such as China and sub-Saharan Africa, mutations in p53

have been implicated in AFB1-induced human liver tumorigenesis. AFB1 produces mutations at the third base of codon 249 in p53, causing a G!T transversion and an amino acid substitution (arginine to serine), and thus a structural alteration of this tumor suppressor protein. This may result in deregulation of the cell cycle and thus loss of tumor suppression by p53. The KRAS proto▶ oncogene, important in ▶ signal transduction, is often implicated in human and mouse lung tumors. AFB1-induced point mutations at specific “hot spots” (e.g., codons 12 and 13) of the KRAS gene, which cause activation of the protein, occur in AFB1-induced mouse lung tumorigenesis and rat hepatocarcinogenesis. Repair In mammals, ▶ nucleotide excision repair (NER) is important for protection against AFB1-induced carcinogenesis. NER is a DNA repair process that deals with a wide array of DNA helix-distorting lesions that affect normal base pairing, thus

Aflibercept

altering transcription and replication. In E. coli, NER is responsible for the repair of both AFB1N7-Gua and AFB1-FAPY. In yeast, NER is also the main repair pathway, although ▶ homologous recombination is also involved in the repair of AFB1-induced damage. In mammals, NER is important in protection against AFB1-induced carcinogenesis. NER is the main repair mechanism for the AFB1-N7-Gua adduct. AFB1-FAPY is repaired less efficiently by mammalian NER than is AFB1-N7-Gua, an effect that is attributed to AFB1-FAPY being less distortive of DNA architecture. Apurinic sites generated by AFB1DNA adduct formation are repaired by base excision repair (BER), although insertion of an incorrect base is a frequent occurrence. Species/Tissue Susceptibility Susceptibility to the toxic and carcinogenic effects of AFB1 varies between species, as well as between different tissue types. In humans, the liver is the main target for this toxin. In rat, duck, and trout, administration of AFB1 results in hepatocarcinogenesis, whereas this is not the case in the mouse, monkey, hamster, and mouse. The reason for this has been attributed to differences in AFB1 biotransformation and DNA repair. For example, the mouse is susceptible to pulmonary carcinogenesis by AFB1, regardless of the route of administration, but does not develop hepatocarcinogenesis. The mouse liver expresses an alpha-class GST with high specific activity toward the exo-epoxide and higher NER activity as compared to the rat liver. On the other hand, mouse lung has lower DNA repair activity than does the liver. AFB1 is able to alter NER activity (by inhibition or elevation) in different animal species and organs, which may contribute to differential susceptibility to the mycotoxin’s carcinogenicity.

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References Bedard LL, Massey TE (2006) Aflatoxin B-induced DNA damage and its repair. Cancer Lett 241(2):174–183 Eaton DL, Groopman JD (eds) (1994) The toxicology of aflatoxins: human health, veterinary, and agricultural significance. Academic Press, San Diego, pp 3–148 Massey TE, Stewart RK, Daniels JM et al (1995) Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B carcinogenicity. Proc Soc Exp Biol Med 208(3):213–227 Massey TE, Smith GBJ, Tam AS (2000) Mechanisms of aflatoxin B lung tumorigenesis. Exp Lung Res 26:673–683 Wogan GN (1973) Aflatoxin carcinogenesis. Meth Cancer Res 7:309–344

Aflibercept Synonyms VEGF trap

Definition Is an ▶ antiangiogenesis agent developed by Regeneron and the Sanofi-Aventis Group; is a fusion protein specifically designed to bind as a soluble decoy receptor all forms of ▶ Vascular Endothelial Growth Factor-A (VEGF-A). VEGF-A is required for the growth of new blood vessels that are needed for tumors to grow and is a potent regulator of vascular permeability and leakage. Disruption of the binding of VEGFs to their cell receptors may result in the inhibition of tumor ▶ angiogenesis, ▶ metastasis, and ultimately lead to tumor regression. In addition, Aflibercept binds ▶ Placenta Growth Factor (PLGF), which has also been implicated in tumor angiogenesis. Breast Cancer Targeted Therapy.

Cross-References Cross-References ▶ Arachidonic Acid Pathway ▶ DNA Oxidation Damage ▶ Homologous Recombination Repair

▶ Angiogenesis ▶ Antiangiogenesis

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▶ Metastasis ▶ Placenta Growth Factor ▶ Vascular Endothelial Growth Factor

AFP ▶ Alpha-Fetoprotein ▶ Alpha-Fetoprotein Diagnostics

Aggressive Fibromatosis ▶ Aggressive Fibromatosis in Children ▶ Desmoid Tumor

Aggressive Fibromatosis in Children Marry M. van den Heuvel-Eibrink Princess Maxima Center for Pediatric Oncology/ Hematology, Utrecht, The Netherlands

Synonyms Aggressive fibromatosis; Desmoid tumor

Definition Aggressive fibromatosis (AF) is a rare soft tissue tumor and rare in childhood with high potential for local invasiveness and recurrence. Primary surgery with negative margins is the most successful primary treatment modality for children with AF. Positive resection margins after surgery indicate a high risk for relapse. Multicenter prospective (randomized) trials are necessary to clarify the role of and best strategy for treatment in pediatric AF after incomplete surgery. For this purpose, ▶ chemotherapy or alternatively radiotherapy can be considered, each with its own potential side effects in consequence.

AFP

Characteristics Aggressive fibromatosis (AF) (▶ Supportive care) is a soft tissue tumor, which arises principally from the connective tissue of muscle and the overlying fascia (aponeurosis). The previously most used synonym is ▶ desmoid tumor. The histological pattern is characterized by elongated fibroblast-like cells. Although AF is a nonmetastasizing tumor with benign histological features, it has a significant potential for local invasiveness (▶ Invasion) and recurrence. The overall incidence of AF in children is 2–4 new diagnoses per 1 million a year. Childhood AF has an age distribution peak at approximately 8 years (range 0–19 years) with a slight male predominance. Clinical Presentation The typical clinical presentation of AF is a painless, slowly growing, deep-seated mass. Predilection sites are shoulder, chest wall and back, thigh, and head/neck. Children with AF of head/neck have shown to be younger at diagnosis than children with AF at other sites. From 1986 until 2004, ten pediatric AF case series reported a total of 206 patients. In 64 of the reviewed patients, site of involvement and age at diagnosis were specified. The children with AF of head/neck had a median age of 3.6 years at diagnosis (range 0.2–9.9 years), whereas the children with AF of trunk/limb had a median age of 7.8 years (range 0.0–15.7 years) (p < 0.01). This difference in age distribution may be influenced by referral and selection bias; however, it may reflect the site distribution in different age groups in children with AF. Diagnostic Approach The diagnosis of AF is based on histology. It arises principally from the connective tissue of muscle and the overlying fascia (aponeurosis). The fibromatosis lesion is characteristically poorly circumscribed and infiltrates the surrounding tissue, which is usually striated musculature. The proliferation consists of elongated fibroblastlike cells of uniform appearance surrounded by and separated from one another by abundant collagen, with little or no cell-to-cell contact. The

Aggressive Fibromatosis in Children

cells lack hyperchromasia or atypia and the mitotic rate is variable. Using immunohistochemistry, the spindle muscle cells stain strongly with vimentin, whereas smooth muscle actin (SMA) and muscle-specific actin stain variable. Rare cases also stain with desmin and S-100. Pathogenesis The pathogenesis of AF is suggested to be multifactorial, i.e., genetic predisposition, endocrine factors, and trauma seem to play an important role. Local physical trauma before developing AF was reported in 20% of 108 reported pediatric AF patients from three studies. Apparent chromosome aberrations and nonrandom X-chromosome inactivation in adult and pediatric AF suggests a true neoplastic character (chromosome translocations). This is supported by a report of eight pediatric AF cases in one study, of which five (63%) had an abnormal karyotype (two at initial diagnosis and three at relapse) with trisomy 8 (n = 4) and trisomy 20 (n = 1) being the only recurrent features (Chromosome Translocations). Sporadic cases of adult AF contain a somatic mutation in either the adenomatous polyposis coli (APC) gene (21%), identified on chromosome 5q22 and associated with familial adenomatous polyposis (FAP), or b-catenin gene and protein expression (52%) (▶ APC Gene in Familial Adenomatous Polyposis; ▶ APC/b-Catenin Pathway). A high prevalence of ▶ desmoid tumor has been reported in 126/880 (14.3%) of adult FAP patients with proven APC gene mutation. Insulinlike growth factor binding protein 6 (IGFBP-6) appears directly downregulated by the b-catenin/ TCF complex in adult AF and implies a role for the IGF axis in the proliferation of AF. In addition, a high prevalence of AF (38%) was reported for patients with Gardner syndrome, a rare hereditary disorder that is characterized by the presence of multiple polyps in the colon. Patients may also develop bone and soft tissue tumors. The coexistence of familial adenomatous polyposis (FAP) with the specific extraintestinal manifestations epidermoid cyst, osteoma, and ▶ desmoid tumor. Advances in the understanding of the genetics of FAP and careful analysis of the phenotype have shown that Gardner syndrome is neither

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genetically nor clinically distinct from FAP. In contrast, in a review of all reported pediatric AF studies no patient with a history of familial AF or FAP, and only two patients with Gardner syndrome was seen. This illustrates that routine karyotyping has a relatively limited value, and the significance of the APC and b-catenin genes in the pathogenesis of childhood AF and their value for differentiating fibroblastic tumors has not yet been established. In adults, a correlation between tumor growth rate and the level of endogenous estrogen was suggested in female patients, because of high amounts of estrogen receptors (ER) in their tumor tissue. These are important findings as the presence of antiestrogen binding sites (AEBS) distinct from ER are suggested to play a role in treatment with antiestrogens in adult AF02254. So far, in two studies, only four children with AF were tested and did not express ER, indicating that the role of expression of ER and AEBS in pathogenesis of childhood AF may be limited. Treatment As these tumors at presentation clinically mimic other more malignant soft tissue tumors like ▶ rhabdomyosarcoma, non-ossyfying ▶ Ewing sarcoma sooner or later pediatric AF patients come to the attention of a pediatric oncologist. However, as the tumor is heterogeneous with regard to site and extension, treatment strategy in each individual patient is ideally determined by a multidisciplinary team which consists of pediatric oncologists, surgeons, and radiotherapists, supported by the diagnostic expertise of pediatric radiologists and pathologists. Aggressive fibromatosis still lacks general recommendations for its clinical management. Although spontaneous regression has been observed in sporadic cases, surgery is generally the primary treatment modality in adults and children with AF, unless there is a risk of significant mutilation and/or functional impairment. Seven of ten pediatric AF studies report treatment of the primary tumor, and all generally treated their patients (n = 168) with initial surgery. The other three series report treatment of recurrent tumor, two of them initially treated their patients (n = 15) with chemotherapy

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(vinblastine (VBL) and methotrexate (MTX)), whereas in the third study (n = 4) radiotherapy 01859 was administered. Relapse rate in the reviewed children with primary AF was approximately 50%. Most relapses (89%) have been observed within 3 years, and nearly all (97%) by 6 years, although relapse after 10 years has been reported. All relapses are local or regional with a pattern consistent with infiltrative growth. Three deaths are reported, caused by invasive tumor destruction of vital organs (▶ Progression), all three located in the head/ neck region. In 85 pediatric patients in whom primary surgery was performed, information on resection margins and relapse was available. Remarkably, only 16% of the patients with free microscopically margins after surgery relapsed, versus 67% of patients with positive margins (p < 0.01). In case of positive resection margins, 74% of patients without additional therapy relapsed, versus 40% of patients who received adjuvant treatment (p = 0.064). Adjuvant treatment consisted of chemotherapy (n = 8) or radiotherapy (n = 2) (▶ Adjuvant therapy). Although this is a retrospective analysis, which implies disadvantages like selection biases, the high risk for relapse in case of positive resection margins may indicate that the role of adjuvant treatment in patients with positive margins needs further exploration. In adults, the standard approach for patients with microscopically positive margins after surgery is adjuvant radiotherapy resulting in a high local control rate of approximately 80%, which is therefore considered to be beneficial regardless of surgical margins. In pediatric patients, the high doses of radiotherapy (55–60 Gy) necessary for tumor control in AF harbors a large risk for growth problems and development of secondary malignancies (▶ Radiation Sensitivity; ▶ Radiation-Induced Sarcomas after Radiotherapy). One pediatric AF study reported 11 children with partially excised or recurrent lesions who received radiotherapy and who had at least 3year follow-up. Four (36%) children relapsed, including two of five who had a dose of 50 Gy. In contrast, another pediatric AF study reported

Aggressive Fibromatosis in Children

11 of 13 (85%) children with relapse after irradiation, including 6 of 8 who had a dose of 50 Gy. The role of radiotherapy in childhood AF as adjuvant treatment in case of SP is not yet established and needs further prospective randomized studies which will not only evaluate response and survival but also late sequelae. The use of chemotherapeutic and other systemic agents might be a reasonable alternative to avoid radiotherapy in the growing child. However, also chemotherapy carries the risk for potentially adverse side effects, like second malignancies, fertility problems, and cardiotoxicity. A review concerning mainly adult AF reported a median overall response rate of 50% (range 17–100%) with combination chemotherapy (doxorubicin, actinomycin-D, methotrexate (MTX), and vinca alkaloids), in 16 single-arm studies. Reviewing all pediatric AF cases treated with chemotherapy in total, 27 out of 187 pediatric patients were treated with chemotherapy only at initial diagnosis (n = 10) or at relapse (n = 17). A combination of VBL and MTX was the most common reported regimen. Response on chemotherapy only was complete remission (CR) in 26%, partial remission (PR) in 18%, whereas stable disease (SD) was found in 30%, progressive disease (PD) in 11%, and response was not reported in 15% of the reviewed cases. Overall relapse rate (RR) after treatment with chemotherapy only was 26%. Comparing the relapse rate (respectively 74% versus 50%) of 46 pediatric patients with positive margins after primary surgery may suggest an advantage in outcome of adjuvant treatment with chemotherapy (n = 8), as compared with patients who did not receive adjuvant treatment (n = 38), however, numbers of cases are small and derived from different series. This illustrates that the role of chemotherapy in childhood AF is not yet established and should be further explored. Currently, a collaborative study of MTX/VBL chemotherapy for children with AF is initiated. Based on the reported experiences, the response of pediatric AF to chemotherapy has shown to be slow and it has been suggested that treatment should be continued for prolonged periods from 12 to 18 months. The chronic and prolonged course

Aggrus

that many of these children with AF endure as a result of these slow-growing lesions suggests that the use of (combinations of) noncytotoxic drugs, like antiestrogens, ▶ nonsteroidal antiinflammatory drugs (NSAIDs), imatinib mesylate, interferon-alpha (IFN-a), and ▶ retinoic acid for part of their treatment might be reasonable treatment options to explore. Side Effects in Survivors So far, information on toxicity of treatment is available from five pediatric AF case series (n = 128) with a median follow-up time of 4 years (range 0–25 years). Two studies reported a limited range of motion of the primary area as the most frequent late complication (42%). Severe short-term toxicity of treatment was reported in three patients, two died of cardiotoxicity after treatment with doxorubicin and one died of severe radiation induced dermatitis with chronic ulcers. During this short median follow-up, one secondary malignancy was reported; a papillary carcinoma of the thyroid gland, which developed 11 years after radiotherapy. Conclusion Primary surgery with negative margins is the treatment of choice for children with AF. In case of unresectable tumors, the use of chemotherapy and/or noncytotoxic drugs in children with AF could be a reasonable alternative. Positive margins after surgery indicate a high risk for relapse. Multicenter prospective (randomized) trials are necessary to clarify the role of and best strategy for adjuvant treatment in pediatric patients with aggressive fibromatosis.

Cross-References ▶ Adjuvant Therapy ▶ APC Gene in Familial Adenomatous Polyposis ▶ APC/b-Catenin Pathway ▶ Chemotherapy ▶ Chromosomal Translocations ▶ Desmoid Tumor ▶ Invasion

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▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Progression ▶ Radiation-Induced Sarcomas After Radiotherapy ▶ Radiation Sensitivity ▶ Retinoic Acid ▶ Rhabdomyosarcoma ▶ Supportive Care

References Buitendijk S, van de Ven CP, Dumans TG et al (2005) Pediatric aggressive fibromatosis, a retrospective analysis of 13 cases and a review of the literature. Cancer 104:1090–1099 Skapek SX, Hawk BJ, Hoffer FA et al (1998) Combination chemotherapy using vinblastine and methotrexate for the treatment of progressive desmoid tumor in children. J Clin Oncol 16:3021–3027 Spiegel DA, Dormans JP, Meyer JS et al (1999) Aggressive fibromatosis from infancy to adolescence. J Pediatr Orthop 19:776–784

See Also (2012) Chromosome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 848. doi:10.1007/978-3-642-16483-5_1145 (2012) Familial adenomatous polyposis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1373. doi:10.1007/978-3-642-164835_2106 (2012) Negative resection margins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2469. doi:10.1007/978-3-642-164835_4000 (2012) Osteoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2663. doi:10.1007/978-3-642-16483-5_4282 (2012) Radiotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3158. doi:10.1007/978-3-642-16483-5_4926 (2012) Recurrence. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3208. doi:10.1007/978-3-642-16483-5_4998 (2012) Surgery. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3574. doi:10.1007/978-3-642-16483-5_5596

Aggrus ▶ Podoplanin

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Aging Cynthia C. Sprenger, Stephen R. Plymate and May J. Reed Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA, USA

Definition Aging is defined at many levels, from the mitotic age of cells to the organismal-wide aging of tissues and organs. The appearance of cancer is only one clinical manifestation of the aging process. Age-associated epithelial cancers, such as ▶ breast cancer, colon cancer, and ▶ prostate cancer, however, contribute significantly to the morbidity and mortality of the elderly and are the second leading cause of death.

Characteristics Aging During an organism’s life span almost every aspect of its phenotype will undergo modification. The complexity of aging has led to a plethora of ideas about the specific molecular and cellular causes and how these alterations lead to age-associated diseases, such as epithelial cancers. Underlying all of these theories is the assumption that aging occurs from the bottomup, beginning with damage to DNA and proteins and ending with organismal frailty, disability, and disease. There is a vast amount of evidence to support the following aging theories: somatic mutation, telomere loss, mitochondrial damage, and altered proteins and waste accumulation. Somatic mutation theory suggests that age-related accumulation of ▶ DNA damage demonstrates a decline in DNA repair mechanisms, while the telomere loss theory argues that telomere shortening confers a finite life span to many human somatic tissues. Shortening of telomeres leads not only to a loss of chromosome replicative ability but also to an increased

Aging

propensity for recombination events, such as chromosomal translocations, that may induce oncogenesis. The mitochondrial theory makes a connection between age-related accumulation of mutations in mitochondrial DNA and impaired ATP production and thus reduced tissue bioenergenesis. Finally, the altered proteins and waste accumulation theory argues that accumulation of damaged proteins, due to either a decline in the function of chaperone proteins or proteosomes, leads to cellular damage, which then contributes to a range of age-related disorders. There is increasing consensus that all of these mechanisms interact to play a role in aging. Epithelial Cancers The body defends itself against epithelial cancers by halting replication of damaged cells either through ▶ apoptosis, in which the cell dies, or by senescence, in which the cell replicatively arrests but remains metabolically active. Both of these mechanisms are important in preventing the formation of epithelial tumors in the young. As one ages, the number of senescent cells increases. The accrual of these senescent cells may alter the microenvironment of the tissue such that cells harboring preneoplastic damage are permitted to proliferate and eventually undergo transformation. Senescent cells may contribute to this milieu, in part, by secreting paracrine factors that compromise tissue structure and function. Consequently, senescence inhibits cancer formation early on, but with time the buildup of senescent cells alters the microenvironment to one that promotes the growth of epithelial cancers. Cellular Senescence Most studies on senescence and cancer focus on the role played by senescent fibroblasts in the transformation of epithelial cells. Fibroblasts can undergo senescence as a result of various processes including: replicative exhaustion (telomere shortening), ▶ oxidative stress, DNA damage, ▶ epigenetic changes to chromatin organization, or activation of ▶ oncogenes, such as ▶ RAS, all of which appear to signal primarily through p53-dependent pathways, although some oncogenes trigger senescence via p16. Once a cell

Aging

has entered senescence, its transcriptome is altered such that genes associated with wound healing (e.g., inflammatory cytokines, epithelial growth factors, and ▶ matrix metalloproteinases (MMPs)) are activated. The alteration in gene expression affects not only the senescent fibroblast itself but the cells surrounding it as well. Senescent fibroblasts that were cocultured with breast or prostate epithelial cells increased the proliferation and tumorigenicity of those epithelial cells, both in vitro and in vivo. Epithelial cells can also undergo senescence due to oxidative stress, DNA damage, epigenetic changes, or activation of oncogenes. The pathways that trigger epithelial senescence include both p53- and p16/pRb-dependent as well as independent pathways. While the specific genes triggered by senescence can vary between the two cell types, the pattern of activation is similar: senescent-associated genes exhibit chromosomal clustering. Genes upregulated in senescent fibroblasts include various cell cycle proteins, interleukins, growth factors, integrins, MMPs, and caspases. Those upregulated in senescent epithelial cells include various cell cycle proteins, epithelial growth factors, transcription factors, integrins, laminins, ▶ fibronectin, MMPs, and ▶ tissue inhibitors of metalloproteinases (TIMPs). It is important to note that not all of these genes were upregulated in all samples or studies, only that these genes have been mentioned in various studies on senescence. In addition, it remains to be seen which genes trigger senescence and which are activated during senescence. Alterations in the Microenvironment Tissue architecture is important for maintaining proper cellular function and thus serves as a protective mechanism against diseases, including cancer. Accordingly, a defining characteristic of epithelial cancers is loss of tissue architecture. The microenvironment, which includes the extracellular matrix (ECM) (collagens, laminins, nidogens, proteoglycans) and soluble factors that are released by the cells or transmitted by other organs (hormones, cytokines, growth factors, enzymes), can serve as a powerful tumor

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suppressor, keeping damaged cells in check. A microenvironment that provides the correct cues can revert cells containing preneoplastic as well as oncogenic mutations back to a normal phenotype. But tissue architecture is not static: it is continually undergoing alterations due to the processes of living. The traditional focus in cancer has been on interactions between cells and various growth factors. However, there is increasing interest in other components of the extracellular space as well as in the bidirectional cross talk between the ECM and cells. The ECM interacts with cells via cognate receptors on the cell membrane, including integrins and syndecans. These receptors are connected to the cytoskelton of the cell, which is connected to the nuclear matrix and chromatin. Thus signals travel back and forth between the ECM and the cell that regulate gene expression and, in turn, protein expression, which then alters the makeup of the microenvironment. This bidirectional interaction between ECM and cells is termed dynamic reciprocity. The appearance of cancer cells disrupts the microenvironment and thereby destroys tissue architecture. Moreover, many oncogenic epithelial cells overexpress matrix metalloproteinases. These enzymes degrade various proteins in the basement membrane, including collagens and laminins. The subsequent disruption of the ECM allows the transformed epithelial cells to migrate into the stroma and form tumors. In breast and prostate carcinomas, the microenvironment consists of transformed epithelial cells, reactive stroma, recruited blood vessels, and infiltrating immune cells such as macrophages, lymphocytes, and leukocytes. Numerous studies also demonstrate that components of the ECM, such as collagen and laminin, are modified by and contribute to further tumor growth. Alterations in ECM protein are mirrored by changes in cell membrane receptors, such as integrins and growth factor receptors. Tumor Progression in Aging Whereas aging confers the greatest risk of developing cancer (as discussed above), it is widely accepted that most histologically similar epithelial tumors behave less aggressively in the aged. This longstanding impression arose from clinical

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studies in humans and was further supported by animal models, in which young and aged mice received identical inocula of tumor cells and were subsequently monitored for tumor growth and aggressiveness. Proposed mechanisms have focused on age-related deficits in immunemediated responses that directly and indirectly promote tumor growth (such as a lack of inflammatory cells and their associated cytokines) and decreased ▶ angiogenesis. It has been argued that the less permissive milieu of tissues is an adaptive response to the greater risk of cancer conferred by senescence and environmentally induced changes in the epithelial and stromal cells. Implications for Treatment A major difficulty with assessment of treatment options in the elderly is that many solid tumor treatment protocols have not been tested and optimized for the elderly. Many ▶ clinical trial phaseII and -III treatment protocols stop recruitment at 75 years of age. This is a problem since bone marrow recovery may be compromised by age and drug dosages may need to be modified due to age-related changes in drug metabolism and clearance. Additionally, standard therapies used for younger individuals may be inappropriate and further contribute to morbidity of the elderly, especially for some cancers, such as prostate, which may have a natural history that extends beyond the patient’s expected life span. Finally, it is important to understand the cell biology of senescence since many chemotherapeutic agents function by halting cell replication through induction of a senescent phenotype. The ability to induce cell senescence in a cancer cell should create a new class of therapeutic agents for cancer treatment in the elderly.

Cross-References ▶ Aging-Associated Inflammation ▶ Angiogenesis ▶ Apoptosis ▶ Breast Cancer ▶ Clinical Trial ▶ Colorectal Cancer

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▶ DNA Damage ▶ E-Cadherin ▶ Epigenetic ▶ Fibronectin ▶ Matrix Metalloproteinases ▶ Oncogene ▶ Oxidative Stress ▶ Prostate Cancer ▶ RAS Genes ▶ Tissue Inhibitors of Metalloproteinases

References Balducci L, Ershler WB (2005) Cancer and ageing: a nexus at several levels. Nat Rev Cancer 5:655–662 Campisi J (2005) Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 120:513–522 Kirkwood TB (2005) Understanding the odd science of aging. Cell 120:437–447 Nelson CM, Bissell MJ (2006) Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 22:287–309 Zhang H, Pan K-H, Cohen SN (2003) Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci. Proc Natl Acad Sci U S A 100:3251–3256

See Also (2012) Epithelial Growth Factors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1292. doi:10.1007/978-3-642-164835_1960 (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Growth Factor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1607– 1608. doi:10.1007/978-3-642-16483-5_2520 (2012) Growth Factor Receptors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1608. doi:10.1007/978-3-642-16483-5_2521 (2012) Inflammatory Cytokines. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1858. doi:10.1007/978-3-642-16483-5_3047 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Laminin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1971– 1972. doi:10.1007/978-3-642-16483-5_3268 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720

Aging-Associated Inflammation (2012) P53 In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/ 978-3-642-16483-5_4331 (2012) PRb In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2967. doi:10.1007/978-3-642-16483-5_4708 (2012) Reactive Stroma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3193. doi:10.1007/978-3-642-16483-5_4968 (2012) Senescence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3370. doi:10.1007/978-3-642-16483-5_5236 (2012) Somatic Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3466. doi:10.1007/978-3-642-16483-5_5408 (2012) Somatic Tissue. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3467. doi:10.1007/978-3-642-16483-5_5413

Aging-Associated Gene 4 Protein (AAG4) ▶ Clusterin

Aging-Associated Inflammation Devanand Sarkar1 and Paul B. Fisher2 1 Department of Human and Molecular Genetics, Virginia Commonwealth University, VCU Medical Center, School of Medicine, Richmond, VA, USA 2 Departments of Urology, Pathology and Neurosurgery, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY, USA

Synonyms Senescence-associated chronic inflammation

Definition Aging: Aging encompasses a set of interconnected processes that contributes to decline in performance, productivity, and health

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ultimately culminating in death with the passage of time. Inflammation: Inflammation is fundamentally a protective response occurring in the vascularized connective tissue to any insult the ultimate goal of which is to eliminate the organism which was the cause of cell injury (such as microbes, toxins) and the consequences of such injury (such as necrotic cells and tissues).

Characteristics A unified theory has been sought to explain how the single physiological process of aging may lead to diverse pathological events culminating in diverse aging-associated pathological conditions in different organs, such as Alzheimer’s, Parkinson’s, and other neurodegenerative disorders, rheumatoid arthritis, atherosclerosis, macular degeneration, etc., The free radical theory of aging, as proposed by Harman, is the most plausible and currently acceptable mechanism to explain the aging process. The central premise of this theory proposes that aging and its related disease processes are the net result of free radical-induced damage and the inability to counterbalance these harmful effects by antioxidative defenses. The generation of reactive oxygen and nitrogen species (ROS and RNS) activates redox sensitive transcription factors leading to the generation of proinflammatory molecules and a state of chronic inflammation. On the other hand, chronic inflammation itself results in the generation of ROS and RNS thus activating a feedback loop that amplifies the process of damage and deterioration. This oxidative stress and the subsequent chronic inflammation have been implicated as a mitigating factor for almost all of agingassociated maladies. The hallmarks of chronic inflammation, infiltration of macrophages, and circulating levels of proinflammatory chemical mediators are observed in aging-associated diseases. Activated macrophages (microglia) are observed in the senile plaques and surrounding tissue in the brain of patients with Alzheimer disease versus similar regions in control brains. Activated

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microglia are also detected in affected regions in Parkinson’s disease and Amyotropic Lateral Sclerosis (ALS). Similarly, many activated macrophages are found in arterial plaques of atherosclerosis and in infarcted heart tissue even years after an acute event. The presence of these activated macrophages/microglia may on one hand be beneficial, and on the other hand be harmful. While the activated macrophages release toxic materials injurious to viable host tissues, they also have phagocytic potential and an ability to destroy invading pathogens. In a state of persistent inflammation, the injurious events overwhelm the protective balance leading to chronic degeneration. ROS and RNS generated from the activated macrophages induce oxidative stress and free radical-induced injuries are evident in AD cortex, PD substantia nigra, and ALS spinal cord in the form of modification of proteins by glycation, the existence of low molecular weight compounds that have been oxidized and nitrated (such as 4-hydroxynonenal, malondialdehyde, 3-nitrotyrosine, 3-nitro-4-hydroxyphenylacetic acid, 5-nitrotocopherol, and 8-hydroxy-deoxyguanosin), and peroxidation of lipids. Additional evidence is the presence of the chemical mediators of inflammation in agingassociated diseases. The tangles and plaques of AD contain activated complement fragments C4d and C3d. The membrane attack complex (MAC) derived from the activation of the complement cascade is evident in dystrophic neuritis in AD brain and in substantia nigra in PD indicating autolytic attack. The mRNAs for complement proteins are sharply upregulated in affected regions of AD and PD brains and also in infarcted heart tissue. Cytokines play important roles as proinflammatory mediators and studies have documented increased blood level of proinflammatory cytokines such as IL-1, IL-6, TNF-a, and IL-8 in aged individuals as compared to young individuals. Plasma levels of TNF-a were positively correlated with IL-6 and acute phase proteins such as C-reactive proteins (CRP) in 126 centenarians, indicating an interrelated activation of the entire inflammatory cascade.

Aging-Associated Inflammation

However, the increase in circulating inflammatory parameters is far from levels seen during acute inflammation indicating that aging is associated with a chronic low-grade inflammatory activity. In a large study of 1,727 elderly Americans aged 70 years or older, age was associated with increased circulating plasma levels of IL-6. Polymorphisms in the promoter and untranslated regions that favor increased expression of proinflammatory genes, such as IL-1b, have been observed in patients with AD and PD. Inheritance of the polymorphic allele of apolipoprotein E4 (apoE4) in combination with the high-risk allele of TNF-a significantly increases the risk of AD. Similarly, simultaneous inheritance of high-risk alleles for IL-1aa889 and IL-1b+3953 significantly increases the odds ratio for developing AD. The association between increased plasma levels of TNF-a and atherosclerosis was demonstrated in 130 humans aged 81 years. The individuals with high TNF-a concentrations showed a significant clinical diagnosis of atherosclerosis. Multiple studies have established an association between elevated levels of IL-6 and diseases of old age. IL-6 induces the production of C-reactive protein (CRP), an important risk factor for myocardial infarction. High concentrations of CRP predict the risk of future cardiovascular disease in apparently healthy men. IL-8 plays a crucial role in initiating atherosclerosis by recruiting monocytes/macrophages to the vessel wall, which promotes atherosclerotic lesions and plaque vulnerability. Type 2 diabetes, atherosclerosis, and cardiovascular diseases have common antecedents. High plasma TNF-a concentrations were shown to predict insulin insensitivity with advancing age in 70 healthy humans with a large age range. Elevated levels of IL-6 and CRP predicted the development of type 2 diabetes in healthy women. In another study, elevated serum IL-6 levels predicted future disability in older adults especially by inducing muscle atrophy. IL-6 and CRP also play a pathogenic role in several diseases such as osteoporosis, arthritis, and congestive heart failure all of which have increasing incidence with age. Moreover, increased serum levels of IL-6 and IL-8 have

Aging-Associated Inflammation Aging-Associated Inflammation, Fig. 1 Schematic representation flowchart showing the proposed involvement of hPNPase old-35 in senescence associated degenerative diseases. See text for details

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hPNPase old-35

Oxidative stress

A NF-κB activation

Pro-inflammatory molecules

Senescence

Senescence-associated degenerative diseases

Chronic inflammation

been detected in patients with chronic obstructive pulmonary diseases and chemokines such as IL-8 and RANTES play important roles in the pathogenesis of these diseases. Various inflammatory mediators, such as IL-1, TNF-a, IL-6, IL-8, RANTES, and MMP-3 are responsible for chronic inflammatory rheumatoid diseases, such as osteoarthritis and rheumatoid arthritis both of which occur during aging. In vitro studies and experiments in animals also establish an intricate relationship between aging and inflammation. Gene expression analysis by microarray in human hepatic stellate cells confirms that replicative senescence in these cells is associated with a pronounced inflammatory phenotype characterized by upregulation of proinflammatory cytokines, including IL-6 and IL-8. An aging-induced proinflammatory shift in cytokine expression profile has been observed in rat coronary arteries. How does the proinflammatory shift occur during aging? A prominent mechanism by which ROS modulates diverse intracellular molecular processes is by regulating the activity of transcription factors, most notably nuclear factor (NF)-kB. By turning on proinflammatory mediators such as TNF-a, IL-1, IL-6, IL-8, IFN-g, iNOS, ICAM-1, VCAM-1, COX-2, and acute phase proteins, NF-kB functions as a central transcription factor for the development of chronic inflammatory diseases. Unfortunately very few studies were carried out in aging humans to establish a clear correlation between NF-kB activation and chronic inflammation. Strong NF-kB DNA binding and COX-2 transcription was detected in aging and in

sporadic AD superior temporal lobe neocortex. An increase in constitutive NF-kB DNA binding in older animals over young animals has been demonstrated in multiple studies. A gradual rise in ROS was evident in kidneys from Fischer rats from 6 to 24 months of age, and this increase correlated with an age-dependent augmentation in binding of NF-kB and elevated expression of cyclogenase-2 (COX-2), an NF-kB-responsive enzyme involved in proinflammatory prostanoid synthesis. Vascular smooth muscle cells from 18-month old rats showed considerably higher NF-kB DNA binding than that from new-born rats, which correlated with increased expression of inducible nitric oxide synthase and intracellular adhesion molecule-1, two proinflammatory molecules, in old smooth muscle cells upon inflammatory stimulation. A similar age-dependent elevation in NF-kB DNA binding has been reported in mouse and rat liver and heart, and in rat brain indicating a potential involvement of NF-kB in regulating aging-associated chronic inflammation. The molecular events leading to the generation of ROS and the development of chronic inflammation during aging are still not deciphered. Studies show that human polynucleotide phosphorylase (hPNPase old-35) might be the key element linking aging with the inflammatory process. hPNPase old-35 is a 30 –50 exoribonuclease involved in mRNA degradation (Fig. 1). Its expression is induced during senescence and ectopic overexpression of hPNPase old-35 induces a senescent phenotype in normal and cancer cells. Overexpression of hPNPase old-35 generates ROS with resultant increase in NF-kB DNA binding

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and increased production of proinflammatory cytokines such as IL-6, IL-8, RANTES, and MMP-3. These effects might be inhibited by an antioxidant N-acetyl-L-cysteine (NAC).

Agnogenic Myeloid Metaplasia

AICDA ▶ Activation-Induced Cytidine Deaminase

References

AID Bruunsgaard H, Pedersen M, Pedersen BK (2001) Aging and proinflammatory cytokines. Curr Opin Hematol 8:131–136 McGeer PL, McGeer EG (2004) Inflammation and the degenerative diseases of aging. Ann NY Acad Sci 1035:104–116 Sarkar D, Fisher PB (2006) Molecular mechanisms of aging-associated inflammation. Cancer Lett 236:13–23 Sarkar D, Lebedeva IV, Emdad L et al (2004) Human polynucleotide phosphorylase (hPNPase): a potential link between aging and inflammation. Cancer Res 64:7473–7478

▶ Activation-Induced Cytidine Deaminase

AIDS-129717 ▶ Temozolomide

AIDS-Associated Cancers Agnogenic Myeloid Metaplasia

▶ AIDS-Associated Malignancies

▶ Primary Myelofibrosis

AIDS-Associated Malignancies Agranulocytosis ▶ Neutropenia

AHNP

Enrique Mesri Viral Oncology Program, Sylvester Comprehensive Cancer Center and Development Center for AIDS Research, Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, USA

▶ Anti-Her2/Neu Peptide Mimetic

Synonyms

AHR ▶ Aryl Hydrocarbon Receptor

AIDS-associated cancers; AIDS-related cancers; HIV/AIDS-associated cancers; HIV-associated cancers; HIV-associated malignancies; HIVrelated cancers

AIB1

Definition

▶ Amplified in Breast Cancer 1 ▶ Steroid Receptor Coactivators

Cancers that are increased in individuals infected with human immunodeficiency virus (HIV). The

AIDS-Associated Malignancies

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AIDS-Associated Malignancies, Table 1 Relative risk, HIV/AIDS, and viral association for human cancers. Relative risk is compared to normal population (Data adapted Cancer type Kaposi sarcoma Non-Hodgkin lymphomas Hodgkin lymphoma Cervical cancer Anal cancer Hepatocellular carcinoma Lung Breast

from Boshoff and Weiss (2002), Grulich et al. (2007), Casper (2011), Mesri et al. (2010))

Relative risk HIV/AIDS >3000 75

Relative risk transplant 200 8

AIDS defining Yes Yes

Viral agent KSHV EBV

% infection viral agent in HIV/AIDS 100 60

10

4

No

EBV

100

5 30 5

2 5 2

Yes No No

HPV HPV HBVHCV

>50 >50 >50

3 1

2 1

No No

– –

– –

most common are ▶ Kaposi sarcoma and a subset of ▶ B-cell lymphomas (non-Hodgkin lymphomas). Other AIDS-associated malignancies are Hodgkin disease and cancers of the cervix, anus, lung, and the gastrointestinal tract.

Characteristics At the beginning of the HIV epidemic, the occurrence of certain cancers was considered as a milestone marking the transition to acquired immunosuppression syndrome (AIDS) in HIV infected individuals (Boshoff and Weiss 2002; Grulich et al. 2007). Those were ▶ Kaposi sarcoma (KS, AIDS-KS), non-Hodgkin lymphomas (NHL, AIDS-NHL), and invasive cervical cancers (Boshoff and Weiss 2002; Grulich et al. 2007; Cavallin et al. 2014). The incidence of HIV-associated cancers have been greatly reduced in the developed world upon the advent of highly active antiretroviral therapy (HAART) to effectively control HIV infection (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). However, AIDS-associated malignancies (AAMs) continue to be a major clinical complication of HIV infection and a major threat in developing countries, where the AIDS epidemic has not been totally controlled and access to HAART and cancer therapies is more restricted (Grulich et al. 2007;

Casper 2011). AIDS-associated malignancies are nowadays classified as: • AIDS-defining cancers: These are ▶ Kaposi sarcoma, non-Hodgkin lymphoma, and invasive cervical cancers (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Cavallin et al. 2014) (Table 1). They are mostly caused by the human oncogenic viruses (▶ virology) ▶ Epstein Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV), and human papillomavirus (HPV) (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Cesarman and Mesri 1999; Mesri et al. 2010). These oncogenic viruses can cause cancer in HIV/AIDS because immunosuppression creates a more permissive host for viral infection, allowing these viruses to express viral oncogenes that promote cell survival and cell proliferation (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2010; Cavallin et al. 2014). The genetic programs that include viral oncogenes tend to be more immunogenic and therefore are not allowed in immunocompetent hosts (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2010; Cavallin et al. 2014). • Non-AIDS-defining cancers: HIV-infected patients are at increased risk of certain other cancers such as ▶ Hodgkin disease, anal and rectal carcinomas, ▶ hepatocellular

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carcinomas, head and neck cancers, and ▶ lung cancer (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011). Some of these are caused by cancer-causing viruses, such as Hodgkin disease (EBV), anal/rectal cancers (HPV), head and neck cancers (HPV), and liver cancers (hepatocellular carcinoma) caused by the hepatitis viruses B and C (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011) (Table 1). AIDS-associated malignancies such as KS and NHL tend to respond favorably to HAART treatment, while others like cervical cancers do not show significant improvements (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). AAMs are generally treated with a combination of antiHIV approaches with systemic ▶ chemotherapy or targeted therapies currently available for non-AIDS-associated cancers. In the last years, rationally designed therapies including approaches targeting oncoviruses and the mechanisms of viral oncogenesis have been clinically tested and are being increasingly implemented. HIV Infection and AIDS Increase the Risk for Certain Cancers The immune system plays a major role in tumor immunosurveillance (see ▶ immunoediting) as well as in the control of oncogenic viruses such as EBV and KSHV. Consequently, immunosuppression and immune deregulation linked to HIV-induced CD4+ T cell depletion, as well as immune activation caused by HIV/AIDS, determine a tumor-prone status in the affected host (Mesri et al. 2014). This specially applies to cancers caused by viruses and cancers affecting cells of the immune system such as KS and NHL. Interestingly, many of the cancers that are increased in HIV/AIDS are the same cancers for which there is an increased risk for immunosuppressed patients such as those receiving organ transplants (Grulich et al. 2007; Casper 2011) (Table 1). This is a very important fact since it implies that preventing HIV to evolve to AIDS could help prevent the development of AAMs, while immune-reconstitution in AIDS patients

AIDS-Associated Malignancies

upon antiretroviral treatment could result in regaining immunity with antitumor consequences (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). Clinical findings consistent with these possibilities have been observed for AIDS-KS and AIDS-NHL: The incidence of these AAMs have decreased since HAART implementation; moreover, both cancers tend to respond favorably to reconstitution of immunity as a consequence of HAART, strongly supporting the idea that they are a consequence of HIV induced immunosuppression (Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). The Human Oncogenic Viruses EBV and KSHV and HIV/AIDS Depending on ethnicity, geographic area, and other factors, KSHV and EBV viruses could have a very high prevalence in the population from 10% (average for KSHV) to almost 90% (average EBV) (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2010; 2014; Cavallin et al. 2014). Fortunately, in immunocompetent hosts, infection with these oncogenic viruses is rarely sufficient to cause cancer, with most of the cancers arising after long periods of incubation and in very low percentages of the population (Cesarman and Mesri 1999; Mesri et al. 2010). This also suggests that factors other than the oncogenic viruses are necessary for full cellular transformation (Mesri et al. 2014). The incidence of AAMs caused by these viruses changes dramatically upon HIV/AIDS. The incidence of KS is thousands higher in HIV/AIDS than in the general population while the incidence of AIDS lymphomas is 70-fold higher (Casper 2011; Cavallin et al. 2014). This indicates that in the context of HIV/AIDS these oncogenic viruses are formidable pathogens. Currently, HIV-related and immune-related mechanisms seek to explain the higher incidence of these cancers in HIV/AIDS. HIV related: This is the immune activation syndrome that leads to changes in cytokine profiles and the presence of HIV accessory proteins such as Tat that were shown to favor KS development through KSHV-related and direct

AIDS-Associated Malignancies

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AIDS-Associated Malignancies, Fig. 1 EBV and KSHV genetic expression programs. EBV lymphomas display three latency programs: I, II, and III. KS tumors display latent and lytic cells. The most oncogenic of these programs tend to be the more immunogenic. Therefore these programs tend to be allowed only in immunosuppression and AIDS

angiogenic mechanisms (Boshoff and Weiss 2002; Mesri et al. 2010; Cavallin et al. 2014). Immunosuppression/AIDS-related mechanisms: The decrease in CD4+ T-helper cells leads to lack of both direct and CD8+ mediated control of KSHV and/or EBV infected cells (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2010; Cavallin et al. 2014). Depending on the stage in the viral life cycle, KSHV could be lytic or latent. During latency, the virus tends to be much less immunogenic by expressing a restricted number of genes necessary for latent virus maintenance (Cesarman and Mesri 1999; Mesri et al. 2010, 2014; Cavallin et al. 2014). In the lytic cycle, the virus expresses the full viral program necessary for replication and assembly of infectious virus with cell lysis. In the case of EBV, this oncogenic virus could exist in three stages of latency displaying increasing number of viral genes expressed and thus oncogenicity (Cesarman and Mesri 1999; Mesri et al. 2014) (Fig. 1). KSHV could exist either in a latent form and a lytic form with increased ▶ angiogenesis (see below) and oncogenicity (Mesri et al. 2010; Cavallin et al. 2014; Mesri et al. 2014) (Fig. 1), both of them simultaneously necessary

A

for KS development (see below) (Mesri et al. 2010). The more oncogenic these patterns of expression are, the more immunogenic (Cesarman and Mesri 1999; Mesri et al. 2010, 2014) (Fig. 1). So in the presence of an immunocompetent host they tend to be controlled by the immune system, and therefore the less immunogenic forms which are also the less oncogenic forms are allowed (Fig. 1). In the absence of immune control both of these viruses would be able to replicate and express their full oncogenic repertoire that include genes able to induce cell proliferation and pro-survival signaling cascades (see below) (Cesarman and Mesri 1999; Mesri et al. 2010; Cavallin et al. 2014). This will progressively lead to cell transformation. Kaposi Sarcoma Herpesvirus and Oncogenesis of AIDS-KS Kaposi sarcoma is an AIDS-defining AIDSassociated cancer (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). It is characterized by the proliferation of spindle-like cells of vascular and lymphatic endothelial origin, intense formation of new blood microvessels (see

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AIDS-Associated Malignancies, Fig. 2 Histological section of an AIDS-KS biopsy stained with hematoxylin and eosin. Note the abundant spindle-like cell proliferation and the blood vessels containing erythrocytes (reddish small cells), which can also be found extravasated within the tumor

▶ angiogenesis) with erythrocyte extravasation, that in the skin tend to give the characteristic purple appearance of the lesions, and cellular inflammatory infiltration (Boshoff and Weiss 2002; Mesri et al. 2010) (Fig. 2). The evolution of the lesion involves a progression through a patch, plaque, and “node” stages, or all these forms could co-exist in the same patient (Mesri et al. 2010). The two main clinical presentations of AIDS-KS are currently classified as per ACTG recommendations (Mesri et al. 2010). T0 – a localized (Cavallin et al. 2014), more indolent disease that tends to respond to initiation of HAART or localized therapies IFN etc. T1 – a disseminated, advanced disease, generally with visceral involvement, that could not respond to HAART and it should be treated with systemic chemotherapy. KS was first described in the late 1800s in Vienna by Dr. Moritz Kaposi as a rare, indolent, type of cancer affecting elder Ashkenazi Jews (Boshoff and Weiss 2002; Mesri et al. 2010). This clinical form is known nowadays as classic KS. It affects mostly old patients of Mediterranean or Jewish origin. KS was later described as a transplant-associated cancer. Another form described prior to the HIV epidemic was the endemic form found in Sub-Saharan Africa. It is in this area where KS continues to be a major health problem, with occurrence of both AIDS-

AIDS-Associated Malignancies

KS as well as HIV negative that can affect men, women, and even children (Boshoff and Weiss 2002; Grulich et al. 2007; Mesri et al. 2010; Cavallin et al. 2014). Finally, the epidemic clinical form was for the first time observed in 1981 as a clustered epidemic among sexually related homosexuals. This observation was, together with the occurrence of lung infections with Pneumocystis carinii, the first clinical manifestations of the upcoming HIV/AIDS epidemic. The increased incidence of AIDS-KS in homosexuals vs. women or i.v. drug patients led in 1990 to the formulation by V. Beral et al. of an infectious hypothesis that proposed the existence of a second infectious sexually transmitted causative agent (Boshoff and Weiss 2002; Mesri et al. 2010; Cavallin et al. 2014). This led in 1994 to a discovery by the Y. Chang and P. Moore lab, that employing genetic techniques identified in AIDS-KS lesions the sequences of a herpesvirus with homology to gamma oncogenic g2herpesviruses (Boshoff and Weiss 2002; Mesri et al. 2010; Cavallin et al. 2014). These sequences were identified as belonging to a new oncogenic herpesvirus: The human herpesvirus-8 (HHV-8) or Kaposi sarcoma herpesvirus (KSHV) (Boshoff and Weiss 2002; Mesri et al. 2010, 2014). It was rapidly established that KSHV fulfilled all Kochlike postulates to be considered as the KS etiologic agent (Boshoff and Weiss 2002; Mesri et al. 2010; Cavallin et al. 2014). It was consistently found in all KS lesions, its infection preceded and correlated with KS, and its genome and further investigations revealed the presence of a formidable oncogenic armamentarium that includes viral oncogenes with potential to cause cancer and to induce an ▶ angiogenesis (Boshoff and Weiss 2002; Mesri et al. 2010, 2014; Cavallin et al. 2014). • Inhibition of tumor suppressor and other cell cycle inhibitors: Tumor suppressors p53 and Rb working in conjunction with the cell cycle inhibitors ▶ p21 and ▶ p27 act as a natural barrier to cell proliferation and transformation. Among the latent KSHV genes, a gene called LANA was shown to be able to inactivate both p53 and Rb, while the virally encoded D-type

AIDS-Associated Malignancies

▶ cyclin (v-cyclin) can constitutively counter act both ▶ p21 and ▶ p27 activities. • Inhibition of ▶ apoptosis: Another barrier that a cell should surpass to become transformed are several mechanisms that compromise cell survival by induction of programmed cell death or ▶ apoptosis. KSHV encodes for a gene termed vFLIP that can constitutively activate NFkB, which is a very well known signaling mechanism that lead to inhibition of ▶ apoptosis and collaborate in cell transformation. • Angiogenic and inflammatory genes: The most singular characteristic of the AIDS-KS lesion is the proliferation of new blood microvessels or ▶ angiogenesis (Fig. 2). KSHV carries genes such as vGPCR and K1 that can activate in the host cell the secretion of growth factors that promote blood microvessel growth such, the so-called angiogenic growth factors, such as ▶ vascular endothelial growth factor (VEGF) and the cytokine interleukin-6 (IL-6). In addition, KSHV encodes a viral IL-6 and other viral angiogenic ▶ chemokines (vMIP-I/III). All these viral genes were shown to be able to induce the KS angiogenic phenotype in experimental cell systems and in laboratory animals. KSHV Oncogenesis and Immunosuppression The presence of this formidable oncogenic armamentarium appears inconsistent with the fact that KSHV is potently oncogenic only in the HIV/AIDS, in the immunosuppression/ transplant setting and in certain endemic areas (Boshoff and Weiss 2002; Mesri et al. 2010, 2014; Cavallin et al. 2014). A paradox pertaining KSHV oncogenesis resides in the fact that KSHV latent infection – the most prevalent in the AIDS-KS lesions – is not totally transforming. On the other hand, lytic infection, expressing the majority of KSHV angiogenic ▶ oncogenes, should lead to cell lysis and thus it cannot theoretically be transforming. Current theories seeking to understand this paradox are based on the occurrence of a minor percentage of lytically infected cells that secrete cytokines and angiogenesis growth factors that help to induce proliferation of

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latently infected cells and induce angiogenesis (Mesri et al. 2010, 2014; Cavallin et al. 2014). Thus KS requires the presence of both latently infected cells, making up the most of the tumor and lytic cells “fueling” tumor proliferation and angiogenesis (Mesri et al. 2010, 2014; Cavallin et al. 2014). A similar case for a paracrine tumor induced by a virus is Hodgkin lymphoma (see below), in which a few transformed cells are thought to drive the lymphoid cell proliferation and tumor formation. These two models serve well to explain why there is more KS in immunosuppression and HIV/AIDS. It has been shown that inflammatory cytokines found in AIDS and the HIV Tat proteins are able to induce lytic reactivation of KSHV leading to expression of lytic oncogenes (Mesri et al. 2010, 2014; Cavallin et al. 2014). Cells lytically infected with KSHV are necessary for KS tumor formation but are immunogenic, and thus they would be eliminated in an immunocompetent host (Fig. 1). Clinical findings consistent with the necessity of lytically infected cells for AIDS-KS tumor formation and its immune control are the response of AIDS-KS to immune reconstitution upon HAART treatment and the fact that some antiviral drugs targeting KSHV replication were able to ameliorate AIDS-KS (Boshoff and Weiss 2002; Grulich et al. 2007; Mesri et al. 2010, 2014; Cavallin et al. 2014). AIDS-Associated Lymphomas Lymphomas are the second most important of the AAMs, with AIDS-NHL being considered an AIDS defining cancer in HIV infected individuals. Most AIDS lymphomas are of B-cell origin and generally have an aggressive clinical presentation with poor prognosis (Boshoff and Weiss 2002; Grulich et al. 2007; Cesarman and Mesri 1999). They are a consequence of immunosuppression and immune deregulation/immune activation caused by HIV/AIDS leading to tumor-prone status for cancers affecting cells of the immune system. These cancers tend to be caused, as a significant number of AIDS lymphomas are, by the oncogenic viruses EBV and KSHV, which are both B-lymphotropic viruses (Boshoff and Weiss 2002; Grulich et al. 2007; Cesarman and Mesri

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1999; Mesri et al. 2014). The most common of lymphomas associated with AIDS are non-Hodgkin lymphomas, this includes ▶ Burkitt lymphoma (BL) and ▶ diffuse large B cell lymphoma (DLBCL), primary CNS lymphomas (PCNSL), ▶ Hodgkin disease (HD), multicentric Castlemans disease (MCD), and primary effusion lymphoma (PEL) (Boshoff and Weiss 2002; Grulich et al. 2007; Cesarman and Mesri 1999). Most of these AIDS lymphomas are causally related to EBV and or KSHV. Sixty percent of NHL, 30% of BL, 100% of PCNSL, and 100% of HD are infected with EBV. All PEL are infected with KSHV and more than a half of them with EBV (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Cesarman and Mesri 1999) (See Table 1). Immunosuppression and immunederegulation were associated to nonviral as a well as to viral NHLs prior to the HIV/AIDS epidemic (Cesarman and Mesri 1999). EBV was first isolated from a case of endemic Burkitt lymphoma. BL was characteristically associated with malariaendemic areas. In this case, latent infection with EBV (Latency I, Fig. 1), in the context of malariarelated chronic B-cell stimulation, increases the chance of aberrant chromosomal translocation that activates c-myc expression (Cesarman and Mesri 1999; Mesri et al. 2014). In the case of transplant associated immunosuppression, EBV causes posttransplant lymphoproliferative disorder (PTLD), a progressively malignant proliferation of B-cells driven by highly oncogenic and immunogenic latency III pattern of EBV gene expression that, only in the context of immunosuppression, can progress to a full malignancy (Cesarman and Mesri 1999; Mesri et al. 2014) (Fig. 1). Similar scenarios of immune deregulation and viral induced proliferation in the context of immunosuppression explain the occurrence of EBV and/or KSHV induced lymphomas in the context of AIDS. • Burkitt lymphoma (latency I transformation): This B-cell lymphoma appears infected with EBV in 30% of the cells of HIV associated BL. In this case, EBV expresses only the

AIDS-Associated Malignancies

gene EBNA1 (Cesarman and Mesri 1999; Mesri et al. 2014). Although this viral gene has shown its ability to cause cancer in cell and animal models, it has very low levels of oncogenicity and thus it requires another major transformation event. In the case of BL, this event is an aberrant chromosomal translocation that places c-myc under the control of the immunoglobulin regulating machinery, thus causing the over expression of this oncogenic transcription factor (Cesarman and Mesri 1999; Mesri et al. 2014). • Hodgkin disease (latency II transformation): HD is associated with AIDS and it is 100% infected with EBV (Grulich et al. 2007; Casper 2011; Cesarman and Mesri 1999; Mesri et al. 2014). HD is a peculiar type of cancer. The tumor is composed of a few transformed EBV-infected cells that are called ReedSternberg cells that drive the proliferation of untransformed lymphocytes forming the bulk of the tumor. In this case, EBV infection displays what is denominated a latency II pattern (Cesarman and Mesri 1999; Mesri et al. 2014). This pattern includes the expression of two powerful EBV oncogenes: LMP-1 and LMP-2A. These EBV oncogenes cause B-cell proliferation by triggering survival and proliferation cascades mimicking two physiological B-cell signals, the CD40 receptor, and the IgG receptor, and leading to activation of important survival and proliferation cascades such as NFkB, MAPK, and AKT (Cesarman and Mesri 1999; Mesri et al. 2014). • Other AIDS-NHL, DBCBL, and PCNSL (latency III transformation): Most of these tumors are infected with EBV. In the case of AIDS-NHL, particularly of large B-cells, the EBV displays the highly oncogenic and highly immunogenic latency III pattern (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2014). In it EBV displays nine oncogenic genes including EBNA 1–6, LMP-1, LMP-2A, and LMP-2B. This is the most oncogenic but also the most immunogenic pattern, and thus it is characteristic of HIV/AIDS immunosuppression as well as organ transplants. It also

AIDS-Associated Malignancies

occurs in immune privileged sites such as the CNS, with PCNSL being 100% infected with EBV (Grulich et al. 2007; Casper 2011; Cesarman and Mesri 1999; Mesri et al. 2014). • KSHV-associated AIDS lymphomas: Multicentric Castlemans disease and primary effusion lymphomas. Two AIDS-associated lymphomas are caused by KSHV infection of B-cells in their later stages of differentiation toward becoming plasma cells (Boshoff and Weiss 2002; Cesarman and Mesri 1999; Mesri et al. 2014). MCD is a polyclonal malignancy driven by infection of plasmalike cells localized to the lymph nodes, while PEL is a clonal lymphoma characterized by its effusion, liquid phenotype (Cesarman and Mesri 1999). Although they could also be co-infected with EBV, KSHV is considered the main transforming virus for these lymphomas. The majority of lymphoma cells are infected with KSHV in its latent stage. All latent KSHV genes, LANA, v-cyclin, and vFLIP (see above) have potential to drive cell proliferation and survival. In particular the gene vFLIP has been shown to be key in promoting PEL transformation by activating the important survival cellsignaling cascade NFkB (Cesarman and Mesri 1999; Mesri et al. 2014). Current Therapies and Clinical Challenges Clinical treatments for AAMs generally involve an anti-HIV treatment, which is generally a HAART regime, with a treatment specific for the type of cancer. As AAMs pose specific clinical problems derived of the HIV/AIDS in combination with generally aggressive cancer presentations, this continues to be a major area of clinical testing and experimentation. In the USA, multicenter cooperative groups carry most of the clinical research in these areas. They are the AIDS Clinical Trials Group (ACTG) and the AIDS Malignancies Consortium (AMC). In the last years rationally designed approaches, including therapies targeting oncoviruses and their mechanisms of viral oncogenesis, have been clinically tested and are being increasingly implemented.

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Treatments for AIDS-Associated Kaposi Sarcoma Reversal of immunosuppression with immune reconstitution with HAART has been associated with the regression of KS lesions and the incidence of KS has decreased over sixfold with the advent of widespread use of HAART in HIV-infected individuals (Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). However, the number of KS cases is still rising in sub-Saharan Africa, where the HIV epidemic is still not controlled (Grulich et al. 2007; Casper 2011). In the vast majority of the cases, AIDS-KS patients respond very favorably to initiation of HAART (Boshoff and Weiss 2002; Grulich et al. 2007; Casper 2011; Mesri et al. 2010; Cavallin et al. 2014). Moreover, certain HAART regimes, in particular those containing protease inhibitors, have been shown to potentially display both preventive and therapeutic activity to AIDS-KS (Mesri et al. 2010; Cavallin et al. 2014). For disseminated KS and HAART resistant KS, chemotherapy is indicated and three FDA-approved agents (pegylated liposomal doxorubicin, liposomal daunorubicin, and paciltaxel) are available (Boshoff and Weiss 2002; Grulich et al. 2007; Mesri et al. 2010; Cavallin et al. 2014). Despite the effectiveness of the available treatments, KS is not totally eliminated for at least half of these advanced patients (Grulich et al. 2007; Mesri et al. 2010; Cavallin et al. 2014). Advances in the understanding of the pathogenesis, and in particular of the mechanism of viral oncogenesis of KS, have uncovered potential targets for KS therapies. Among the most promising approaches are those geared to intervene the powerful viral and host mechanism that mediate the angiogenic response in KS (Mesri et al. 2010; Cavallin et al. 2014). The small molecule inhibitors of the PDGF and VEGF ▶ receptor tyrosine kinases ▶ Imatinib and Sunitinib have been and are being tested as KS therapies for their combined antitumor and anti-angiogenic activities. Another promising antitumor target in KS is the PI3K/AKT/mTOR signaling pathway. This is a pathway triggered by KSHVoncogenes such as K1 and the vGPCR that

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lead to secretion of VEGF and other growth factors and ▶ cytokines that in turn act on inducing neighbor cell proliferation by activating receptors that also lead to activation of PI3K/AKT/mTOR (Mesri et al. 2010; Cavallin et al. 2014). Inhibition of the mTOR pathway by ▶ Rapamycin was shown to be highly effective in transplant KS and therefore Rapamycin and other similar new generation drugs are being actively tested in AIDS-KS. Treatments for AIDS Lymphomas AIDS lymphomas have been shown to ameliorate with HAART therapy, but this treatment has always to be provided in addition to antilymphoma therapy (Grulich et al. 2007). Importantly, the use of HAART has increased the survival for patients with AIDS-related lymphoma to a level comparable to the outcome in the general population. Moreover, the use of HAART has also allowed the use of similar chemotherapy regimes for AIDS-related lymphomas than in non-HIV patients. Among the ▶ chemotherapy regimes that are used are the cyclophosphamidedoxorubicin-vincristine-prednisone combination (CHOP), the methotrexate-bleomycindoxorubicin-cyclophosphamide, vincristinedexamethasone (m-BACOD), and infusional cyclophosphamide, doxorubicin, and etoposide. Many times these cytotoxic regimes could affect the bone marrow blood and immune systemrepopulating cells. Therefore they should be administered with growth factors that compensate these effects with very aggressive regimes even needing bone marrow transplant. Since many B-cell lymphomas express the CD20 surface marker, the use of an anti-CD20 monoclonal antibody Rituximab (Mabthera) have been also implemented. Among potential mechanisms of viral oncogenesis that can be intervened, the survival pathway NFkB and its viral activators continue to concentrate most of the interest (Cesarman and Mesri 1999). An emerging concept that seeks to target lymphomas that are latently infected with EBV and KSHV is the so-called induction therapy, which combines an agent able to induce lytic replication of the virus (EBV and/or KSHV), with an antiviral or another

AIDS-Associated Malignancies

agent able to inhibit the virus and/or potentiate killing of the lytically infected cells. Several combinations have been or are being tested such as AZT/aIFN, butyrate/ganciclovir, and AZT/ganciclovir. Conclusion In spite of the sharp decrease in the incidence for the main AAMs upon introduction of antiretroviral therapies, AAMs continue to be a major complication for HIV infection and to be a major health problem for developing countries, particularly Sub-Saharan Africa, where the AIDS epidemic is not completely controlled and access to HIV and cancer diagnosis and therapies are more restricted. In the last years, rationally designed therapies, with many based on mechanisms of viral oncogenesis, are being clinically tested showing prowess for the treatment of these diseases both in resource-rich and resource-limited settings.

Cross-References ▶ Epstein-Barr Virus ▶ Kaposi Sarcoma

References Boshoff C, Weiss R (2002) AIDS-related malignancies. Nat Rev Cancer 2:373–382. http://www.ncbi.nlm.nih. gov/pubmed/12044013 Casper C (2011) The increasing burden of HIV-associated malignancies in resource-limited regions. Annu Rev Med 62:157–170. http://www.ncbi.nlm.nih.gov/ pubmed/20868276 Cavallin LE, Goldschmidt-Clermont P, Mesri EA (2014) Molecular and cellular mechanisms of KSHVoncogenesis of Kaposi’s sarcoma associated with HIV/ AIDS. PLoS Pathog. 10:e1004154. http://www.ncbi. nlm.nih.gov/pubmed/25010730 Cesarman E, Mesri EA (1999) Virus-associated lymphomas. Curr Opin Oncol 11:322–332. http://www.ncbi. nlm.nih.gov/pubmed/10505767 Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM (2007) Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet 370:59–67. http://www. ncbi.nlm.nih.gov/pubmed/17617273

Akt Signal Transduction Pathway Mesri EA, Cesarman E, Boshoff C (2010) Kaposi sarcoma and its associated herpesvirus. Nat Rev Cancer 10:707–719. http://www.ncbi.nlm.nih.gov/pubmed/ 20865011 Mesri EA, Feitelson MA, Munger K (2014) Human viral oncogenesis: a cancer hallmarks analysis. Cell Host Microbe 15:266-82. http://www.ncbi.nlm.nih.gov/ pubmed/24629334

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AIM1 ▶ Aurora Kinases

AIM-1 ▶ Aurora Kinases

AIDS-Related Cancers ▶ AIDS-Associated Malignancies

AIE2 ▶ Aurora Kinases

AIF-Mediated Cell Death ▶ Caspase-Independent Apoptosis

AIK

Akt Signal Transduction Pathway George Z. Cheng1, Santo V. Nicosia2 and Jin Q. Cheng3 1 Harvard Medical School, Boston, MA, USA 2 H. Lee Moffitt Cancer Center, Tampa, FL, USA 3 Molecular Oncology Program and Research Institute, H. Lee Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, FL, USA

Definition Akt, also called protein kinase B, represents a serine/threonine protein kinase subfamily. Three members of this family have been cloned to date, namely, AKT1/PKBa, AKT2/PKBb, and AKT3/ PKBg. The overall homology between these three isoforms is >85% at amino acid level.

▶ Aurora Kinases

Characteristics

AIK2 ▶ Aurora Kinases

AIK3 ▶ Aurora Kinases

AKT1, AKT2, and AKT3 share a very similar structure, which contains an N-terminal pleckstrin-homology (PH) domain, a central kinase domain, and a serine/threonine-rich C-terminal region (Fig. 1). The PH domain and C-terminal region between these three isoforms are more diverse (homology 73–84% at amino acid level) as compared to the kinase domain (90–95%), suggesting that PH and C-terminal regions may represent functional difference between AKT1, AKT2, and AKT3. All three

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Akt Signal Transduction Pathway

Akt Signal Transduction Pathway, Fig. 1 Akt signaling. Activation of Akt involves recruitment of the Akt to the cell membrane by means of PH domain binding to product of PI 3-kinase, PI(3,4,5)P3, promoting a conformational change in Akt which results in phosphorylation of Thr308 and Ser473 by PDK1 and mTORC2, respectively.

In addition, IKBKE functions as Akt kinase by direct phosphorylation of Thr308 and Ser473. Upon its release from the membrane, Akt would become available to phosphorylate a number of molecules to induce cell growth, survival, and angiogenesis (KD kinase domain, RD regulatory domain)

members of Akt localize to the cytoplasm; however, they could translocate to the nucleus upon activation. In addition, they are located on different human chromosomes (AKT1 on 14q32, AKT2 on 19q13.1-13.2, and AKT3 on 1q44).

molecules occurs in up to 50% of all human tumors, and thus Akt is a critical target for anticancer drug discovery. Increasing evidence suggests that AKT members have different cellular functions. Of note, knockout of individual AKT member resulted in distinct phenotypes. AKT1-deficient mice exhibited a uniform reduction in organ size, while AKT2-null mice develop typical type II diabetes, and AKT3-deficient mice displayed a selective impairment of brain development. Moreover, although AKT1- and AKT3-deficient brains are reduced in size to comparable degree, the absence of AKT1 reduces neuronal cell number, whereas the lack of AKT3 results in smaller and fewer cells. In tumor biology and invasion process, overexpression of only AKT2, not AKT1 or AKT3, recapitulated the invasive effects of PI3K in breast cancer cells. Additionally, only the expression of dominant negative AKT2, not its counterparts, inhibited invasion induced by either activation of PI3K or overexpression of Her2/Neu. These observations suggest that

Akt in Human Malignancy and Different Functions of Akt Family Members Although AKT1, AKT2, and AKT3 display high sequence homology, there are clear differences between these three members in terms of biological and physiological function: (1) AKT1 expression is relatively uniform in various normal organs whereas high levels of AKT2 and AKT3 mRNA are detected in skeletal muscle, heart, placenta, and brain; and (2) overexpression of wildtype AKT2, but not AKT1 and AKT3, transforms NIH 3 T3 cells. Amplification of the AKT2 has been observed in 15% of human ovarian carcinomas and 20% of human pancreatic cancers. Infrequent mutations of the Akt have been also detected in human cancer. However, activation of Akt kinase due to alterations of its upstream

Akt Signal Transduction Pathway

AKT1 and AKT3 may be acting more in a cellular growth and survival role, while AKT2 may be more involved in regulating cellular metabolism, mobility, invasion, and metastasis. Signal Transduction Akt is activated by a variety of stimuli, including growth factors, protein phosphatase inhibitors, and cellular stress in a PI3-kinase dependent manner. Activation of Akt depends on the integrity of the pleckstrin-homology (PH) domain, which mediates its membrane translocation, and on the phosphorylation of Thr308 and Ser473. Phosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3, produced by PI3K bind directly to the PH domain of Akt, driving a conformational change in the molecule, which enables the activation loop of Akt to be phosphorylated by PDK1 at Thr308. Full activation of AKT1 is also associated with phosphorylation of Ser473 within a C-terminal hydrophobic motif characteristic of kinases in the AGC kinase family. Although the role of PDK1 in Thr308 phosphorylation is well established, the mechanism of Ser473 phosphorylation is controversial (Fig. 1). A number of candidate enzymes responsible for this modification have been put forward, including the rictor-mTOR (mTORC2) complex, ILK, and DNA-dependent kinase. The activity of Akt is negatively regulated by tumor suppressor PTEN, which is frequently mutated in human malignancy. PTEN encodes a dual-specificity protein and lipid phosphatase that reduces intracellular levels of PtdIns-3,4,5-P3 by converting them to PtdIns-4,5-P2, thereby inhibiting the PI3K/Akt pathway. Studies have shown that IKBKE directly phosphorylates Thr308 and Ser473 to activate Akt in a PI3K/PTEN/mTORC2 independent manner. Sirt1 and TRAF6 also trigger Akt activation by deacetylation of Akt and PDK1 and induction of K63 ubiquitination of Akt, respectively. In addition, PHLPP phosphatase dephosphorylates the Ser473 leading to inactivation of Akt. Akt phosphorylates and/or interacts with a number of molecules to exert its normal cellular functions, which include roles in cell proliferation, survival, angiogenesis, and differentiation. A couple dozen of molecules have been identified to be downstream targets of Akt,

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including TSC2, XIAP, Bad, FOXO, IKKa, ASK, EZH2, etc. The vast majority of Akt substrates contain Akt phosphorylation consensus sequence RXRXXS/T (R is arginine; S/T is serine/threonine, Fig. 1). Akt Pathway as a Target for Cancer Intervention Since Akt functions as a cardinal nodal point for transducing extracellular (growth factor and insulin) and intracellular (receptor tyrosine kinases, Ras, and Src) oncogenic signals, it presents an exciting new target for molecular therapeutics. Lipid-based inhibitors of Akt were the first to be developed, including perifosine, PX-316, and phosphatidylinositol ether lipid analogues, which were designed to interact with the PH domain of Akt. In addition, several Akt antagonists have been identified using high-throughput screening of chemical libraries and rational design. These inhibitors include Akt/PKB signaling inhibitor-2 (API-2), 9-methoxy-2-methylellipticinium acetate, indazole-pyridine A-443654, and isoformspecific canthine alkaloid analogues. Following its identification in a screen of the NCI diversity set, API-2 was shown to inhibit Akt kinase activity and stimulate apoptosis of xenografts of human cancer cells exhibiting high Akt activity. API-2 is a tricyclic nucleoside that previously showed antitumor activity in phase I and phase II trials conducted, but multiple toxicities, including hepatotoxicity, hyperglycemia, thrombocytopenia, and hypertriglyceridemia, precluded further development. The identification of Akt inhibition as a mechanism underlying API-2 activity has provided new interest in studying this drug and raises the possibility that lower doses may inhibit Akt and induce tumor cell apoptosis without the previously associated side effects.

Cross-References ▶ Angiogenesis ▶ Mammalian Target of Rapamycin ▶ PI3K Signaling

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References Bellacosa A et al (1991) A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254:274–277 Brognard J et al (2007) PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol Cell 25:917–931 Cheng JQ et al (1992) AKT2, a putative oncogene encoding a member of a novel subfamily of serine-threonine protein kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A 89:9267–9271 Cheng JQ et al (2005) The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 245:7482–7492 Dummler B et al (2006) Life with a single isoform of Akt: mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies. Mol Cell Biol 26:8042–8051 Guo JP et al (2011) IKBKE activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and PH domain to sustain transformation. J Biol Chem 286:37389–37398

Alcohol Consumption Helmut K. Seitz1,2 and Sebastian Mueller1 1 Centre of Alcohol Research (CAR), University of Heidelberg, Heidelberg, Germany 2 Department of Medicine, Salem Medical Center, Heidelberg, Germany

Definition Alcohol is a widely used stimulant, toxin and nutrient, depending on doses and drinking pattern. Its chronic abuse damages almost all cells in the human body and results in organ injury, including the development of certain cancers.

Characteristics Alcohol is responsible for 390,000 cancer cases worldwide, representing 3.6% of all cancers (5.2% in men and 1.7% in women). In February 2007, the International Agency for Research on Cancer (IARC) invited 26 scientists from 15 countries to evaluate the evidence for ethanol and

Alcohol Consumption

ethanol-containing beverages as a cancer causing agent. The experts reviewed all epidemiological and experimental studies covering this topic and came finally to the following conclusion: “Regular alcohol consumption is associated with an increased risk for cancer of the oral cavity, pharynx, larynx, esophagus, liver, breast and colorectum. There is substantial mechanistic evidence in humans deficient in aldehyde dehydrogenase that acetaldehyde derived from the metabolism of ethanol contributes to causing maligant esophageal tumors. The studies demonstrate that ethanol and not the type of alcoholic beverage is responsible for the tumor risk.” Epidemiology Cancer of the Upper Aerodigestive Tract

A large number of prospective and case-control studies have shown that the risk for upper aerodigestive tract (UADT) cancer is significantly dose-dependent, increased two- to threefold at a daily consumption of 50 g of ethanol or more. Smoking has an additionally synergistic effect. A carefully performed French study showed an 18-fold increased risk for esophageal cancer when 80 g of ethanol were consumed daily. Twenty cigarettes per day increased cancer risk by a factor of 5. However, drinking and smoking were associated with 44-fold increased cancer risk. Other factors which increase the alcoholmediated cancer risk are oral bacterial overgrowth (poor oral hygiene and dental status) as well as gastroesophageal reflux disease (GERD). Hepatocellular Cancer (HCC)

HCC develops in 1–2% per year of patients with alcoholic liver cirrhosis of the liver every year. Cirrhosis is a consequence of chronic liver disease characterized by the replacement of liver parenchyma by fibrotic tissue and regenerative nodules, leading to progressive loss of liver function. Cirrhosis is most commonly caused by excessive consumption of alcohol and viral infections but has many other possible causes. Cirrhosis has a high mortality due to various

Alcohol Consumption

complications. The risk for HCC is between 4.4and 7.3-fold at an alcohol dose of 80 g/day. HCC in a non-cirrhotic liver is extremely rare. Chronic alcohol consumption also increases HCC risk in patients with other liver diseases such as chronic hepatitis B and C, hereditary hemochromatosis, and non-alcoholic fatty liver disease (NAFLD). Patients with chronic hepatitis C have a threefold increased risk when they consume 80 g of ethanol or more as compared to ▶ hepatitis C alone. In hepatitis B patients, ethanol in doses of 40 g or more shortens the development of a HCC by approximately 10 years. Breast Cancer

A clear cut dose-dependent association between alcohol intake and breast cancer has been reported in more than 100 publications. The risk starts at a dose of 18 g of alcohol per day. According to a meta-analysis of 38 studies, one, two, or three drinks increase breast cancer risk by 10, 20, and 40%. Every additional 10 g of alcohol increase breast cancer risk by 7%. At 50 g of alcohol daily, cancer risk is enhanced by 50%. In the United States, it has been calculated that 4% of all newly diagnosed breast cancer cases are due to alcohol, resulting in a total of approximately 8,000 cases per year. Colorectal Cancer

More than 50 prospective- and case-control studies found a positive association between colorectal cancer and alcohol consumption. According to pooled data from eight cohort studies and data from a meta-analysis, a 1.4-fold increased cancer risk was found in patients with an alcohol intake of more than 50 g as compared to non-drinkers. Excessive alcohol consumption also favors high risk polyp or colorectal cancer occurrence among patients with adenomas. Five out of six studies also showed an increased risk for colorectal polyps following chronic alcohol consumption as compared to abstinence. Epidemiologic studies also underline the importance of the lack of dietary factors such as methionine and folate which modulate the ethanol-associated colorectal cancer risk.

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Mechanisms of Alcohol-Mediated Carcinogenesis Acetaldehyde

Acetaldehyde is the first metabolite of ethanol oxidation. Acetaldehyde binds to proteins and DNA; it has been found to be mutagenic and carcinogenic in animal experiments. The most convincing evidence for the role of acetaldehyde as cancer causing agent comes from genetic linkage studies in populations who accumulate acetaldehyde following alcohol consumption. Fifty percent of Japanese have a mutation of the acetaldehydehydrogenase (ALDH)2 gene which codes for an ALDH enzyme with low activity. When these individuals drink alcohol, acetaldehyde accumulates in the blood, and they develop a flush syndrome with tachycardia, nausea, and vomiting. In addition, acetaldehyde also accumulates in the saliva, rinses the mucosa of the upper aerodigestive tract, and may enter the mucosal cells, resulting in DNA adduct formation. Ten percent of the Japanese population, who have zero ALDH activity, are incapable of consuming alcohol, even in small doses. Despite the unpleasant side effects of flushing, however, heterozygotes of the ALDH2 2/1, 40% of the Japanese population with low ALDH activity, may consume alcohol. These individuals have a significant increased cancer risk for upper aerodigestive tract cancer, in particular esophageal cancer and for colorectal cancer. This gene mutation does not exist in Caucasians. However, Caucasians have a gene polymorphism for the ADH1B and ADH1C gene. While the ADH1B*2 allele encodes for an ADH enzyme with a 40-fold increased acetaldehyde production as compared to the ADH1B*1 allele, the ADH1C*1 allele encodes for an enzyme with a 2.5-fold increased ADH activity as compared to the ADH1C*2 allele. Thus, heavy drinkers who are homozygous for the ADH1C*1 allele not only have an increased concentration of acetaldehyde in their saliva, but also seem to have an increased risk for upper aerodigestive tract cancer. Considerable amounts of acetaldehyde can also be produced from ethanol by microorganisms in the oral cavity and in the colon. Therefore, poor

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oral hygiene leading to bacterial overgrowth is a risk factor in the alcoholic for cancer of the oral cavity. Oxidative Stress

▶ Reactive oxygen species (ROS) are generated during the oxidation of ethanol via ▶ cytochrome P-450 2E1 and during intramitochondrial reoxidation of NADH generated by ethanol oxidation through alcoholdehydrogenase. This is especially relevant in the liver. ROS cause lipid peroxidation and lipid peroxidation products such as 4-hydroxynonenal can bind to DNA, forming exocyclic DNA-etheno adducts with mutagenic and carcinogenic properties. Under normal conditions ROS are neutralized by the antioxidative defense system, which, however, is severely altered by chronic ethanol consumption. Altered Methyl Transfer

Chronic ethanol consumption results in a significant reduction of S-adenosyl methionine (SAMe), the active methyl donor. This is due to multiple effects of ethanol and acetaldehyde on enzymatic reactions leading to the generation of SAMe, including folate deficiency. The lack of SAMe results in a reduction of all methylation processes. With respect to ▶ carcinogenesis, the most important methylation process is the methylation of cytosine bases within the DNA. This DNA hypomethylation results in a diminished silencing of oncogenes and therefore favors carcinogenesis. Reduced Retinoic Acid

Chronic ethanol consumption results in a decrease of retinol and ▶ retinoic acid (RA) in the liver, associated with an activation of the AP-1 gene resulting in an increased expression of c-jun and c-fos and finally hepatocellular hyperproliferation associated with increased cancer risk. The decrease of RA is predominantly due to the ethanol-mediated induction of CYP2E1, since CYP2E1 is also responsible for the metabolism of RA and retinol. An enhanced metabolism of RA and retinol induced by CYP2E1 results in the generation of metabolites with apoptotic properties. In this context, it is important to note that the

Alcohol Consumption

concomitant administration of ß-carotin for the prevention of bronchial cancer and the use of alcohol in a dose of more than 12 g/day increases, instead of decreasing, the risk of bronchial carcinomas in smokers. Specific Mechanisms (Cirrhosis, Gastroesophageal Reflux Disease, Estrogens)

In the liver, cirrhosis caused by chronic ethanol consumption is a prerequisite for the development for a HCC due to mechanisms not clearly understood, but predominantly due to chronic inflammation with inflammation-driven oxidative stress and proliferative changes during the development of cirrhosis. HCC in a non-cirrhotic alcoholic liver is extremely rare. Gastroesophageal reflux disease (GERD) is an additional factor, which favors carcinogenesis in the esophagus due to acid-mediated chronic inflammation of the esophageal mucosa. GERD is favored by alcohol, since alcohol decreases the tonus of the lower esophageal sphincter which facilitates GERD. Increased estrogens levels due to alcohol consumption, even in small quantities, is most likely an important pathophysiologic factor to explain the increased risk of breast cancer in regular drinkers. The mechanism by which alcohol increases estradiol levels is not known.

Cross-References ▶ Carcinogenesis ▶ Cytochrome P450 ▶ Hepatitis C Virus ▶ Reactive Oxygen Species ▶ Retinoic Acid

References Baan R, Straif K, Grosse Y et al (2007) WHO International Agency for Research on Cancer Monograph Working Group. Carcinogenicity of alcoholic beverages. Lancet Oncol 8:292–293 Bofetta P, Hashibe M (2006) Alcohol and cancer. Lancet Oncol 7:149–156 Seitz HK, Stickel F (2007) Molecular mechanisms in alcohol mediated carcinogenesis. Nat Rev Cancer 7:599–612

Alcoholic Beverages Cancer Epidemiology Vasiliou V, Zakhari S, Seitz HK, Hoek JB (eds) (2015) Biological basis of alcohol – induced cancer in advances in experimental medicine and biology 815, Springer Cham Heidelberg New York Dordrecht London Zakhari S, Vasiliou V, Gua QM (eds) (2011) Alcohol and cancer, Springer New York, Dordrecht, Heidelberg, London

See Also (2012) Acetaldehyde. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 16. doi:10.1007/978-3-642-16483-5_22 (2012) Acetaldehydehydrogenase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 16. doi:10.1007/978-3-64216483-5_23 (2012) Alcohol dehydrogenase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 120. doi:10.1007/978-3-64216483-5_6732 (2012) Alcohol-mediated cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 126. doi:10.1007/978-3-642-16483-5_170 (2012) Chronic liver disease. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 849. doi:10.1007/978-3-642-16483-5_1152 (2012) Cirrhosis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 869. doi:10.1007/978-3-642-16483-5_1184 (2012) Colorectal cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 916. doi:10.1007/978-3-642-16483-5_1265 (2012) Estrogens. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1333. doi:10.1007/978-3-642-16483-5_2019 (2012) Gastroesophageal reflux disease. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1511. doi:10.1007/978-3-642-16483-5_2334

Alcoholic Beverages Cancer Epidemiology Paolo Boffetta1 and Mia Hashibe2 1 Icahn School of Medicine at Mount Sinai, New York, NY, USA 2 University of Utah, Salt Lake City, UT, USA

Definition Alcoholic beverages are drinking beverages that contain ethanol such as wine, beer, or hard liquors.

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Other alcoholic beverage types are specific to certain geographic regions or countries such as rice wine in East Asia or arrack in India. In some cultures, alcoholic beverages are also made locally or in the home.

Characteristics A causal link has been established between alcohol drinking and cancers of the oral cavity, pharynx, esophagus, liver, and breast. For other cancers, a causal association is suspected. The importance of alcohol as a human carcinogen is often underestimated. There is increasing evidence of an important role of genetic susceptibility to alcohol-related cancer, and knowledge on possible mechanisms of the carcinogenic action of alcohol has evolved. The major nonneoplastic diseases caused by alcohol drinking are alcoholic polyneuropathy, alcoholic cardiomyopathy, alcoholic gastritis, depression and other mental disorders, hypertension, hemorrhagic stroke, liver cirrhosis and fibrosis, as well as acute and chronic pancreatitis. In addition, alcohol drinking is a major cause of several types of injuries, and alcohol consumption during pregnancy is associated with various adverse effects including fetal alcohol syndrome, spontaneous abortion, low birth weight, prematurity, and intrauterine growth retardation. On the other hand, there is strong evidence that moderate consumption of alcohol reduces the risk of ischemic heart disease, ischemic stroke, and cholelithiasis. Epidemiology of Alcohol-Related Cancer A causal relationship between elevated alcohol drinking and oral squamous cell carcinoma and that of pharynx, larynx, and esophagus have been demonstrated. In epidemiological studies of this group of tumors, an effect of heavy alcohol intake and a linear relationship with both duration and amount of drinking have been consistently shown. A synergism between alcohol drinking and tobacco smoking has been demonstrated and has become since a paradigm of interaction of two environmental factors in human carcinogenesis.

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Studies on the association of alcohol drinking and adenocarcinoma of the esophagus have not been consistent. Some studies reported risk estimates for adenocarcinoma of the esophagus and gastric cardia together on the order of 1.5- to 4fold increases in risk. Many of the studies that have reported risk estimates for adenocarcinoma of the esophagus have tended to be small, while the larger studies have reported no association with ever alcohol consumption and no indication of dose–response relations. Heavy alcohol intake increases the risk of ▶ hepatocellular carcinoma. Dose–response relations between the amount of alcohol consumed and the risk of hepatocellular carcinoma have been demonstrated. The most likely mechanism of alcohol-related liver carcinogenicity is through the development of liver cirrhosis, although alternative mechanisms such as alteration in the hepatic metabolism of carcinogens may also play a role. Alcoholic liver cirrhosis is probably the most important risk factor for hepatocellular carcinoma in populations with low prevalence of HBV and HCV infection, such as North America and northern Europe. Synergistic interactions on the risk of liver cancer are also thought to occur between tobacco and alcohol and between HBV/HCV and alcohol (hepatitis virus-associated hepatocellular carcinoma). The association between alcohol consumption and the risk of breast cancer has been reported fairly consistently in numerous studies, though the risk is thought to be moderate. The association is observed among both premenopausal and postmenopausal women, though it is unclear whether the period of life in which drinking occurs modifies the carcinogenic effect of alcohol. Although the magnitude of the excess risk of breast cancer due to alcohol drinking is not very large, the high incidence of this cancer results in a large number of cases. Several studies have provided evidence, although not fully consistent, of an association between elevated intake of alcohol and increased risk of colorectal adenoma and adenocarcinoma. Dietary factors such as low folate intake are thought to increase the risk of colorectal cancer by two- to fivefold, and alcohol adversely affects

Alcoholic Beverages Cancer Epidemiology

folate metabolism. There may be a synergistic interaction between alcohol consumption and low folate intake, or alcohol may be acting through folate metabolism to increase colorectal cancer risk. Since risk estimates reported suggest a moderate association between alcohol drinking and the risk of colorectal cancer, residual confounding by such dietary factors or other strong risk factors for colorectal cancer is of concern. However, it is doubtful that residual confounding is entirely responsible for the observed increases in colorectal cancer risk due to alcohol consumption. Though the effects may be moderate, there does appear to be a causal relationship between alcohol consumption and colorectal cancer risk. There is no consistent evidence that alcohol drinking influences the risk of cancers of the stomach, pancreas, lung, endometrium, bladder, or prostate. In the case of ovarian and kidney cancers, the evidence from epidemiological studies is of a possible protective effect, but further investigation is necessary to clarify the relationships. The risk of non-Hodgkin lymphoma was reported to be reduced among alcohol drinkers: this effect, if real, might differ by lymphoma type, which may explain the inconsistencies in results of earlier studies of alcohol and lymphoma. Mechanisms of Alcohol Carcinogenicity The mechanisms by which alcoholic beverages exert their carcinogenic effect are not fully understood, and, as in the case of other multisite carcinogen, they are likely to differ by target organ. Table 1 lists the main mechanistic hypotheses, together with a subjective assessment of the strength of the available supporting evidence. The table is restricted to mechanisms known or suspected to operate in cancers with an established association with alcohol drinking. Ethanol in its pure form does not act as a carcinogen in experimental models, and one explanation is that alcoholic beverages act as a solvent for penetration of carcinogens through the mucosa of upper aerodigestive organs. Although this mechanism would explain the synergistic effect of tobacco smoking and alcohol drinking,

Alcoholic Beverages Cancer Epidemiology Alcoholic Beverages Cancer Epidemiology, Table 1 Possible mechanisms of carcinogenicity of alcoholic beverages Mechanism Strong evidencea DNA damage by acetaldehyde Increased estrogen level Moderate evidencea Solvent for other carcinogens Production of reactive oxygen and nitrogen species Alteration of folate metabolism Weak evidencea DNA damage by ethanol Nutritional deficiencies (e.g., vitamin A) Reduced immune surveillance Carcinogenicity of constituents other than ethanol a

Potential target organs Head and neck, esophagus, liver Breast Head and neck, esophagus Liver, others? Colon and rectum, breast, others? Head and neck, esophagus, liver Head and neck, others? Liver, others? Head and neck, esophagus, liver, others?

Subjective assessment of strength of supportive evidence

it would not account for the increased risk observed among never smokers. The primary metabolite of ethanol, acetaldehyde, is a plausible candidate for the carcinogenic effect of alcoholic beverages although direct evidence linking acetaldehyde as a cause of cancer in humans is lacking. Acetaldehyde forms ▶ adducts to DNA in human cells in vitro, as well as in rats chronically exposed to ethanol. In experimental models, acetaldehyde inhalation has been shown to cause tumors of the respiratory tract, particularly adenocarcinomas and squamous cell carcinomas of the nasal mucosa in rats and laryngeal carcinomas in hamsters. It also damages hepatocytes, leading to increased proliferation. Autoantibodies against acetaldehyde-modified proteins have been detected in blood and bone marrow of alcohol abusers. Overall, studies strongly suggest that DNA damage occurs in humans following heavy alcohol consumption, and acetaldehyde can be responsible for it. The increasing evidence of a role of polymorphism in enzymes implicated in the oxidation of ethanol and acetaldehyde in

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modulating alcohol-related cancer risk further supports the hypothesis of a mechanistic role of acetaldehyde. Production of ▶ reactive oxygen species and nitrogen species is an additional possible mechanism of alcohol-related carcinogenesis. ▶ Oxidative stress leads to ▶ lipid peroxidation, whose products are reactive electrophilic compounds reacting with DNA to form exocyclic DNA adducts and reactive aldehydes. This mechanism can be particularly relevant to liver carcinogenesis and might explain the synergistic effect of alcohol and viral infection. In the liver, oxidative stress is induced by alcohol via induction of CYP2E1, stimulation of parenchymal cells in response to cytokines, and activation of Kupffer cells. Heavy alcohol intake may lead to nutritional deficiencies by reducing the intake of foods rich in micronutrients, by impairing intestinal absorption, and by altering metabolic pathways. The most relevant effect appears to be on folate metabolism, resulting in alteration in DNA ▶ methylation and, hence, control of genes potentially involved in carcinogenesis. Intake, absorption, and metabolism of vitamin B12 and vitamin B6 may also be affected by alcohol intake, resulting in further alterations of DNA methylation pathways. Vitamin A deficiency has also been proposed as alcohol-mediated carcinogenic mechanism. Alcoholics have a lower level of serum vitamin A and b-carotene, and vitamin A metabolism is altered by chronic alcohol intake. Alcohol drinking can reduce immune surveillance, thus favoring cancer development as well as metastatic potential. This hypothesis is supported by experimental data showing reduced resistance to metastasis of alcohol-exposed mice. Components of alcoholic beverages other than ethanol, including impurities and contaminants, have been proposed to increase risk of cancer among drinkers. ▶ Polycyclic aromatic hydrocarbons have been found in dark hard liquors, and N-nitrosamines have been detected in beers, but, in general, information on composition of alcoholic beverages, and in particular hard liquors, is limited. If components in alcoholic beverages represented an important factor contributing to carcinogenicity, one would predict a role of type of beverage in determining the risk.

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These mechanisms are mainly relevant to the head and neck, liver, and colorectal carcinogenesis; in the case of breast cancer, the main hypothesis to explain alcohol carcinogenicity is increased estrogen level. The evidence is strongest for postmenopausal women using ▶ hormone replacement therapy, but the available data suggest an effect also in other groups of women. Additional possible mechanisms include increased susceptibility to endogenous and exogenous carcinogens and greater invasiveness potential. An effect mediated by folate metabolism, mentioned above for colorectal cancer, would be also relevant to breast carcinogenesis.

Alcoholic Pancreatitis

References Boffetta P, Hashibe M (2006) Alcohol and cancer. Lancet Oncol 7:149–156 Boyle P, Autier P, Bartelink H et al (2003) European code against cancer and scientific justification: third version. Ann Oncol 14:973–1005 International Agency for Research on Cancer (1988) Alcohol drinking. IARC monographs on the evaluation of carcinogenic risks to humans, vol 44. IARC, Lyon Thakker KD (1998) An overview of health risks and benefits of alcohol consumption. Alcohol Clin Exp Res 22: S285–S298

Alcoholic Pancreatitis Conclusions Alcohol drinking is one of the most important known causes of human cancer, second only to tobacco smoking, chronic infections, and possibly overweight/obesity (obesity and cancer risk). With the exception of ▶ aflatoxin, for no single dietary factor, there is such a strong and consistent evidence of carcinogenicity. In the case of breast and colorectal cancer, two major human neoplasms, a causal association with alcohol drinking has been established, and the public health implications of these associations have not been not fully elucidated. In many countries, people of lower socioeconomic status or education consume more alcohol, which contributes to social inequalities in cancer burden. Given the linear dose–response relationship between intake of alcohol drinking and the risk of cancer, control of heavy drinking remains the main target for cancer control. For example, the European Code Against Cancer recommends keeping daily consumption within two drinks (about 20–30 g alcohol) for men and one drink for women. Total avoidance of alcohol, although optimal for cancer control, cannot be recommended from a broader public health perspective, in particular in countries with high incidence of cardiovascular diseases.

Cross-References ▶ Estrogenic Hormones

Dahn L. Clemens and Katrina J. Schneider Research Service, Veterans Administration Medical Center, Omaha, NE, USA

Definition Pancreatitis associated with alcohol abuse.

Characteristics The pancreas is a dual-function abdominal organ that produces both proteins that aid digestion (digestive enzymes) and hormones responsible for the regulation of sugar in the blood. These two functions are carried out by distinct populations of cells. Pancreatic digestive enzymes are produced by cells known as acinar cells. Approximately 90% of the pancreas is made up of these cells, which comprise what is known as the exocrine pancreas. Dispersed throughout the exocrine pancreas are distinct “islands” of specialized cells known as the islets of Langerhans. The islets of Langerhans are clusters of cells that produce and secrete insulin and other hormones involved in regulating the levels of sugar in the blood. The islets of Langerhans comprise what is known as the endocrine pancreas. Pancreatitis is an inflammatory disease of the exocrine pancreas that is initiated by the premature activation and intracellular release of the

Alcoholic Pancreatitis

digestive enzymes produced in acinar cells. The release of these enzymes causes destruction of acinar cells and a robust inflammatory response. Pancreatitis can be caused by a variety of factors. One of the most common factors associated with pancreatitis is alcohol abuse. Pancreatitis associated with alcohol abuse is known as alcoholic pancreatitis. Alcoholic pancreatitis has been recognized for well over 100 years, yet it remains one of the least understood alcohol-associated diseases. Pancreatitis in general, and alcoholic pancreatitis specifically, has historically been classified as either acute (of short duration) or chronic (persisting for a long time or constantly recurring). Incidence In the western world, the annual incidence of acute pancreatitis ranges from 5 to 35 per 100,000 people. It appears that the incidence of acute pancreatitis is on the rise. It is thought that this increase is the result of increased ▶ alcohol consumption combined with more sensitive, sophisticated diagnostic capabilities. In the United States alone, acute pancreatitis accounts for over 220,000 hospital admissions yearly. Acute pancreatitis can be a very painful and potentially fatal condition. The majority of episodes of acute pancreatitis are mild, self-limiting, and normally subside within 3–5 days. Unfortunately, a minority of cases of acute pancreatitis (up to 20%) result in severe clinical disease. These severe episodes are associated with considerable mortality. In developing countries, chronic alcohol abuse is the second most common factor associated with acute pancreatitis, accounting for approximately one third of reported cases. It is generally thought that acute pancreatitis can progress to chronic pancreatitis. It is unknown whether the pancreas completely heals after the initial attack of acute pancreatitis, or what circumstances lead to the progression of the disease from acute to chronic. Although it is not clear what factors are involved in the progression of acute pancreatitis to chronic pancreatitis, the progression of acute pancreatitis to chronic pancreatitis is associated with the frequency and severity of acute episodes.

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The most common factor associated with chronic pancreatitis is alcohol abuse, which is associated with approximately 70% of reported cases. Chronic pancreatitis is thought to develop after years of pancreatic ▶ inflammation. The tissue damage associated with chronic pancreatitis is thought to start prior to the onset of clinical symptoms. Because of this, the diagnosis of chronic pancreatitis is normally made after the pancreas is severely damaged and the disease is well established. Symptoms The most common symptom associated with chronic pancreatitis is severe abdominal pain. This pain is normally recurrent, much like the pain associated with acute pancreatitis, although in some cases the pain is constant and more prolonged. Initially, the pain can normally be treated with pain medication, but most patients with severe chronic pancreatitis eventually require surgery for pain relief. Many times this pain is associated with food intake. To avoid this pain, some patients do not eat properly. This can lead to weight loss and malnutrition. To complicate matters, one of the major consequences of chronic pancreatitis is impairment of the production of the digestive enzymes produced by the exocrine pancreas. This can lead to maldigestion and malabsorption of fats. Fat malabsorption results in the excretion of excessive fats in the feces (steatorrhea) and deficiencies in fat-soluble vitamins, namely vitamin A, vitamin D, vitamin E, and vitamin K. Microscopically, chronic pancreatitis is characterized by changes in the normal architecture of the pancreas. These changes include fibrotic scarring, blockage of the pancreatic ducts, atrophy and loss of acinar cells, as well as infiltration of inflammatory cells. Endocrine insufficiency may develop in the later stages of the disease resulting in diabetes. These changes are generally considered to be irreversible. Therefore, the prognosis for improvement from chronic pancreatitis is not good. Treatment Treatment for chronic pancreatitis is dependent on the specific symptoms experienced by the patient.

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In general, providing pancreatic enzymes, limiting fats in the diet, and abstinence from alcohol consumption is recommended. Additionally, stents may be inserted to bypass the blocked ducts and, as mentioned above, surgery for pain relief may also be required. Chronic pancreatitis is itself a serious condition. Furthermore, if one suffers from chronic pancreatitis the risk of developing pancreatic cancer increases 20-fold compounding the seriousness of this disease. Etiology Alcohol abuse is the major factor associated with the development of chronic pancreatitis, accounting for approximately 70% of reported cases. The mechanism(s) by which alcohol abuse induces alcoholic pancreatitis is not well understood. It has been estimated that, on average, consumption of 80 g of ethanol a day (approximately 10–11 drinks or bottles of beers) for a period of 6–12 years is required to cause clinically overt alcoholic pancreatitis. Although the risk of developing pancreatitis increases with both increased consumption and prolonged duration of alcohol abuse, only about 5% of alcoholics develop clinically detectable alcoholic pancreatitis. The fact that relatively few alcohol abusers develop alcoholic pancreatitis indicates that alcohol alone is not sufficient to cause alcoholic pancreatitis; thus, other factors are required. Although it is evident that alcohol abuse can play an important role in the development of pancreatitis, it does not appear that alcohol abuse alone can cause pancreatitis nor is it responsible for the development of this disease. Rather, it appears that ethanol alters the normal physiologic responses of pancreatic cells to injury, and environmental factors are required to actually develop alcoholic pancreatitis. A number of factors, including cigarette smoking, high lipid diet, genetics, and infections, have been suggested as possible cofactors for alcoholic pancreatitis. How alcohol abuse sensitizes the pancreas to environmental factors is not known. It has been proposed that alcohol abuse is not sufficient to cause alcoholic liver disease, but that the breakdown or metabolism of alcohol sensitizes or predisposes the liver to damage. The metabolism of alcohol causes many changes in cells. It is thought

Alcoholic Pancreatitis

that these changes interact with, or amplify the actions of, factors that normally would not cause tissue damage and that these interactions result in tissue injury. Like the liver, the pancreas possesses the ability to metabolize alcohol. Because of this, it has been suggested that alcohol metabolism also sensitizes the pancreas to damage from factors that normally would not cause clinical pancreatitis. Alcohol can be metabolized by pathways that require oxygen (oxidative) or by pathways that do not require oxygen (nonoxidative). Two proteins, alcohol dehydrogenase and ▶ cytochrome P450 2E1, primarily carry out the oxidative metabolism of ethanol. Metabolism of ethanol by either of these proteins results in the production of the intermediate acetaldehyde and the production of ▶ reactive oxygen species. Both acetaldehyde and reactive oxygen species can bind to and alter DNA and proteins, and in this manner cause damage to cells. Although the pancreas expresses both alcohol dehydrogenase and cytochrome P450 2E1, these proteins are not expressed at high levels. Because of this, the capacity for oxidative metabolism of ethanol by the pancreas is significantly less than that of the liver. Nonoxidative metabolism of ethanol is carried out by a number of enzymes, the most important being the fatty acid ethyl ester synthases. Metabolism of ethanol by these enzymes results in the formation of compounds known as fatty acid ethyl esters (FAEEs). Although the capacity for oxidative metabolism of alcohol in the pancreas is much lower than in the liver, the capacity for nonoxidative metabolism of alcohol in the pancreas is much higher because the pancreas possesses high fatty acid ester synthetic activity. Because the oxidative metabolism of ethanol in the pancreas is relatively low, the contribution of the nonoxidative metabolism of ethanol, and the production of FAEEs, may be more important in the pancreas than in the liver. In animal models of alcoholic pancreatitis, FAEEs have been shown to activate trypsin, one of the key digestive enzymes in the pancreas. FAEEs also cause alterations in acinar cells and have been shown to increase the activity of proteins that are involved in the activation of the inflammatory response in the pancreas. Additionally, it has also been shown that FAEEs can inhibit the breakdown

Aldehyde Dehydrogenases

of proteins that are involved in fibrotic scarring of the pancreas. Therefore, the production of FAEEs may have a role in initiating tissue damage, the inflammatory response, and the fibrotic scarring characteristic of chronic alcoholic pancreatitis. Even though the oxidative metabolism of alcohol may not be as prominent as the nonoxidative pathway in the pancreas that does not mean that the oxidative metabolism of ethanol has no role in the alcohol-mediated sensitization of the pancreas. Acetaldehyde, a reactive byproduct of the oxidative metabolism of ethanol, has been shown to cause some detrimental effects in the pancreas. Much like FAEEs, acetaldehyde treatment of pancreatic acinar cells has been shown to be involved in the regulation of proteins that initiate the inflammatory response. Treatment of these cells with antioxidants, compounds that neutralize reactive oxygen species, inhibits the activation of some of these proteins. Thus, reactive oxygen species also have a role in the activation of these proteins. Additionally, acetaldehyde has been shown to be capable of activating proteins that are involved in the replication of pancreatic stellate cells. Pancreatic stellate cells are the cells in the pancreas that synthesize the vast majority of the fibrotic proteins. Taken together, these results indicate that the production of acetaldehyde and reactive oxygen species may have a role in the inflammation and fibrosis associated with chronic alcoholic pancreatitis. Summary In summary, alcoholic pancreatitis is an inflammatory disease of the exocrine pancreas that can result in severe morbidity or mortality. Alcoholic pancreatitis can manifest either as acute pancreatitis or chronic pancreatitis. Although it does not appear that alcohol abuse is sufficient to cause pancreatitis, there is a very close association between alcohol abuse and pancreatitis. Alcohol abuse is the most common factor associated with chronic pancreatitis, and the second most common factor associated with acute pancreatitis. It is not known how alcohol abuse predisposes the pancreas to disease but, like the liver, the pancreas is able to metabolize ethanol. Many of the byproducts of ethanol metabolism have been shown to have detrimental effects on

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pancreatic acinar cells. It is thought that these detrimental effects may not be sufficient to themselves cause pancreatitis, but they predispose the pancreas to more severe injury from factors that may not normally cause clinical pancreatitis. Because of the lack of knowledge of the specific mechanisms by which alcohol abuse predisposes the pancreas to disease, the only current preventive measure is abstinence.

Cross-References ▶ Alcoholic Beverages Cancer Epidemiology ▶ Inflammation ▶ Inflammatory Response and Immunity ▶ Pancreatitis

Aldehyde Dehydrogenases Jan S. Moreb Department of Medicine, Division of Hematology/Oncology, College of Medicine, University of Florida, Gainesville, USA

Synonyms ALDH

Definition A group of NAD(P)+-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding acids. Nineteen forms exist in humans and they are present in all tissues. Aldehydes are abundant in nature and can be generated during normal metabolism or from metabolism of exogenous drugs and environmental substrates. Several of these enzymes are important in detoxification of anticancer drugs.

Chracteristics Aldehyde dehydrogenase (ALDH) isoenzymes are found in all cell types and play an essential

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role in the removal of toxic aldehydes as well as the production of active molecules. Aldehydes are abundant in nature and come from normal endogenous metabolism or from ingested materials or environmental sources. Examples include the removal of aldehydes produced from alcohol ingestion and toxic aldehdyes from smoke. Some ALDH isoenzymes are involved in the synthesis of retinoic acid (from Vitamin A) and purines as well as the metabolism of corticosteroids and catecholamines, which are amines derived from the amino acid tyrosine – examples include epinephrine (adrenaline), norepinephrine (noradrenaline), and dopamine – that act as ▶ hormones or neurotransmitters. Updates on all ALDH genes can be found in www.aldh.org hosted by Dr Vasilis Vasiliou’s laboratory (Black and Vasiliou 2009). Nomenclature System The ALDH isoenzymes superfamily is divided into multiple separate families and given names according to system approved by the Ninth International Workshops on Enzymology and Molecular Biology of Carbonyl Metabolism in 1998. The root symbol ALDH is followed by a number for the family, then letter to designate the subfamily, then another number following the letter to denote the individual gene within the subfamily. Genes within the subfamily should exhibit >60% amino acid identity. For example, ALDH1 family has 3 subfamilies ALDH1A, ALDH1B, and ALDH1L, and each may contain few related genes such as ALDH1A1, ALDH1A2 and ALDH1A3. ALDH Activity and Other Assays Different assays for the measurement of ALDH isozymes have been available including Western blot analysis, RT-PCR, spectrophotometric assay for enzyme activity, and immunohistochemistry. ALDH activity measurement is one of the main methods to detect the presence of ALDH isoenzymes and has become one of the identifying markers of stem cells, both normal and malignant (Moreb 2008). A relatively new flow cytometrybased method, aldefluor staining, has the advantage of measuring ALDH activity in viable cells.

Aldehyde Dehydrogenases

With the introduction and marketing of the Aldefluor assay (StemCell Technologies, Inc.), it has become more feasible to study the significance of ALDH expression in multiple cell types. ALDH Activity as a Marker for Stem Cells The hypothesis of stem cell plasticity which means that somatic stem cells can regenerate and repair different types of tissues, and that cancer behaves like an organ with its own sustaining cancer stem cells (CSC), has intensified the search for a more practical way of defining stem cell. Stemness markers or genes are badly sought after. ALDH has been known to be highly expressed in hematopoietic stem cells (HSC) for years. The use of ALDH activity as the basis of flow cytometry-based method to sort hematopoietic progenitors has opened the way to study high ALDH activity as a marker for stem cells in different tissues. This method has allowed the isolation of viable progenitors that can now be studied for their functional characteristics in vitro and in vivo. Several publications have shown the existence of ALDH positive cells in several cancers including multiple myeloma, leukemia, head and neck, lung, pancreas, colon, liver, breast, cervix, ovaries, bladder, and prostate, which possess some stem cell characteristics and ability to initiate tumors in immunodeficient mice. ALDH in Retinoic Acid Synthesis ▶ Retinoic acid (RA) is a small rapidly diffusing molecule that is essential for growth and development of the embryo. It is produced by two-step process that involves the oxidation of retinols such as vitamin A into retinaldehyde and then to retinoic acid by the ALDH1 family members. RA is involved in gene regulation and cell differentiation. Retinoic acids such as all-trans retinoic acid (ATRA) are used as differentiation agents in stem cell research and as differentiating therapy for ▶ acute promyelocytic leukemia (APL). Studies in the mouse revealed that the enzyme retinaldehyde dehydrogenase 1 (Raldh1) has similar tissue specificity and developmental control as the human ALDH1. Studies by Elizondo et al. demonstrated that mouse Raldh1

Aldehyde Dehydrogenases

transcription is under the regulation of a negative feedback mechanism (Elizondo et al. 2009). As RA levels increase, a cascade of signalling events results in inhibiting the transactivation of Raldh1 and decrease in RA synthesis. It has been shown previously that by administering ATRA, as well as 9-cis and 13-cis RA, ALDH1A1 and ALDH3A1 in human lung cancer cells are downregulated, thereby decreasing the proliferation rate and viability of cells in vitro, as well as increasing the sensitivity of various lung cancer cell lines to chemotherapy, mainly cyclophosphamide derivatives that are usually inactivated by these enzymes (Moreb et al. 2005). ALDH8A1, a cytosolic enzyme (~53.4 kDa subunits), is currently considered to be a retinaldehyde dehydrogenase (Raldh 4) that oxidizes retinaldehyde to retinoic acid. Alcohol Metabolism Most of the consumed alcohol is metabolized in the liver. The first step is converting the alcohol into acetaldehyde by alcohol dehydrogenase (ADH) and other liver P450 enzymes. The acetaldehyde is then removed by ALDH isoenzymes including ALDH1A1 and ALDH2. ALDH1A1 has been implicated in several alcohol-related phenotypes including alcoholism, alcohol-induced flushing, and alcohol sensitivity. Acetaldehyde formed during ethanol metabolism is efficiently metabolized by ALDH1A1 to nontoxic metabolites. Due to the role of ALDH1A1 in acetaldehyde metabolism, ALDH1A1 inhibition by the antialcoholic medication, disulfiram (>90%) or low ALDH1A1 activity due to polymorphisms contribute to alcohol sensitivity and alcohol-induced flushing. ALDH2 has a broad expression pattern and is most notably involved in the second step of ethanol metabolism, the oxidation of acetaldehyde. A large portion of individuals of Far East Asian descent (~40%) have a functional polymorphism in the gene encoding ALDH2 (ALDH2*2) leading to a partially inactive form of the enzyme. This results in acetaldehyde accumulation and an alcohol-induced flushing reaction, as well as an increased level of response to alcohol and lower rates of alcoholism in this population.

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ALDH and Drug Metabolism The oxazaphosphorines, ▶ alkylating agents, are a group of frequently used anticancer drugs that include cyclophosphamide (CP) and ifosphamide (IFO); however resistance to these drugs can be an obstacle to achieving cancer control. These drugs were designed to be activated by oxidase enzymes in the liver. The main active metabolites include 4-hydroxycyclophosphamide and aldophosphamide, both can be inactivated by ALDH isoenzymes thus leading to drug resistance. The main effect of CP is due to its metabolite phosphoramide mustard. This metabolite is only formed in cells that have low levels of ALDH. Stem cells and different types of cancers that express high levels of ALDH activity show resistance to oxazaphosphorines. ALDH1A1 and/or ALDH3A1 are thought to be the main contributors to such resistance. Overexpression of ALDH1A1 and ALDH3A1 in hematopoietic progenitors, leukemic cells, and other cancer cells results in resistance to 4hydroperoxycyclophosphamide (4-HC), an active derivative of CP. On the other hand, the downregulation of either one of these isoenzymes by RNA antisense, ATRA, or ▶ siRNA results in increased sensitivity of lung cancer cell lines to 4-HC. ALDH3A1 overexpression has been associated with resistance to other chemotherapeutic drugs such as ▶ mitomycin C and ▶ etoposide through ALDH3A1 mediated protection against oxidative damage. ALDH2 has been identified as a major enzyme responsible for the bioactivation of nitroglycerin, used to treat angina and heart failure (Chen and Stamler 2006). ALDH Related Diseases Many disparate aldehydes are ubiquitous in nature and are toxic at low levels because of their chemical reactivity. Thus levels of metabolicintermediate aldehydes must be carefully regulated which explains the existence of several distinct ALDH families in most studied organisms with wide constitutive tissue distribution. Indeed, many allelic variants within the ALDH gene family have been identified, resulting in pharmacogenetic heterogeneity between individuals which, in most cases, results in distinct phenotypes (Moreb 2008) including intolerance to

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alcohol and increased risk of ethanol-induced cancers (ALDH2 and ALDH1A1), Sjogren-Larsson Syndrome (ALDH3A2), type II hyperprolinemia (ALDH4A1), succinic semialdehyde dehydrogenase deficiency with 4-hydroxybutyric aciduria, mental retardation and seizures (ALDH5A1), developmental delay (ALDH6A1), hyperammonemia (ALDH18A1), pyridoxinedependent epilepsy (ALDH7A1), and late onset of Alzheimer disease (ALDH2). Changes in ALDH activity have also been observed during experimental liver and urinary bladder carcinogenesis and in a number of human tumors. Furthermore, knockouts of ALDH1A2 and ALDH1A3, catalyze the irreversible oxidation of retinal to retinoic acid, in mouse are embryonic lethal and newborn lethal, respectively. ALDH1A1 and ALDH3A1 null mice develop cataracts. ALDH5A1 knockout mice die at age of 3–4 weeks due to tonic-clonic seizures and suffer from a variety of biochemical abnormalities. ALDH2 knockout mice are more sensitive to acetaldehyde and have increased formation of DNA adducts. ALDH dysfunction could also be acquired and caused by substrate inhibition, drugs and environmental substances, as well as metabolic and oxidative stress.

ALDH Chen Z, Stamler JS (2006) Bioactivation of nitroglycerin by the mitochondrial aldehyde dehydrogenase. Trends Cardiovasc Med 16:259–265 Elizondo G, Medina-Diaz IM, Cruz R, Gonzalez FJ, Vega L (2009) Retinoic acid modulates retinaldehyde dehydrogenase 1 gene expression through the induction of GADD153-C/EBPbeta interaction. Biochem Pharmacol 77:248–257 Moreb JS (2008) Aldehyde dehydrogenase as a marker for stem cells. Curr Stem Cell Res Ther 3:237–246 Moreb JS, Gabr A, Vartikar GR, Gowda S, Zucali JR, Mohuczy D (2005) Retinoic acid down-regulates aldehyde dehydrogenase and increases cytotoxicity of 4hydroperoxycyclophosphamide and acetaldehyde. J Pharmacol Exp Ther 312:339–345

See Also (2012) Immunodeficient NUDE MICE. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1816. doi: 10.1007/978-3-642-164835_2986 (2012) Neurotransmitters. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2505. doi: 10.1007/978-3-642-16483-5_4049 Johnson BA (2015) Disulfiram. In: Stolerman IP, Price LH (ed) Encyclopedia of psychopharmacology. Springer, Berlin/Heidelberg, pp 531–534. doi: 10.1007/978-3642-36172-2_172 http://ghr.nlm.nih.gov/condition/hyperprolinemia http://ghr.nlm.nih.gov/condition/pyridoxine-dependentepilepsy http://ghr.nlm.nih.gov/condition/sjogren-larssonsyndrome http://ghr.nlm.nih.gov/condition/succinic-semialdehydedehydrogenase-deficiency

Cross-References ▶ Acute Promyelocytic Leukemia ▶ Alkylating Agents ▶ Antisense DNA Therapy ▶ Detoxification ▶ Etoposide ▶ Hepatic Ethanol Metabolism ▶ Hormones ▶ Mitomycin C ▶ Retinoic Acid ▶ SiRNA

ALDH ▶ Aldehyde Dehydrogenases

Aldo-Keto Reductases ▶ Reductases

References

ALK

Black W, Vasiliou V (2009) The aldehyde dehydrogenase gene superfamily resource center. Hum Genomics 4:136–142

▶ Activin Receptors ▶ ALK Protein

ALK Protein

ALK Protein Karen Pulford Nuffield Division of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK

Synonyms ALK; Anaplastic lymphoma kinase; CD246; Ki-1

Definition Anaplastic lymphoma kinase (ALK) is a ▶ receptor tyrosine kinase with an essential role in early neural and muscle development. ALK phosphorylates intracellular molecules for the transduction of signals from the exterior of the cell to the nucleus. Aberrant expression of full-length ALK receptor protein has been reported in ▶ neuroblastoma, rhabdomyosarcoma, and glioblastoma while the presence of ALK fusion proteins in ▶ anaplastic large cell lymphoma (ALCL) has resulted in the identification of the tumor entity ALK-positive ALCL. ALK fusion proteins have also been reported in ALK-positive diffuse large B-cell lymphoma DLBCL, a subset of non–small cell lung carcinomas (NSCLC) and a variety of other tumors. ALK is a rare example of a receptor tyrosine kinase that is expressed in both hematopoietic and nonhematopoietic tumors.

Characteristics The anaplastic lymphoma kinase (ALK) gene (HUGO approved name anaplastic lymphoma kinase (Ki-1)) was originally identified on chromosome 2 at position p23 in the t(2;5)(p23;q35) translocation associated with anaplastic large cell lymphoma. The ALK protein product is a 200 kDa ▶ Receptor tyrosine kinase protein and a member of the ▶ insulin receptor superfamily bearing significant homology to leucocyte tyrosine kinase (LTK). Other members of the insulin

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receptor subfamily include: insulin growth-1 receptor (IGF-1R), TRK neurotrophin receptors, MET, and cFOS. ALK is a highly conserved single-chain transmembrane protein of 1,620 aminoacids in the human (Fig. 1), 1,621 aminoacids in the mouse and 1,701 aminoacids in Drosophila. The ALK protein was given the designation of CD246 at the VIIth Leucocyte Typing Workshop. Full details on ALK can be obtained from the following websites: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=gene&cmd=Retrieve&dopt=Graphics&lis t_uids=238 and at http://biogps.org/#goto= genereport&id=238. Initial studies described the presence of ALK mRNA in human fetal liver, brain, testis, placenta, and the enteric innervation. Subsequent studies also identified Alk mRNA in the central and peripheral nervous system, as well as in testis, ovary, and midgut of fetal rats and mice while DAlk was detected in the brain, ventral nerve, and gut musculature of Drosophila during embryonic development. ALK homologues have also been identified in a range of other organisms e.g., chicken and C. elegans. The expression of both Alk mRNA and Alk protein decreased rapidly in rodent neonates while ALK protein was detected in only rare scattered cells in the brain in humans. Both of these findings are suggestive of a role for ALK in early neural development. The use of DAlk mutants also provided evidence for its role in the development of the ventral mesoderm in Drosophila. The ligand(s) for full-length ALK are, at present, still unknown. Possible candidates include a neurotrophic factor and the two heparin-binding growth factors pleiotrophin and midkine. There is also evidence of ALK activation via a ligand independent pathway. In vivo experiments using flies expressing loss-of-function mutant DAlk genes have identified Jelly belly protein (Jeb) as a ligand for DAlk in Drosophila. The expression of both Jeb and dAlk proteins were essential for activation of the RB protein pathway in visceral gut muscle development. In common with other receptor tyrosine kinases, binding of ligand to the extracellular receptor of ALK results in dimerization of the ALK proteins permitting the

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1

ALK Protein Extracellular domain

Cytoplasmic region

1620

Tyrosine kinase domain

1058 IRS-1 CD30 Signal sequence

Glycine rich region

Putative PTN binding sites

Transmembrane domain

LDL-A domain

Phosphorylation sites

MAM domain

Breakpoint of ALK protein for production of ALK fusion proteins

PI3-K SHC PLC-γ

ALK Protein, Fig. 1 Diagram of the human full-length ALK and ALK fusion proteins. The extracellular region contains 16 N-glycosylation sites. The presence of these increases the size of ALK from a predicted 170 kDa up to 200 kDa. Recognition sites of some of the major

intracellular interacting proteins are indicated. The arrow at aminoacid 1,058 indicates the site of cleavage of the ALK protein occurring as a result of the t(2;5)(p23q35) translocation

subsequent autophosphorylation and creation of binding sites on the intracellular regions of ALK protein for downstream signaling molecules. Interactions between full-length ALK and members of the MAP-kinase pathway, IRS-1, and c-Cbl have been identified in the differentiation of neurites. Other proteins involved in ALK signaling pathways are discussed below with reference to the ALK fusion proteins.

signaling pathways. There is, however, evidence that point mutations and amplification of ALK is a major cause of neuroblastoma.

ALK Protein and Cancer ALK is a receptor tyrosine kinase that has been implicated in the development of both nonhematopoietic as well as hematopoietic tumors. Full-Length ALK

The expression of full-length ALK protein has been reported in a number of mesenchymal tumors such as malignant peripheral nerve sheath tumors and leiomyosarcoma. ALK mRNA has also been detected in cell lines arising from rhabdomyosarcoma, neuroblastoma, melanoma, glioblastoma, and breast cancer as well as primary neural tumors such as glioblastoma and neuroblastoma. The precise role of wild type ALK protein in oncogenesis is uncertain at present although it has been implicated in the RAS/MAPK and the glycogen synthase 3/Wnt

ALK Fusion Proteins

In vivo and in vitro studies in both hematological and solid tumors have led to the conclusion that ALK fusion proteins play a primary role in tumor development. Indeed the aberrant expression of ALK fusion proteins is a marker of malignancy. Structure Translocations affecting the ALK gene result in the production and expression of chimeric ALK fusion proteins. The most common translocation is the t(2;5)(p23;q35), involving the ALK gene at 2p23 and the nucleophosmin (NPM) gene at 5q35, resulting in the expression of the NPM-ALK fusion protein. At least 16 other variant ALK fusion proteins have been identified and the most common examples are listed below in Table 1. All of these fusion proteins consist of the N-terminal of the partner proteins and the intracytoplasmic region of ALK containing the tyrosine kinase domain (Fig. 2a). With the exception of MSN-ALK and MYH9-ALK, all of the fusion proteins contain the final 563 amino acids of ALK while MSN-ALK and MYH9-ALK contain the final 567 and 566 amino acids, respectively.

ALK Protein

175

ALK Protein, Table 1 Characteristics and distribution of ALK fusion proteins Fusion protein NPM-ALK

Chromosomal translocation t(2;5)(p23;q35)

Subcellular location Nucleus, nucleolus and cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Granular cytoplasmic

Size fusion protein (kDa) 80

TPM3-ALK TFG-ALKS TFG-ALKL TFG-ALKXL ATIC-ALK CLTC-ALK

t(1;2)(p25;p23) t(2;3)(p23;q21)

MSN-ALK

Cell membraneassociated Cytoplasm Cytoplasm Nuclear periphery

125 95–105 ND 160

ALCL, IMT ALCL IMT

Cytoplasm Cytoplasm

220 130

ALCL IMT

SEC31L1-ALK EML-4

t(2;X)(p23; q11–12) t(2;19)(p23;p13.1) t(2;17)(p23;q25) t(2;2)(p23;q13) or inv(2)(p23q11–13) t(2;22)(p23;q11.2) t(2;11;2)(p23;p15; q31) t(2;4)(p23;q21) inv(2)(p21;q21)

ALCL ALCL, B cell lymphoma, IMT ALCL

Cytoplasm Cytoplasm

Not known 119–122

KIF5B-ALK

t(2;10)(p23;q22.1)

Cytoplasm

168

IMT, ALK + DLBCL NSCLC, breast and colorectal cancer NSCLC

TPM4-ALK ALO17-ALK RANBP2-ALK MYH9-ALK CARS-ALK

inv(2)(p23q35) t(2;17)(p23;q23)

104 85 97 113 96 250

Expression in tumors ALCL, B cell lymphoma ALCL, IMT ALCL

NPM nucleophosmin, TPM3 tropomyosin 3, TFG TRK-fused gene, ATIC 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), also known as PurH, CTLC clathrin heavy chain, MSN moesin, TPM4 tropomyosin 4, ALO17 unknown gene, ALK lymphoma oligomerization partner on chromosome 17, RANBP2 RAN binding protein also known as Nup358, MYH9 nonmuscle myosin heavy chain, CARS cysteinyl-tRNA synthetase enzyme, SEC31L-ALK SEC31 homologue A (Saccharomyces cerevisiae), EML4-ALK echinoderm microtubuleassociated protein-like 4, KIF5B kinesin family member 5B, ALCL anaplastic large cell lymphoma, DLBCL diffuse large B-cell lymphoma, IMT inflammatory myofibroblastic tumor, NSCLC non–small cell lung cancer

Distribution Partner proteins of 14 ALK fusion proteins all contain an oligomerization domain in their amino-region. The presence of these domains permits the formation, not only of homodimers of ALK fusion proteins, but also heterodimers of the ALK fusion protein and the normal wild type partner protein. Variations of this mechanism may occur with the MSN-ALK and MYH9-ALK proteins. The ability of the ALK-fusion proteins to dimerize results in each of the ALK fusion proteins having a characteristic subcellular distribution. NPM-ALK, for example, has a nuclear, nucleolar, and cytoplasmic localization due to the presence of NPM-ALK homodimers in the cytoplasm of the cell while the presence of a nuclear localization motif present in wild type NPM results in a nuclear and nucleolar distribution of NPM/NPM-ALK

heterodimers (Fig. 2b). Other “variant” ALK fusion proteins exhibit a variety of distribution patterns again determined by the identity of the partner protein (Table 1 and Fig. 2b). Function Another consequence of an oligomerization domain in the ALK fusion proteins is that it mimics ligand-mediated aggregation of the fulllength ALK protein with the subsequent constitutive activation of the ALK tyrosine kinase domain. This results in the aberrant activation of multiple downstream signaling pathways involved in mitogenesis and ▶ apoptosis. Examples of these pathways include the ▶ AKT signal transduction pathway, Janus kinase, and signal transducer and activator of transcription (JAK/STAT), BCL2, GRB2, JNK, FOX03A, phospholipase Cg (PLC-g), phosphatidylinositol

A

176

ALK Protein

a

Protein X

ALK Tyrosine kinase domain Protein X-ALK Tyrosine kinase domain

b

NPM-ALK

MSN-ALK

ALK Protein, Fig. 2 Structure and distribution of ALK fusion proteins. (a) The general mechanism of translocations affecting genes encoding ALK and a partner protein (Protein X). As a result of the translocation (shown by the small arrows and dotted lines), the N-terminus of Protein X is joined to the intracytoplasmic region of ALK to produce a chimeric protein, Protein X-ALK. (b)

Immunoperoxidase labeling of tissue sections from cases of anaplastic large cell lymphoma to illustrate the different subcellular distribution patterns of ALK fusion proteins. NPM-ALK is present in the nucleus, nucleolus, and cytoplasm of the tumor cells (white arrow) while MSN-ALK is present at the cell membrane (black arrow)

3-kinase (PI3K), and MAP Kinase. NPM itself may also play a role in tumor development through activation of p53. Proteomics-based studies have confirmed the complexity of NPM-ALK signaling pathways in cell proliferation, cellular structure and migration, protein synthesis and the ability of cells to evade apoptosis. Proteins identified in this way include additional adaptor molecules (suppressors of cytokine signaling, ▶ Rho family proteins, and RAB35), kinases (such as MEK kinase 1 and protein kinase C), and phosphatases (meprin, PTPK, and protein phosphatase 2 subunit). One potential role of the ALK fusion proteins in oncogenesis is the relocation of interacting proteins away from their normal site of activity within the cell. FOX03A, for example, is redirected to the cytoplasm rather than to the nucleus. Further studies are, however, necessary to understand fully the mechanisms employed by ALK proteins in cell proliferation, differentiation, and survival in both normal and disease states.

Tumor Types Although representing only 5% of non-Hodgkin lymphomas (NHL), ALK-positive anaplastic large cell lymphoma constitutes 40% of pediatric large cell tumors. These CD30-positive tumors are of T- and null-cell phenotype. NPM-ALK is the most common ALK fusion protein being expressed in 60–80% of the cases, TPM3-ALK is detected in about 15% of cases, while CLTC-ALK fusion proteins are present in approximately 8% of tumors. The other ALK fusion proteins are present in the remaining 2% of ALK-positive lymphomas. The differential diagnosis of ALK-positive anaplastic large cell lymphoma is important since these lymphomas are associated with a good prognosis with an overall 5-year survival of 71–80% compared to only 15–46% for ALK-negative anaplastic large cell lymphoma. ALK fusion proteins may also be implicated in the development of other tumors. NPM-ALK and CLTC-ALK have been reported in a small subset

ALK Protein

of CD30-negative B cell lymphoma, while TPM3-ALK, TPM4-ALK, RanBP2-ALK, and CLTC-ALK fusion proteins have been identified in inflammatory myofibroblastic tumors. Importantly, the oncogenic EML4-ALK and KIFB5ALK fusion proteins have also been described in a significant subset (3–7%) of NSCLC. Evidence for the presence of ALK fusion proteins in other tumors is also increasing, e.g., in breast and renal cell carcinomas. Therapeutic Options

Current treatments for ALK-positive lymphomas include the use of various combination chemotherapy protocols originally developed for T-cell lymphoblastic tumors and high-grade B-cell non-Hodgkin lymphomas. Autologous and allogeneic stem cell transplantation techniques have also been utilized. However, 20–30% of patients fail to respond to current treatment regimens, and so improved therapeutic options still continue to be sought. One approach is to use ALK as a specific target through the use of ALK specific tyrosine kinase inhibitors (a paradigm is the ABL kinase inhibitor imatinib mesylate or Gleevec used in chronic myeloid leukemia). Crizotinib, a small molecule tyrosine kinase inhibitor, has been approved by the Food and Drugs Administration (FDA) for use in lung cancer and its efficacy in ALK-positive lymphoma is under investigation. Recognition of ALK as an immunogenic tumor-associated antigen has also highlighted its use as a potential target for ▶ immunotherapy, either via antibody-based therapies for treatment of tumors expressing fulllength ALK protein or through the use of T-cell mediated immunity in the case of tumors bearing intracellular ALK fusion proteins. Another avenue that has shown promise is the use of small molecule inhibitors affecting proteins involved in the ALK signaling pathways; examples of this include the ansamycin class of natural ▶ HSP90 inhibitors. In conclusion, the ALK receptor tyrosine kinase and ALK fusion proteins have been implicated in a diverse range of cellular functions. However, despite major advances, in depth analysis of the signaling pathways is necessary to

177

unravel the full role of this RTK in both normal and neoplastic cells and tissues.

A Cross-References ▶ AKT Signal Transduction Pathway ▶ Anaplastic Large Cell Lymphoma ▶ Apoptosis ▶ Hsp90 ▶ Immunotherapy ▶ Insulin Receptor ▶ Insulin-Like Growth Factors ▶ MYB ▶ Neuroblastoma ▶ RANK–RANKL Signaling ▶ Receptor Tyrosine Kinases ▶ Rho Family Proteins

References Chiarle R, Martineo C, Mastini C et al (2008) The anaplastic lymphoma kinase is an effective oncoantigen for lymphoma vaccination. Nat Med 14:676–680 Delsol G, Jaffe E, Falini B et al (2008) Anaplastic large cell lymphoma (ALCL), ALK-positive. In: Swerdlow SH, Campo E, Harris NL et al (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. International Agency for Research on Cancer, Lyon, pp 312–316 Duyster J, Bai RY, Morris SW (2001) Translocations involving anaplastic lymphoma kinase. Oncogene 20:5623–5637 Hallberg B, Palmer RH (2013) Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer 10:685–700 Janoueix-Lerosey I, Lequin D, Brugieres L et al (2009) Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455:967–970 Pulford K (2014) ALK anaplastic lymphoma kinase. In: Gelman EP, Sawyers CL, Rauscher RJ III (eds) Molecular oncology – Causes of cancer and targets for treatment. Cambridge University Press, New York, pp 162–189

See Also (2012) ALK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 128. doi:10.1007/978-3-642-16483-5_178 (2012) AML-1/ETO/CBFβ/TEL in chromosomal translocations. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 157. doi:10.1007/ 978-3-642-16483-5_232

178 (2012) CBP/p300. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 684. doi:10.1007/978-3-642-16483-5_898 (2012) Clathrin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 880. doi:10.1007/978-3-642-16483-5_1207 (2012) CTL. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1012. doi:10.1007/978-3-642-16483-5_1406 (2012) FOXO 3A. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1447. doi:10.1007/978-3-642-16483-5_2257 (2012) Glioblastoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1554. doi:10.1007/978-3-642-16483-5_2421 (2012) MSC. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2383. doi:10.1007/978-3-642-16483-5_3859 (2012) Non-Hodgkin lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4110 (2012) NPM. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2565. doi:10.1007/978-3-642-16483-5_4133 (2012) NPM-ALK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2565–2566. doi:10.1007/978-3-642-16483-5_4134 (2012) Tropomyosin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3785. doi:10.1007/978-3-642-16483-5_5991

Alkylating Agents Lakshmaiah Sreerama Department of Chemistry and Biochemistry, St. Cloud State University, St. Cloud, MN, USA Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar

Definition Alkylating agents (al-ka-LAYT-ing AY-jints) are a family of anticancer drugs that interfere with cell’s DNA and inhibit cancer cell growth. They are so named because of their ability to add alkyl groups to negatively charged groups on biological molecules such as DNA and proteins. Alkylating agents are among the first group of chemicals determined to be useful in cancer ▶ chemotherapy. They remain to be the most important

Alkylating Agents

components of modern chemotherapeutic protocols (individually or in combination with other drugs) because of their proved and significant clinical anticancer activities.

Characteristics Discovery of alkylating agents as anticancer drugs has its origin in the use of sulfur mustard gas for warfare during World War I. Sulfur mustard gas was not only fatal but it also showed ▶ myelosuppression/immunosuppression in its victims as well as in animal models. The latter observation led to the development of less volatile mustargen (mechlorethamine) with strong antitumor activity against lymphomas and other cancers. Eventually mustargen (nitrogen mustard) was developed for clinical use to treat ▶ Hodgkin disease. Following the discovery of mustargen, less toxic and more clinically effective nitrogen mustard derivatives, e.g., ▶ cyclophosphamide, and other alkylating agents in clinical use today were developed (Table 1). Cyclophosphamide is a bifunctional nitrogen mustard that is a most commonly used drug in combination chemotherapy and is a DNA ▶ alkylating agent that is used as an immunosuppressive drug. It acts by killing rapidly dividing cells. Alkylating agents, as suggested by their names contain reactive alkyl groups. An alkyl is an univalent reactive group containing only carbon and hydrogen atoms arranged in a chain with a general formula of Cn H2n+1, e.g., methyl, CH3 (derived from methane) and butyl C4H9 (derived from butane). Alkylating agents used as anticancer drugs are cable of reacting with biological molecules such as DNA and proteins, and disrupt cellular function by either killing the cell or by preventing its growth. The most common biological functional moiety alkylated by these compounds is guanine, a nucleobase. The anticancer activities of alkylating agents are caused in two ways: (i) through cross-linking two different DNA strands via the reaction with guanine nucleobases present on the opposing strands of DNA and (ii) preventing/affecting the activities of critical DNA processing enzymes and thereby

Alkylating Agents

179

Alkylating Agents, Table 1 Classification of clinically used alkylating agents Class Nitrogen mustards

Aziridines and epoxides Alkyl sulfonates Nitrosoureas

Hydrazine and triazine derivatives

Clinically used agents Cyclophosphamide Ifosfamide 4-Hydroxycyclophosphamide Mafosfamide Melphalan Chlorambucil Thiotepa Mitomycin C Dianhydrogalactitol Busulfan BCNU [N,N0 -bis(2-chloroethyl)N-nitrosourea] CCNU [N-(2-chloroethyl)N0 -cyclohexyl-N-nitrosourea] MeCCNU [N-(2-chloroethyl)-N0 (4-methylcyclohexyl)N-nitrosourea] Procarbazine Dacarbazine Temozolomide

stimulating apoptosis via the reaction with guanine nucleobases on a single DNA strand. The cross-linking of DNA makes it impossible to uncoil DNA during cell division thus preventing its growth. Based on the reactivity, alkylating agents are of two types: (i) monofunctional (monoalkylating – alkylate nucleobases on one DNA strand); and (ii) bifunctional (dialkylating – alkylate nucleobases on both DNA strands and cross-link them). Classification Alkylating agents currently used as anticancer drugs are divided into five major classes. The examples of the clinically used agents (most common) under each of these classes and their clinical utility are shown in Table 1. Mechanism of Action Alkylating agents are a diverse group of chemical compounds with a common characteristic of forming positively charged (electrophilic – electron poor) alkyl groups in

Cancer/other disease treated ▶ Breast cancers, most lymphomas, and ▶ childhood cancers High dose therapies in conjunction with bone marrow transplantation Multiple myeloma, melanoma, and sarcomas B-cell chronic lymphocytic leukemia and immunosuppressive therapy for autoimmune diseases Breast, ovarian, and ▶ bladder cancers ▶ Esophageal, breast, and bladder cancers Breast, cervical, and brain cancers Bone marrow transplantation for chronic myelogenous leukemia ▶ Brain tumors (glioma, glioblastoma, medulloblastoma, and astrocytoma), multiple myeloma, and lymphoma

Hodgkin lymphoma and certain brain cancers such as glioblastoma multiforme astrocytoma, and ▶ melanoma

aqueous solutions under physiological conditions. The positively charged alkyl groups are capable of reacting with basic/negatively charged (nucleophilic – electron rich) groups present in DNA and proteins/peptides. Such reactions lead to adding alkyl groups at oxygen, nitrogen, phosphorous, or sulfur atoms (nucleophilic centers), thus altering the biological function of DNA and proteins. The most important reaction of alkylating agents with regard to their antitumor activity is their reactions with DNA nucleobases. The most preferred DNA nucleobase for alkylation is guanine and the alkylation preferentially occurs at N7 position on guanine (Fig. 1). Other nucleobases alkylated and the atomic positions at which alkylation occurs in order of preference include N1 and O6 positions of guanine; N1, N3, and N7 positions on adenine; N3 position on cytosine; and O4 position of thymidine. DNA Cross-Links Various techniques used to elucidate the reactions of alkylating agents with DNA and the possible

A

180

Alkylating Agents

Guanine base O N R = Deoxyribose residue

N

NH N

NH2

Cl N

R Cl N Cl

+

−Cl

O N

Cl

N N

G N

NH NH2

R

Nitrogen mustard (Mechlorethamine)

Alkylated guanine Alkylating Agents, Fig. 1 Reaction between nitrogen mustard and guanylate residue on DNA at N7 position of guanine

basis for their anticancer activities has led to identifying at least four different types of ▶ DNA adducts (DNA cross-links) (Fig. 2). Nitrogen mustard (mustargen) and its derivatives, e.g., cyclophosphamide, as well as alkylsulfonates, e.g., busulfan, produce interstrand cross-links in -G-X-C/C-X-G- configuration of DNA double helix in greater frequency. The cross-link involves the N7 atoms of the guanylates in the -G-X-C/C-X-G- configuration of the DNA double helix (cross-link 1; Fig. 2). Aziridine and epoxide alkylating agents produce DNA cross-links in -G-C/C-G- configuration of DNA. Agents such as thiotepa and dianhydrogalactitol in this class drugs react with N7 position of the guanylate groups. Whereas mitomycin C reacts with the extracyclic N2 atom of the amino group in guanylates (cross-link 2, Fig. 2). Nitrosoureas such as BCNU produce DNA cross-links between a guanine and a cytidine in a -G/C- base-pair configuration of the DNA double helix (cross-link 3; Fig. 2). Hydrazine and triazine derivatives such as procarbazine, dacarbazine, and temozolomide decompose to produce a methyl diazonium ion which in turn will methylate guanines on DNA at O6 position (cross-link 4, Fig. 2). Other types of guanylate-alkyl cross-links of type 4, e.g., O6-ethylguanine and O6-benzylguanine, have also been observed.

Molecular Pharmacology, Drug Resistance, and Clinical Efficacy Metabolism

Alkylating agents are strong electrophiles and react with many biological nucleophiles within the tumor cells. Many of these reactions result in inactivation/detoxification of alkylating agents and thus lead to drug resistance. The most abundant and principal nucleophile in the cell is glutathione (GSH – concentrations in mM levels). The cysteine sulfhydryl (nucleophile) reacts with alkylating agents both in enzyme and no-enzyme catalyzed reactions resulting in glutathione conjugates. The glutathione conjugates of alkylating agents are generally nontoxic. The enzyme catalyzed conjugation of alkylating agents to GSH is catalyzed by ▶ glutathione S-transferases (GSTs). Tumor cells resistant to alkylating agents commonly have increased levels of GSTs. Inhibitors of GSTs such as sulfasalazine and inhibitors of gamma-glutamylcysteine synthase (a rate limiting enzyme in the synthesis of GSH) such as buthionine sulfoximine have been shown to reverse the resistance originating due to elevated levels of GSH in both in vitro and in vivo settings. GSH conjugates of some alkylating agents, e.g.,melphalan and chlorambucil, are good substrates for absorption (membrane transporter

Alkylating Agents

181 DNA

Alkylating Agents, Fig. 2 Schematic representation of alkylation (interstrand cross-links and O-alkylation) of DNA by alkylating agents

5'

3'

A

T

G

C

C

1

G

C

G

T

A C

G 2 C

G

A

T

G

4

3

C

C

G

T

A

G

C

A

T

3'

5'

multidrug resistance proteins, MDR, ▶ P-glycoprotein), and modulation of these enzyme systems is also believed to improve clinical efficacy of alkylating agents. Thiol groups in metallothionein enzymes have been shown to sequester alkylating agents such as chlorambucil, melphalan, and phosphoramide mustard (activated cyclophosphamide) and cause resistance. This has been proved by transfection and overexpression, as well as induced expression of genes coding of metallothioneins in tumor cells. Modulation of this enzyme system is also expected to increase the efficacy of alkylating agents. Cyclophosphamide and its analogs (nitrogen mustard derivatives) are prodrugs and undergo extensive metabolism. During their metabolism three aldehyde intermediates, viz., aldophosphamide, acrolein, and chloroacetaldehyde are formed. Although all three aldehydes are toxic to cells, the pivotal aldehyde

A 1 = G-X-C / C-X-G Interstrand crosslink caused by nitrogen mustards. e.g., cyclophosphamide

2 = G-C / C-G Interstrand crosslink caused by azridines and epoxides, e.g. mitomycin C 3=G/C Interstrand crosslink caused by nitrosoureas, e.g. BCNU

4 = O6-Alkylation or Methylation caused by hydrazine and triazine derivatives, e.g., Procarbazine

metabolite of the three is aldophosphamide as it gives rise to the DNA alkylating mustard that is finally responsible for the anticancer activity of these agents. ▶ Aldehyde dehydrogenases catalyze NAD-dependent oxidation of aldehydes in tumor cells. These enzymes have also been shown to oxidize aldophosphamide and cause resistance to cyclophosphamide and its derivatives in various tumor cell models in both in vitro and in vivo settings. Inhibitors of aldehyde dehydrogenases have been shown to reverse resistance to cyclophosphamide and its analogs, as well as increase their efficacy in vitro. Relatively large concentrations of aldehyde dehydrogenases are naturally present in critical normal cells such as bone marrow stem cells, intestinal progenitor cells, and the liver cells. Accordingly, these normal cells are protected from toxicities due to cyclophosphamide and its analogs. The main mechanism by which alkylating agents present their anticancer properties is via

182

alkylation of DNA. Alkylation further leads to the formation of various DNA adducts (Fig. 2) which in turn are responsible for the inhibition of tumor cell growth. Removal of such adducts is yet another mechanism by which tumor cells become resistant to alkylating agents. O6-Alkylguanine-alkyltransferase has been shown to remove alkyl groups from the O6 position of guanine. This process leads to alkylation of the enzyme alkyltransferase and the alkylated enzyme is rapidly degraded. This mechanism has been shown to be very effective against DNA methylating agents such as procarbazine and temozolomide. The same enzyme has also been shown to remove other alkyl and aryl groups, e.g., dealkylation of O6-ethylguanine and debenzylation of O6-benzyguanine. Inhibitors of O6-alkylguanine-alkyltransferase have been successfully used to prevent resistance to certain clinically used alkylating agents, e.g., BCNU. DNA cross-links of type 1–3 (Fig. 2) have been shown to be removed via ▶ nucleotide excision repair and poly(adenosine diphosphate-ribose) polymerase pathways; however, the exact mechanism by which this is achieved is not clear.

Alkylating Agents

4.

5.

6.

7. Toxicology The most common toxicities associated with administering alkylating agents to treat cancers are as follows. 8. 1. Hematopoietic toxicity – In general, the clinical dose-limiting toxicity for alkylating agents is hematopoietic toxicity, particularly suppression of granulocytes and platelets exhibited for 8–16 days after treatment. The toxicity usually disappears after 20 days and granulocytes and platelets return to their normal levels. 2. Gastrointestinal toxicity (nausea and vomiting) – Damage to the gastrointestinal tract is a toxicity that frequently occurs with high-dose regimens of alkylating agents. These toxicities are characterized by mucositis, stomatitis, esophagitis, and diarrhea. This toxicity can be managed by administering corticosteroids and antiemetics. 3. Gonadal toxicity – Treatments with alkylating agents have been shown to cause testicular

lesions leading to depletion of sperm in male patients and decrease in ovarian follicles in female patients. Pulmonary toxicity – Pulmonary toxicities characterized by interstitial pneumonitis and fibrosis leading to dyspnea and nonproductive cough that may lead to cyanosis, pulmonary insufficiency, and death have also been observed in patients treated with alkylating agents. Alopecia – Although the association between alkylating agents and alopecia was first described with busulfan therapy, this toxicity is predominantly associated with cyclophosphamide and ifosfamide therapy. Alopecia is caused by introduction of nicks in the hair fibers due the temporary stoppage in synthesis of hair in hair follicles by alkylating agents. Teratogenicity – All therapeutically used alkylating agents cause teratogenicity (developmental defects) in animal models. Fetal malformations have been observed in mothers receiving alkylating agents in the first trimester of pregnancy but not second and third trimesters. Carcinogenicity – Reports of the incidence of leukemia and increased frequency of incidence of solid tumors have been reported in patients receiving therapies that include alkylating agents. Immunosuppression – Alkylating agents have been shown to inhibit antibody production. All alkylating agents produce some degree of immunosuppression; however severe immunosuppression is caused by cyclophosphamide and its analogs, and chlorambucil. Accordingly, therapies that include high-dose cyclophosphamide or chlorambucil without bone marrow transplantation are now being used to treat some autoimmune diseases.

Cross-References ▶ Acute Myeloid Leukemia ▶ Adducts to DNA ▶ Aldehyde Dehydrogenases ▶ Alkylating Agents

Allergic Asthma

▶ Bladder Cancer ▶ Brain Tumors ▶ Breast Cancer ▶ Chemotherapy ▶ Childhood Cancer ▶ Cisplatin ▶ Cyclophosphamide ▶ Esophageal Cancer ▶ Glutathione S-Transferase ▶ Hodgkin Disease ▶ Mitomycin C ▶ Myelosuppression ▶ Nucleotide Excision Repair ▶ P-Glycoprotein ▶ Toxicological Carcinogenesis

183 (2012) Metallothionein enzymes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2259. doi:10.1007/978-3-642-164835_3667 (2012) Nitrogen mustards. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2528. doi:10.1007/978-3-642-16483-5_4092 (2012) O6-alkylguanine-alkyltransferase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2595. doi:10.1007/978-3-642-164835_4182

ALL ▶ Acute Lymphoblastic Leukemia

References

Allele Imbalance Berger NA (1996) Alkylating agents. Cancer Chemother Biol Response Modif 16:28–38 Colvin OM, Friedman HS (2005) Alkylating agents. In: DeVita VT, Hellman S, Rosenberg SA (eds) Cancer: principles and practice of oncology. Lippincott Williams & Wilkins, New York, pp 332–344 Sladek NE (1994) Metabolism and pharmacokinetic behavior of cyclophosphamide and related oxazaphosphorines. In: Powis G (ed) Anticancer drugs: reactive metabolism and drug interactions. Pergamon, Oxford, pp 79–156

Definition Alteration of the normal 1:1 ratio of the two alleles at a given genetic locus. The altered ratio can be secondary to increased copy number of one allele (due to amplification or aneuploidy) or decreased copy number of one allele (also known as loss of heterozygosity).

See Also (2012) Combination chemotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 952. doi:10.1007/978-3-64216483-5_6902 (2012) DNA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1129. doi:10.1007/978-3-642-16483-5_1663 (2012) Gamma-glutamylcysteine synthetase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1494. doi:10.1007/978-3-64216483-5_2314 (2012) Glioblastoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1554. doi:10.1007/978-3-642-16483-5_2421 (2012) Glutathione. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1559. doi:10.1007/978-3-642-16483-5_2438 (2012) Intestinal absorption. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1900. doi:10.1007/978-3-642-16483-5_3114 (2012) Leukemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2005. doi:10.1007/978-3-642-16483-5_3322

See Also (2012) Loss of heterozygosity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2075–2076. doi: 10.1007/978-3642-16483-5_3415

Allelic Association ▶ Linkage Disequilibrium

Allergic Asthma ▶ Allergy

A

184

Allergic Conjunctivitis

Allergic Conjunctivitis

IgE-mediated hypersensitivity

hypersensitivity;

Type-1

▶ Allergy

Definition

Allergic Rhinitis ▶ Allergy

Allergy Erika Jensen-Jarolim1,2, Sophia N. Karagiannis3,4 and Michelle C. Turner5,6,7,8 1 Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria 2 The Interuniversity Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria 3 St. John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences and Medicine, King’s College London, London, UK 4 NIHR Biomedical Research Centre at Guy’s and St. Thomas’ Hospitals, Guy’s Hospital, King’s College London, London, UK 5 McLaughlin Centre for Population Health Risk Assessment, University of Ottawa, Ottawa, ON, Canada 6 ISGlobal, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain 7 Universitat Pompeu Fabra (UPF), Barcelona, Spain 8 CIBER Epidemiología y Salud Pública (CIBERESP), Madrid, Spain

Synonyms Allergic asthma; Allergic conjunctivitis; Allergic rhinitis; Asthma; Atopic dermatitis; Atopy; Eczema; Hay fever; Hypersensitivity;

This field of study refers to the evaluation of the association between allergy and cancer, specifically the role of allergy-related so-called Th2 immune responses and IgE immunoglobulins in cancer. Whereas previously the main hypothesis of a positive history of allergy as a protective factor in cancer development was predominant, the new field of AllergoOncology evaluates all opportunities, but also potentially negative effects due to biological Th2-type mechanisms.

Characteristics Immunoglobulin E (IgE) is expressed in mammalians, but its overall biological role and the apparent increase in IgE-mediated allergies has so far not been fully understood. In approximately 30% of the population globally, the encounter and uptake of actually harmless environmental, insect, or food allergens leads to IgE formation, which is fixed to inflammatory cells in skin and mucosa. Upon allergen re-exposure, a cascade of events occurs leading to an immediate, followed by a late inflammatory response. Additionally, atopic patients have an inherited predisposition to produce even higher total and allergenspecific levels of IgE. The overwhelming IgE and Th2 dominance in allergics can only be counteracted by allergen immunotherapy, typically rendering immunomodulation that is characterized by a Th1 shift, appearance of T-regulatory cells, and interestingly increasing IgG4 antibody levels. Soon after the discovery of IgE as major player in specific hypersensitivity reactions in 1966–1967, the possible function of IgE in cancer was approached. Although atopic disorders have been commonly assumed to offer little benefit to the individual, it has been hypothesized that this atypical, Th2-dominated, immune

Allergy

response may in fact represent a state of enhanced immune function of possible relevance for cancer etiology. The immune system has been recognized as playing an essential role in cancer development, evidenced by the fact that immunosuppressed or immunodeficient individuals tend to display higher rates of certain types of malignancies. Immune surveillance theory suggests the ongoing search for and eradication of cancer cells by the immune system as a self-protective mechanism against cancer development. Traditionally, it has been the Th-1 immune response, however, that has been thought to play a predominant role. Today it is accepted that the sum of all immune versus tolerance mechanisms in cancer determines the progression of disease. In this context, the great relevance of infiltrating T- and B-regulatory cells has been recognized, and publications report that IgG4 levels correlate positively with progression of melanoma. Immunologically, allergy and oncology are thus truly complementary fields. Whereas tolerance is desired against allergens, it is detrimental in cancer; whereas IgE is detrimental in allergy, it may be harnessed against cancer antigens. It is crucial that in AllergoOncology studies, all possible facets are considered to foster both fields and result in clinically relevant recommendations. Epidemiological Studies The association between allergy and cancer has been examined in numerous epidemiological studies conducted over the past several decades. Inverse associations between allergy and cancer have been reported overall and at specific sites including pancreatic cancer, brain cancer (glioma), and childhood leukemia as reported in several meta-analyses ranging from approximately 20% to 45% reductions in risk associated with histories of allergy or specific allergic conditions (Pancreas cancer, basic and clinical parameters; brain tumors; childhood cancer; leukemia). However, potential methodological limitations remain, including in the assessment of lifetime history of allergic disorders, and results at other cancer sites are mixed.

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The majority of previous studies have relied on self-reported history of specific allergic disorders as an indicator of allergic status. Results from a large prospective cohort study revealed a significant 12% reduction in cancer mortality overall and 24% reduction in colorectal cancer mortality specifically associated with a history of selfreported physician-diagnosed asthma and hay fever at enrolment. Results for total cancer mortality attenuated somewhat when considering never smoking participants only. Studies have examined various biomarkers of allergic status including IgE. There were inverse associations between levels of both total or specific IgE antibodies and subsequent cancer risk in several studies including studies examining cancer risk overall as well as at specific sites including glioma, melanoma, female breast, and gynecological cancers though further research is required to confirm these findings. There are also studies of cohorts of skin prick tested patients, though no clear associations were observed. There was an inverse trend between both absolute and relative eosinophil count and subsequent colorectal cancer risk in one study with an approximately 40% reduction in risk in the highest tertile category. There are also several studies based on linkage of administrative data and hospital discharge records with one study reporting an inverse association between an allergy/atopy history of at least 10 years and brain cancer risk. Several studies have also reported various associations between allergy-related genetic polymorphisms and glioma risk which require confirmation. There is also concern surrounding potential increased cancer risk in asthmatics treated with anti-IgE therapy; large cohort, long-term followup studies are however still required to ascertain a link between anti-IgE therapy and the development of cancer in this patient group. Further research is needed to clarify these findings and to investigate possible roles for IgE and IgE-mediated immune responses in protection from carcinogenesis and in cancer therapy. Biological Details The atopic immune response involves antigen/ allergen presentation, and activation of CD4+

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Th2 T cells, associated with section of cytokines such as IL-4 and IL-13. These lead to class switching and production of allergen-specific IgE by affinity-matured and clonally expanded B lymphocytes. Upon allergen exposure, IgE-allergen complexes bound to Fce receptors on mast cells trigger the release of a number of factors including histamine, leukotrienes, and chemotactic factors among others. The resulting response initially involves smooth muscle contractions, mucus secretion, vasodilation, and a loss of microvascular integrity which is then followed by the infiltration and activation of eosinophils, neutrophils, Th2-type CD4+ T cells, and macrophages at the sites of allergen challenge. Both IgE cell surface receptors, FceRI and CD23, which are known to be upregulated by IgE and Th2 environments, are involved in such allergic inflammatory processes. The defined multidisciplinary field of AllergoOncology seeks to understand the role of IgE and Th2 immunity and IgE-mediated immune responses in cancer prevention, cancer development, and treatment. Immune effector cells such as macrophages, dendritic cells, CD4+ T cells, B cells, and mast cells infiltrate tumor lesions and tumor-associated inflammatory signals appear to share features with Th2 immune responses. These signals are thought to be immunosuppressive and render host immune effector cells less effective in mounting antitumor responses. Studies demonstrate that tumorassociated inflammatory environments provide an alternative Th2-biased cytokine milieu which may divert cancer patient B cells away from production of IgE and in favor of expressing less immune activatory antibody isotypes such as IgG4. These alternative Th2 conditions may be associated with worse clinical prognosis in patients with cancer. The findings point to a tumor-associated immunological bias which does not favor IgE and support the notion that IgE antibody production may not be compatible with the growth of tumors. Indeed, results from in vitro studies and studies in animal models have revealed that IgE antibodies engineered to

Allogeneic Bone Marrow Transplantation

recognize cancer antigens or triggered by specific active immunization approaches can be effective antitumor agents. Eosinophils, dendritic cells, monocytes, and macrophages have been shown to become activated in response to IgE engagement through its Fc domain. These processes have been shown to be associated with reduced tumor growth or protection from tumor challenge in preclinical models. Therefore, the concept of recruiting and activating immune effector cells with IgE antibodies is gaining substantial ground as a potential tumor inhibition strategy, which may be able to overcome tumor-induced immunosuppressive signals. Translation of these strategies and their relevance treating patients with cancer is awaited.

References Jensen-Jarolim E, Achatz G, Turner MC, Karagiannis S, Legrand F, Capron M et al (2008) AllergoOncology: combat cancer with IgE antibodies. Allergy 63:1255–1266 Karagiannis SN, Josephs DH, Karagiannis P, Gilbert AE, Saul L, Rudman SM et al (2012) Recombinant IgE antibodies for passive immunotherapy of solid tumors: from concept towards clinical application. Cancer Immunol Immunother 61:1547–1564 Karagiannis P, Gilbert AE, Josephs DH, Ali N, Dodey T, Saul L et al (2013) IgG4 subclass antibodies impair antitumor immunity in melanoma. J Clin Invest 123:1457–1474 Turner MC (2012) Epidemiology: allergy history, IgE, and cancer. Cancer Immunol Immunother 61:1493–1510 Wulaningsih W, Holmberg L, Garmo H, Karagiannis SN, Ahlstedt S, Malmstrom H et al (2016) Investigating the association between allergen-specific immunoglobulin E, cancer risk and survival. Oncoimmunology (in press). doi:10.1080/ 2162402X.2016

Allogeneic Bone Marrow Transplantation ▶ Allogeneic Cell Therapy

Allogeneic Cell Therapy

Allogeneic Cell Therapy Wolfgang Herr Universitätsklinikum Regensburg, Regensburg, Germany

Synonyms Allogeneic bone marrow transplantation; Allogeneic cellular immunotherapy; Allogeneic hematopoietic stem cell transplantation

Definition Allogeneic cell therapy consists of chemoradiotherapeutic conditioning therapy followed by transplantation of hematopoietic stem cells and lymphocytes isolated from allogeneic healthy donors to generate an effective graftversus-malignancy immune response in patients with treatment-refractory malignant disorders.

Characteristics Rationale Allogeneic hematopoietic stem cell transplantation (HSCT) aims to break autologous immunotolerance toward malignant cells in tumor-bearing patients. The treatment approach is based on the alloreactive graft-versus-malignancy effect that is mainly mediated by T cells of donor origin. These donor T cells are infused together with allogeneic hematopoietic stem cells (HSC) at the time of transplantation or originate from donor HSC in the patient thereafter. Allogeneic HSCT is capable of inducing long-term disease control in patients with chemotherapy-refractory leukemias and other ▶ hematological malignancies. Procedure Allogeneic HSCT requires chemoradiotherapeutic conditioning therapy to allow the

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engraftment of subsequently infused allogeneic HSC and lymphocytes of donor origin. Preclinical as well as clinical research has demonstrated that the long-term leukemia control following allogeneic HSCT depends on the immunological graftversus-leukemia (GVL) effect rather than on the intensity of the pre-transplant ▶ chemoradiotherapy. This important observation led to a change of paradigm, shifting the antileukemic effect of the allotransplantation procedure from the preparative cytostatic drugs to alloreactive immune effector cells. Consequently, reduced-intensity conditioning (RIC) regimens were developed that do not irreversibly destroy recipient hematopoiesis, but are sufficiently immunosuppressive to permit the engraftment of allogeneic HSC. This results in an initial coexistence of donor and recipient hematopoiesis (“mixed hematopoietic chimerism”) that can be gradually shifted to complete hematopoietic donor chimerism by modulating the posttransplant immune system using immunosuppressive agents or donor lymphocyte infusions (DLI). The majority of these non-myeloablative RIC regimens are combinations of 1–3 different chemotherapeutic drugs and low-dose total body irradiation. Compared to conventional myeloablative conditioning protocols based on high-dose radiochemotherapies, RIC regimens carry a much lower treatment-related morbidity and mortality allowing the use of allogeneic cell therapy in patients until 60–70 years of age or in patients with significant comorbidities. The allogeneic HSC donors are healthy-related and unrelated volunteers who are matched with the patients for human leukocyte antigen (HLA) class I and II molecules. Although transplantation of donor HSC with single or multiple HLA allele or antigen disparities is feasible, this increases the risk of immune-mediated graft rejection and graftversus-host disease (GVHD). The HSC can be harvested either by bone marrow aspiration during general anesthesia or by apheresis. The apheresis procedure requires the prior mobilization of HSC into the peripheral circulation by a 3–5-day treatment course with recombinant human

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granulocyte-colony stimulating factor (G-CSF). HSC express the hematopoietic ▶ stem cell marker CD34 that enable their detection in clinical samples by ▶ flow cytometry. After conditioning therapy and transplantation of the HSC allograft, the patient enters a 1–3week-long period with a low neutrophil cell count (▶ neutropenia) in which the patient is susceptible to bacterial and fungal infections. The hematopoietic engraftment is indicated by the reoccurrence of circulating neutrophils. These neutrophils are of donor origin which can be verified by analyzing the proportion of donor- and patient-derived DNA in chimerism assays. Early graft rejection is prevented by treating the patient with immunosuppressive medication, mainly consisting of the calcineurin inhibitors cyclosporine A and tacrolimus, the antimetabolite methotrexate, and T-cell depletion antibodies. The strong suppression of T-cell immunity increases the risk of infections with opportunistic agents of which herpes family viruses (e.g., cytomegalovirus, varicella zoster virus, ▶ Epstein-Barr virus) and Pneumocystis carinii have the greatest clinical significance during the first 1–2 years after transplantation. The drug-induced immunosuppression is also necessary to lower the incidence and severity of GVHD. GVHD is a life-threatening complication of allogeneic HSCT in which donor T cells attack the tissues of the transplant recipient after perceiving host tissues as antigenically foreign. GVHD is mainly directed against epithelial tissues of the skin, liver, and gastrointestinal tract. Other GVHD target organs include the hematopoietic tissues such as the bone marrow and thymus and the lungs in the form of idiopathic pneumonitis. Clinically, GVHD is divided into acute and chronic forms. The acute form is observed within the first 100 days post-transplant, and the chronic form occurs following day 100 after HSCT. Chronic GVHD damages the abovementioned organs, but also causes changes to the connective tissues including the skin and exocrine glands. If the GVHD is severe and requires intense immunosuppressive treatment, the patient may develop serious infections as a result of the immunosuppression and may die of infection. Moderate forms

Allogeneic Cell Therapy

of GVHD are associated with a lower incidence of relapse of the underlying malignant disease and, therefore, require no escalated immunosuppressive treatment. If the patient develops disease relapse after allogeneic HSCT, donor lymphocyte infusions (DLI) can be administered to augment the GVL effect. The DLI are collected from the original stem cell donor by apheresis without prior G-CSF treatment. DLI therapy is most efficient in patients with low disease burden. Moreover, DLI carry a superior GVL effect in chronic leukemias compared with acute leukemias. This may rely on disease-inherent factors such as the growth kinetic and immunogenicity of leukemic blasts that favors efficient immune reactions in chronic leukemias over their acute counterparts. The major complication of DLI therapy is an accompanying severe GVHD reaction, particularly if high lymphocyte doses are administered. Mechanisms The therapeutic success of allogeneic HSCT relies on the GVL immune effect that is closely linked to GVHD (Fig. 1). However, there are a considerable number of patients who develop efficient GVL reactions in the absence of GVHD. The main effectors that induce the GVL reaction as well as the GVHD are T lymphocytes of donor origin. In allogeneic HLA-identical HSCT, the donor lymphocytes generate a ▶ T-cell response against a group of proteins (called minor histocompatibility antigens, minor Hag) that are genetically polymorphic between donor and recipient. The peptide epitopes derived from minor Hag are presented by HLA molecules on recipient cells, and there are well-described examples of HLA class I and II associated minor Hag recognized by CD8+ and CD4+ donor T cells, respectively. It is comprehensible that minor Hag exclusively expressed in the hematopoietic tissue lineage promotes the engraftment of donor hematopoiesis as well as the GVL effect, while minor Hag with a ubiquitous expression pattern including epithelial tissues will facilitate the development of GVHD. There is also increasing evidence that donor T cells can recognize non-polymorphic antigens that are de novo expressed or overexpressed on

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Allogeneic Cell Therapy, Fig. 1 Donor-derived T lymphocytes infused into the leukemia patient are key mediators of the alloreactive graft-versusleukemia effect (GVL) and the graft-versus-host disease (GVHD). Main GVHD target organs are the skin, gut, and liver

Graft-versusleukemia (GVL)

Graft-versushost disease (GVHD)

Healthy donor

leukemic cells of the recipient. Hematopoietic minor Hag and leukemia-associated antigens are ideal candidates to redirect donor immunity specifically against the hematopoietic recipient cells including leukemia, either by vaccination with ▶ cancer vaccines or by ▶ adoptive immunotherapy. A great deal of current research on allogeneic HSCT involves attempts to separate the undesirable GVHD aspects of T-cell pathophysiology from the desirable GVL effect. For many leukemia patients lacking an HLA-matched hematopoietic stem cell donor, transplantation of HLA-incompatible HSC remains the only curative treatment option. In haplo-identical transplantation, the donor shares only one haplotype with the recipient. Because disparate HLA alleles are strongly immunogenic targets of alloreactive T cells, these regimens require concomitant T-cell depletion to prevent graft rejection and severe GVHD. Several research groups have demonstrated that in HLA-mismatch transplantation settings incorporating extensive T-cell depletion, the main immunological effector cells are ▶ natural killer cells of donor origin that recognize recipient hematopoietic (including leukemia) cells lacking the expression of natural killer cell inhibitory receptors. Clinical Aspects Allogeneic HSCT is a curative treatment modality for patients with insufficient hematopoietic stem cell function such as aplastic anemia and for patients with chemotherapy-refractory forms of hematological malignancies including ▶ chronic

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myeloid leukemia, ▶ acute myeloid leukemia, and ▶ acute lymphoblastic leukemia. Ongoing studies explore the role of allogeneic HSCT in patients with ▶ Hodgkin disease, non-Hodgkin lymphoma, and ▶ chronic lymphocytic leukemia. With the development of less-toxic RIC regimens, many groups are currently trying to establish allogeneic HSCT for the treatment of diseases with a dysfunctional immune system, e.g., autoimmune disorders and solid tumors such as renal carcinoma. The general idea is to first generate stable hematopoietic donor chimerism in the patient as a platform allowing in a second step the redirection of immunity using adoptively transferred donor lymphocytes with beneficial specificity.

Cross-References ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Histocompatibility Antigens ▶ Natural Killer Cell Activation

References Baron F, Storb R (2004) Allogeneic hematopoietic cell transplantation as treatment for hematological malignancies: a review. Springer Semin Immunopathol 26:71–94 Bleakley M, Riddell SR (2004) Molecules and mechanisms of the graft-versus-leukaemia effect. Nat Rev Cancer 4:371–380 Ferrara JL, Reddy P (2006) Pathophysiology of graftversus-host disease. Semin Hematol 43:3–10

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190 Kausche S, Wehler T, Schnurer E et al (2006) Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8 + T cells. Cancer Res 66:11447–11454 Kolb HJ, Schmid C, Barrett AJ et al (2004) Graft-versusleukemia reactions in allogeneic chimeras. Blood 103:767–776

Allogeneic Cellular Immunotherapy ▶ Allogeneic Cell Therapy

Allogeneic Hematopoietic Stem Cell Transplantation ▶ Allogeneic Cell Therapy

Alpha1-Fetoglobulin ▶ Alpha-Fetoprotein Diagnostics

Alpha-Fetoprotein Karel Kithier Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA

Synonyms AFP; Carcinofetal proteins; Feto-specific proteins; Oncodevelopmental proteins; Oncofetal antigens; Tumor markers; a-Fetoprotein

Definition AFP or alpha-fetoprotein is a serum protein of mammalian fetuses that is hardly detectable in healthy adults. Its reoccurrence in serum of adults may often attest to specific malignancy especially in high-risk patients, such as those with hepatocellular carcinomas (▶ hepatocellular carcinoma,

Allogeneic Cellular Immunotherapy

▶ hepatoblastoma) and chronic hepatitis B or C virus infection (▶ hepatitis B virus x antigen associated hepatocellular carcinoma). It also serves in evaluation (▶ serum biomarkers, ▶ surrogate endpoints) of therapy and disease progress in patients with embryonal carcinomas (germ cell tumors, ▶ platinum-refractory testicular germ cell tumors).

Characteristics The studies of fetal serum proteins came from different corners: from researchers interested primarily in the development of proteins and from those studying proteins of tumor-bearing laboratory animals. These two groups were at the beginning not very aware of each other’s results. The fetal protein history began with the physicochemical and biochemical studies of serum proteins, which depended, as this often happens in the laboratory endeavor, on the development, improvement, and refinement of laboratory methods. In the field of serum proteins, the electrophoretic and immunochemical techniques (▶ proteomics) were of crucial importance, especially in the case of fetal proteins, where usually only minute volumes of sera were available. Studies of electrophoretic patterns of serum proteins in human fetuses showed some considerable differences when compared with the sera of adults. Thus, in 1956 Bergstrand and Czar, using filter paper electrophoresis, reported on the special fetal band (called substance X), which was located between albumin and alpha-1 globulins. Substance X was absent from maternal sera and from sera of healthy adults. Also, Halbrecht and Klibanski reported similar findings in the same year. The first immunochemical studies of the substance X were done by Muralt and by Masopust in 1961 and 1962, respectively. Using antisera to fetal serum proteins (rabbits were immunized with the human fetal sera), an additional precipitin line with alpha globulin mobility was observed on immunoelectrophoresis (IEP) of human fetal serum; however, it was not present in adult sera. This fetal component was called independently “alpha-foeto-proteine” by Muralt and “fetoprotein” by Masopust. These findings

Alpha-Fetoprotein

resembled older observations in large animals; in 1944 Pedersen studied bovine fetal sera by ultracentrifugation and found a distinct gradient, not present in sera of adult animals. The fraction was named fetuin. Thus, it was believed by some that the human fetoprotein was related to fetuin, and the term “human fetuin” was used in some papers on human fetoprotein. Physicochemical properties of fetuin, which was found to be a typical glycoprotein, were studied by a number of workers; its physiological and pathologic properties attracted much less interest. Because fetuin and fetoprotein were present in higher concentrations in fetuses and undetectable in adults, they were sometimes called “feto-specific proteins.” Immunochemical Techniques For the detection of feto-specific proteins, the immunochemical techniques became the methods of choice in the 1960s. Antisera to these proteins were prepared by the immunization of animals, usually rabbits, with fetal sera. To obtain specific antisera to feto-specific proteins, the antisera were absorbed with the sera of adult men or animals. The absorbed antisera should contain only the antibodies directed to the feto-specific protein (s) of a given species. In some cases, the absorbed antisera showed two to three precipitin lines on IEP of fetal serum. Sometimes, in human fetal sera, two lines with the absorbed antiserum were observed. The line in alpha zone of IEP was that of human fetoprotein, the other line, in beta position, was sometimes incorrectly, without justification, called beta-fetoprotein. The lines showed no antigenic relationship each to other. For this reason, the original term “fetoprotein” was changed to “alpha-fetoprotein” and consequently the abbreviation of AFP came to life. The term “betafetoprotein” ceased to be used since the beta protein was later identified as fetal ferritin. AFP in Pathology In 1964, a study of a possible occurrence of AFP in sera of patients was started. The putative presence of AFP was tested by double radial immunodiffusion (Ouchterlony test). After hundreds of negative results, a patient was identified, who had a definitely detectable serum concentration of AFP. The

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diagnosis of this patient, confirmed histopathologically at the autopsy, was that of hepatocellular carcinoma. In 1966 and 1967, the occurrence of AFP in four children with a malignant growth of embryonic character was reported. One of them was a 5-year-old boy with embryonal cell carcinoma of the left testicle (testis cancer, ▶ childhood cancer, ▶ germinoma) and another patient was a 14-year-old girl with malignant teratoblastoma of the right ovary (▶ ovarian cancer, ▶ ovarian tumors during childhood and adolescence). Also, Abelev published in 1967 the finding of “alpha fetal globulin” in patients with embryonal testicular cancer. Several pediatric patients with noncancerous liver diseases, such as infectious hepatitis and some unspecified hepathopathies, were identified, who had detectable AFP serum levels. A highly sensitive technique, radioactive single radial immunodiffusion (employing the second, 125 Iodine-labeled antibodies to the primary antiAFP immunoglobulin fraction), enabled to quantify previously undetectable levels of AFP in various body fluids. By such means, AFP serum levels of patients with hepatocellular carcinomas were studied in a correlation with their individual histopathologic findings. A further increased sensitivity of AFP quantitation was facilitated by the development of a radioimmunoassay. This technique made the quantitation of AFP in healthy persons such as pregnant women a routine test in clinical laboratories. In the 1970s, a number of reviews on AFP were published along the studies of AFP physicochemical properties. The first studies on serum concentrations of AFP and their changes in the course of diseases were done in those years. Thus, the impact of the therapy could be evaluated and monitored in some malignancies. Fetuin Versus AFP In the early years, AFP was considered by some investigators to be a protein similar to bovine fetuin and therefore called “human fetuin.” Fetuin was isolated from fetal calf serum and the antisera were prepared to fetuin, and to serum proteins of human and bovine fetuses. With the use of absorbed antiserum to calf serum, an additional protein component was detected in alpha zone of bovine fetal serum, which was not detectable in sera of adult

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animals. This component could be considered as a “bovine fetoprotein.” Antiserum to this protein did not react with isolated fetuin and conversely the specific antiserum to fetuin did not react in immunodiffusion experiments with the “bovine fetoprotein.” The protein was not detected in adult healthy animals; it was, however, found in sera of two, out of four, adult cows with hepatocellular carcinomas (▶ comparative oncology). No antigenic relationship was observed in double radial immunodiffusion and the precipitin lines of fetuin and “bovine fetoprotein” crossed each other, showing thus the pattern of antigenic nonidentity. AFP in Laboratory Animals Rat sera were studied electrophoretically already in the 1950s. A “fetal” protein was detected by Beaton (1961) in the macroglobulin fraction of starch gel electrophoresis. This protein migrated as an alpha-2 globulin in electrophoretic media without molecular sieve effect (filter paper) and slowly in starch gel. Therefore, it was called “alpha-2 slow globulin.” The protein was found in sera of rat fetuses and newborns, as well as in pregnant rats, but not in healthy, nonpregnant adult rats. It was present, however, in sera of tumor-bearing rats and in animals with various inflammatory processes, e.g., with turpentine abscess. Another alpha globulin was found by Darcy in fetal rat sera; it was also present in sera of pregnant animals and adult rats with tumors and/or with inflammations. Protein was also detectable in much lower concentrations in healthy, nonpregnant rats. Wise in 1963, using two-dimensional electrophoresis (filter paperstarch gel), demonstrated in rat fetal sera special proteins, named “fetal postalbumins” (two electrophoretic bands), which were not present in sera of adult animals. Altogether, at least three fetal components were reported in rats. To address this question, rabbit antiserum directed to rat fetal serum proteins was prepared. The absorbed antiserum (with the serum proteins of adult, healthy, nonpregnant animals) did not react with sera of adult, healthy nonpregnant rats or with the protein described by Darcy. It did react, however, with three different proteins on IEP of fetal rat sera; two of them located in alpha-2 and one in alpha-1

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globulin zone. The antibody to the protein in alpha-2 zone could be absorbed with the serum from an adult rat with turpentine abscess. This protein was also detected immunochemically in extracted proteins from macroglobulin position in starch gel electrophoresis of fetal serum. The protein obviously corresponded to alpha-2 slow globulin of Beaton. The other precipitin line in alpha-2 globulin zone was stainable with lipid stains (Red Oil and Sudan Black B) and represented most probably a lipoprotein-esterase found by Stanislawski–Birencwajg in fetal rat serum. The precipitin line in alpha-1 zone, present in sera of fetuses, absent from sera of adult rats, either healthy or with the acute inflammation, was considered to be a typical feto-specific protein, probably related to human AFP. However, no crossreaction was seen by immunodiffusion between human AFP and antiserum to rat fetal proteins. To prepare a monospecific antiserum to alpha Ft protein, it was important to remove the antibodies to alpha-2 slow globulin, e.g., by using sera of adult rats with some inflammatory pathology. In 1963, Abelev reported the finding of “embryonal alpha globulin” in serum of adult mouse with transplantable hepatoma; the globulin was also present in sera of fetal mice (▶ mouse models). Much progress has been done since the early modest beginnings of AFP research. Presently, June 2015, a review of AFP literature shows 21,158 papers related to the topic.

Cross-References ▶ Alpha-Fetoprotein Diagnostics ▶ Childhood Cancer ▶ Comparative Oncology ▶ Germinoma ▶ Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma ▶ Hepatoblastoma ▶ Hepatocellular Carcinoma ▶ Mouse Models ▶ Ovarian Cancer ▶ Ovarian Germ Cell Tumors ▶ Ovarian Tumors During Childhood and Adolescence

Alpha-Fetoprotein Diagnostics

▶ Platinum-Refractory Testicular Germ Cell Tumors ▶ Proteomics ▶ Serum Biomarkers ▶ Surrogate Endpoint ▶ Testicular Cancer ▶ Testicular Germ Cell Tumors

References Abelev GI (1971) Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Adv Cancer Res 14:295–358, PubMedCrossRef Kithier K, Poulik MD (1972) Comparative studies of bovine alpha-fetoprotein and fetuin. Biochim Biophys Acta 278:505–516, PubMedCrossRef Kithier K, Prokes J (1966) Fetal alpha-1 globulin of rat serum. Biochim Biophys Acta 127:390–399, PubMedCrossRef Kithier K, Houstek J, Masopust J et al (1966) Occurrence of a specific foetal protein in a primary liver carcinoma. Nature 212:414, PubMedCrossRef Masopust J, Kithier K, Radl J et al (1968) Occurrence of fetoprotein in patients with neoplasms and non-neoplastic diseases. Int J Cancer 3:364–373, PubMedCrossRef

See Also (2012) Germ cell tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905

Alpha-Fetoprotein Diagnostics David E. Kaplan Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA, USA

Synonyms AFP; Alpha1-fetoglobulin; Embryonal serum alpha-globulin; Embryonal serum a-globulin; Embryo-specific alpha-globulin; Embryo-specific The entry “Alpha-Fetoprotein Diagnostics” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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a-globulin; a1-Fetoglobulin; Fetuin; Fetuin-A; Foeto-protein

a-Feto-protein;

A Definition ▶ Alpha-fetoprotein (AFP) is a 68.7 kDa plasma protein synthesized primarily by the fetal liver and embryonic yolk sac that is highly homologous with human albumin. Widely expressed in the fetal liver, AFP mRNA is down-regulated in postnatal hepatocytes. Serum AFP levels are used clinically for detection, confirmation, and follow-up of human ▶ hepatocellular carcinoma (HCC) and nonseminomatous germ cell tumors, although lack of sensitivity and specificity complicate its use.

Characteristics Alpha-fetoprotein (AFP) is a 590 amino-acid plasma protein that shares 40% amino acid and 40–44% nucleotide sequence homology with human serum albumin and is a member of the albumin gene superfamily. The AFP gene covers approximately 22 kB of DNA and has 15 exons and 14 introns. The human albumin gene lies 14.5 kB upstream to its AFP homologue. Regulation of AFP protein production occurs mainly at the transcriptional level. In human cells, the AFP enhancer region contains binding sites for several liver-enriched transcription factors (HNF1-4, C/EBP) which control tissue specific expression. Expression of AFP also appears to be positively regulated by NFkB, by steroids via retinoid X receptors as well as by interactions with extracellular matrix. AFP is normally expressed by villous trophoblasts in the human placenta during pregnancy and by fetal hepatoblasts. In fetal and newborn rats, AFP mRNA can be detected at low levels in the kidney, pancreas, heart, and gastrointestinal tracts as well. In early postnatal life, AFP production is repressed in normal hepatocytes and silenced in nonhepatic parenchymal cells. The mechanisms for the repression or silencing of AFP expression have largely been characterized. In mice, an unlinked locus called alpha-

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fetoprotein regulator 1 (Afr1) on chromosome 15 appears to interact with the AFP promoter region; repression of Afr1 appears to be associated with postnatal repression of AFP expression. The AFP promoter may also interact with Ku inducing a hairpin tertiary structure that may abrogate HNF1 binding to the promoter. Postnatal repression of AFP expression in the liver has also been shown to be ▶ p53- and ▶ TGFb1dependent whereas genetic silencing primarily involves epigenetic mechanisms that concomitantly silence the upstream albumin gene. In the adult liver, AFP expression is present but repressed. In situ hybridization studies confirm the presence of minute quantities of AFP mRNA, but at levels generally below the sensitivity of immunohistochemical detection. In the setting of hepatocyte regeneration, e.g., ischemic injury, surgical resection, and chronic viral hepatitis, in ▶ hepatoblastoma as well as in a subset of hepatocellular carcinoma (HCC) (and rarely ▶ cholangiocarcinoma), AFP expression is de-repressed. AFP production also occurs in nonseminomatous germ cell tumors such as choriocarcinoma, mixed germ cell tumors, and teratomas. In fetal and newborn rats, AFP mRNA can be detected at low levels in the kidney, pancreas, heart, and gastrointestinal tracts as well. Rarely in adults, nonhepatic/ non–germ cell malignancies such as ▶ gastric cancer, ▶ pancreatic cancer, ▶ endometrial cancer, ▶ colon cancer, and ▶ ovarian cancer are associated with loss of silencing of AFP expression. The critical activities of AFP in vivo remain poorly defined. Many cell types including vascular endothelium and T-cells express receptors for AFP. AFP administration in human cell lines has been associated with differential expression of FasL and ▶ TRAIL relative to fas and TRAIL Receptor, leading to postulation of a role for AFP in escape from tumor immunosurveillance. AFP also appears to inhibit TNF receptor 1-signalling-mediated tumor cell apoptosis. Paradoxically, some studies suggest a pro-apoptotic role for AFP in tumor cells lines via interactions with X-linked inhibitor of apoptosis protein (XIAP). Other studies postulate that AFP may mediate anti-inflammatory effects that suppress autoimmunity and anti-fetal immune responses during pregnancy, possibly via inhibition of CD4 T-cell

Alpha-Fetoprotein Diagnostics

proliferation. Tumor-derived AFP has been shown to impair dendritic cell activation and reduce the allostimulated T-cell proliferation in vitro. Serum AFP determinations has two main clinical uses. First, it is used to screen women during pregnancy for fetal developmental abnormalities. Second, AFP is used as a tumor marker for hepatocellular carcinoma (HCC) and nonseminomatous germ cell tumors. Serum AFP determinations have been used since the late 1960s to detect hepatocellular carcinoma despite limitations in its sensitivity and specificity. While AFP levels greater than 400 ng/ml are considered diagnostic of HCC, such elevations are rarely present. The sensitivity and specificity of AFP determinations also appears to be dependent on the underlying cause of liver disease that results in HCC development. Using a cutoff of 20 ng/ml, sensitivity ranges from 41% to 65% and specificity ranges from 80% to 94%. In chronic hepatitis C, AFP levels vary in relation to transaminase levels limiting the specificity of AFP for detection of HCC in patients with active inflammation. The role of serum AFP in screening programs for HCC in patients with cirrhosis remains controversial. It remains unclear if the addition of AFP determinations to routine imaging examinations, e.g., ultrasound every 6 months, provides any incremental benefit. Current guidelines from the United Network of Organ Sharing (UNOS) in the United States support the use of AFP levels greater than 400 ng/ml to confirm the presence of HCC when a hypervascular lesion on CT or MRI imaging is seen. Exception points may be petitioned from UNOS to provide the rare individual patients with AFP levels greater than 400 ng/ml but no visible tumor to increase the priority of such patients for liver transplantation. Several glycoforms (AFP-L1, AFP-L2, and AFP-L3) of AFP have been resolved based on differences in glycosylation groups. Lectinreactive AFP (AFP-L3) in some studies has been associated with intrahepatic cholangiocarcinoma. In other studies, a high percentage of total AFP made up of the L3 fraction has been associated with hepatocellular carcinomas. Measurement of specific glycoforms is not in routine clinical use.

Alu Elements

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Cross-References

Alternative Reading Frame ▶ Alpha-Fetoprotein ▶ Cholangiocarcinoma ▶ Endometrial Cancer ▶ Gastric Cancer ▶ Hepatoblastoma ▶ Hepatocellular Carcinoma ▶ Ovarian Cancer ▶ Ovarian Germ Cell Tumors ▶ p53 Family ▶ Pancreatic Cancer ▶ Testicular Germ Cell Tumors ▶ TNF-Related Apoptosis-Inducing Ligand ▶ TP53 ▶ TRAIL Receptor Antibodies ▶ Transforming Growth Factor-Beta ▶ Tumor Necrosis Factor

References Abelev GI, Eraiser TL (1999) Cellular aspects of alphafetoprotein reexpression in tumors. Cancer Biol 9:95–107 Gupta S, Bent S, Kohlwes J (2003) Test characteristics of a-fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. Ann Intern Med 139:46–50 Nahon JL (1987) The regulation of albumin and a-fetoprotein gene expression in mammals. Biochimie 69:445–459 Pardee AD, Shi J, Butterfield LH (2014) Tumor-derived a-fetoprotein impairs the differentiation and T cell stimulatory activity of human dendritic cells. J Immunol 193:5723–5732 Richardson P, Duan Z, Kramer J et al (2012) Determinants of serum alpha-fetoprotein levels in hepatitis C-infected patients. Clin Gastroenterol Hepatol 10:428–433

See Also (2012) Germ cell tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747X. doi:10.1007/978-3-642-16483-5_4331 (2012) TGF–ß. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3661. doi:10.1007/978-3-642-16483-5_5753 Möslein G (2009) Colon cancer. In: Schwab M (ed) Encyclopedia of cancer, 2nd edn. Springer, Berlin/Heidelberg, pp 722–727. doi:10.1007/978-3-540-476481_1265

▶ ARF Tumor Suppressor Protein

Alu Elements Christine M. Morris Cancer Genetics Research, University of Otago, Christchurch, New Zealand

Definition The most abundant class of dispersed repeat elements in the human genome and one member of the family of short interspersed repeat elements (SINEs). An estimated one million copies comprise about 10% of DNA in human cells.

Characteristics Structure Alu elements are 280 bp in length and consist of two similar monomers that have homology to, and were originally derived from, the 7SL RNA gene (a component of the signal recognition particle) (Fig. 1). Individual Alu elements are flanked by direct repeats and end in a 30 A-rich tract, and the left monomer contains an internal RNA polymerase III promoter that directs transcription initiation to the first residue of the element. Alu are retrotransposable elements, and several subfamilies, mobilized from different “source” genes at different evolutionary times, can be recognized on the basis of their sequence divergence and diagnostic bases. Because Alu has no coding machinery, it depends on LINE-1 (▶ LINE-1 Elements) and other cellular processes to obtain the factors needed for retrotransposition, and these elements are therefore regarded as non-autonomous. However, the vast majority of Alu copies in the human genome are not retrotranspositionally active, with only a few likely to be active Alu source elements. Alu activity is evidenced by some of the

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Alu Elements 7SL-specific sequence 7SL RNA

300 bp

Left monomer

Right monomer

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(A)n

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Alu Elements, Fig. 1 Alu elements have a dimeric structure that originated from 7SL RNA. Colored areas show 7SL sequences present in the Alu repeat consensus

integrated subfamilies, primarily AluY, which are polymorphic, occupying regions on some chromosomes that are not occupied at the same locus on others. The current estimate for Alu retrotransposition activity in humans is 1 insertion for every 20 births. Across the genome, Alu distribution is nonrandom and concentrated in GC-rich regions. Function The function of Alu elements has been subject to intense investigation, debate, and speculation over the past three decades. Proposed roles include modulation of chromosome structure and packaging of DNA around nucleosomes, initiation or switch sites for DNA replication, regulation of gene transcription through Alu-specific protein binding domains, RNA editing as preferential templates for adenosine to inosine (A-to-I) substitution by the ADAR family of enzymes, and regulation of translation by RNA transcribed from Alu elements. Although Alu expression increases in cells stressed by chemical agents or viral infection, most human Alu repeats are silent in somatic cells, with only the minor, evolutionarily younger subgroups actively transcribed. Consistent with these observations, CpG sites in the majority of Alu sequences are normally fully methylated (▶ Methylation) in most somatic cell types, a state which is considered to suppress

expression and therefore transposition. However, methylation status of Alu is reported to vary in different tissues. For example, at least a subset of the integrated Alus are almost completely unmethylated in sperm DNA relative to other somatic tissues. Differences in Alu methylation have also been found mosaically in the same tissue, such as has been reported in the brain. Overall, analysis of Alu expression is complex, and their ubiquity presents technical challenges. For this reason, and excepting germ line expression, there are currently minimal data available on differential expression of Alu elements in somatic tissue or during development. Role in Human Cancer Alu-mediated gene rearrangement underlies several important constitutional diseases, including familial cancers. Different mechanisms for these rearrangements include recombination between homologous or nonhomologous regions of Alu elements at different locations within a gene, or on the same or different chromosomes, expansion of 30 polynucleotide tracts to form fragile sites, or disruption of coding regions of functional genes by transpositional insertion of actively transcribed Alu elements. Instability of 30 polynucleotide tracts may also indicate a DNA mismatch repair deficiency.

AME Transcription Factor

Because of their high density in the human genome, nonrandom chromosomal distribution, and the high degree of homology between individual elements, Alu repeats are also recognized candidates to mediate somatically acquired gene rearrangements with neoplastic potential. Specific underlying mechanisms for involvement of Alu in somatic rearrangements have begun to be explored, with possibilities including promotion of DNA exchange by sequences within Alu that share homology with known recombinogenic translin DNA-binding motifs or the w-like Alu core sequence, preferential recombination between DNA regions that are localized within Alu-rich clusters on the same or different chromosomes, or otherwise unknown features of individual Alu elements that predispose to recurrent recombination events associated with some breakpoint cluster regions.

197 (2012) x-(Chi)-like sequence. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 796. doi:10.1007/978-3-642-164835_1085 (2012) Translin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5940

AME Transcription Factor Vitalyi Senyuk Department of Medicine (M/C 737), College of Medicine Research Building, University of Illinois at Chicago, Chicago, IL, USA

Synonyms AML1/EVI-1; RUNX1/MDS1/EVI1

Cross-References ▶ LINE-1 Elements ▶ Methylation

References Ade C, Roy-Engel AM, Deininger PL (2013) Alu elements: an intrinsic source of human genome instability. Curr Opin Virol 3(6):639–645 Deininger P (2011) Alu elements: know the SINEs. Genome Biol 12(12):236 Kolomietz E, Meyn MS, Pandita A, Squire JA (2002) The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors. Genes Chromosomes Cancer 35(2):97–112 Konkel MK, Batzer MA (2010) A mobile threat to genome stability: the impact of non-LTR retrotransposons upon the human genome. Semin Cancer Biol 20(4):211–221 Wang C, Huang S (2014) Nuclear function of Alus. Nucleus 5(2):131–137

See Also (2012) ADAR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 43. doi:10.1007/978-3-642-16483-5_77 (2012) Breakpoint cluster region. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 485. doi:10.1007/978-3-642-164835_716

Definition AME is an aggressive oncoprotein (chimeric transcription factor) associated with several types of ▶ acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative disorders (MPD).

Characteristics The legendary discovery of chromosomal translocations by Janet D. Rowley in 1972 has revolutionized leukemia research and therapy by allowing biological interrogation and classification of these disorders. Several recurring translocations have been identified and the participating genes cloned and characterized at the molecular level. One such recurring abnormality is the balanced translocation between the long arms of chromosomes 3 and 21, t(3;21)(q26;q22), originally discovered in a patient with therapyrelated chronic myelogenous leukemia (CML) which is classified as an MPD. The t(3;21) is a complicated chromosomal rearrangement that employs a mechanism of

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AME Transcription Factor

PR

ZnF1

ZnF2

MDS1/EVI1 Runt RUNX1 AME

AME Transcription Factor, Fig. 1 Diagram of ME, RUNX1, and the fusion protein AME. The Runt, PR, and two zinc finger (ZnF) domains are shown. The vertical dashed line indicates the breakpoint fusion

intergenic splicing to generate several ▶ fusion genes of which AME is perhaps the best characterized and the most important. Among the less frequent translocations involving RUNX1 (also known as AML1, CBFA2, and PEBP2), AME is the only fusion gene that has been cloned and characterized at the molecular level. AME, obtained by in-frame fusion of the truncated RUNX1 and MDS1/EVI1 (ME) genes, is controlled by the RUNX1 promoter which becomes active during the execution of multiple steps of hematopoietic program, especially during the development of myeloid lineage. The t(3;21) is a relatively rare translocation infrequently seen in de novo leukemias. It was observed in ~1% of AML, MDS, and MPD cases and often associated with secondary leukemia that arises in patients previously treated with ▶ alkylating agents or topoisomerase inhibitors for other malignancies. In particular, the t(3;21) was detected in the patients after administration of cytostatic drugs such as busulfan, teniposide, etoposide, hydroxyurea, ▶ fludarabine, ▶ 5-fluorouracil, and others. There is no unique clinical picture of t(3;21)-associated leukemias such as restriction to a certain FAB (FrenchAmerican-British classification) category, and it has been classified as M1, M2, M4, and M7 subtypes. The common morphologic feature of t (3;21)-positive AML is minimally differentiated blasts with prominent nucleoli and scant cytoplasm. There is no age or gender specificity for t (3;21)-associated diseases, but, as for many other ▶ cancers, older individuals are at higher risk. In contrast to many other translocations, the t(3;21) causes a very aggressive myeloid leukemia/

▶ blast crisis of CML characterized by a low response to the existing therapeutic treatments and a poor prognosis. In the largest clinical investigation of t(3;21) patients published to date, the majority of AML patients died between 1 week and 8.5 months (median 2 months) after presentation, whereas MPD patients survived 1–21 months (median 6.5 months) after presentation. RUNX1 is a DNA-binding subunit of the transcription factor CBF which is essential for hematopoiesis and is involved in several chromosomal abnormalities associated with human leukemias. RUNX1 consists of an N-terminal DNA-binding domain called Runt with homology to the product of the Drosophila segmentation gene Runt and a C-terminal activation domain. ME is a zinc finger transcription factor related to the leukemiaassociated protein ecotropic viral integration site 1 (EVI1) of unknown function. ME contains a conserved N-terminal region, called PR domain, two sets of DNA-binding zinc finger domains, a proline-rich central domain, and an acidic C-terminal domain. AME consists of the DNA-binding domain Runt of RUNX1 fused to almost the entire ME (Fig. 1). Forced expression of AME upregulates the cell cycle and blocks granulocytic differentiation of the murine hematopoietic cell line 32Dcl3 and delays the myeloid differentiation of normal murine bone marrow progenitors in vitro. The exact mechanisms of AME oncogenic activation are unknown and several possibilities exist. Also, as with many other oncoproteins, most probably AME alone is insufficient to transform a healthy normal cell into a leukemic one, and additional cooperating genetic abnormalities are necessary.

AME Transcription Factor

It has been shown that the majority of AME-positive patients have, in addition to t (3;21), several other chromosomal abnormalities readily detected by cytogenetic analysis, translocations, deletions, and duplications, the most common of which is t(9;22)(q34;q11) found in CML patients. One of the first investigated properties of AME was its effect on a subset of target promoters regulated by both parent proteins. RUNX1 is generally considered a transcription activator through its C-terminus, which interacts with several transcription coregulators and regulates critical genes in hematopoiesis. ME is also considered a transactivator, and both parent proteins act as antagonists of AME. Therefore, it was suggested that AME could act as a bifunctional transcription factor possessing the ability to bind to and repress/ deregulate both the RUNX1- and ME-dependent promoters. In support of this hypothesis, it was shown that AME directly interacts with the corepressors C-terminal-binding protein (CtBP) and histone deacetylase 1 (HDAC1) which are often a part of big repressor complexes transiently formed at the promoter sites. AME has distinct regions for HDAC1 and CtBP binding, and, taking in consideration that both corepressors are able to dimerize and interact to each other, one AME molecule can recruit several molecules of the corepressors. AME represses the target promoters by CtBP-dependent and CtBPindependent mechanisms, probably reflecting the dual nature of this protein. In vitro CtBP enhances not only AME repression potential but also the ability of AME to upregulate growth and deregulate differentiation in murine hematopoietic cells, suggesting that AME repression is necessary for its oncogenic activity. However, the transcription properties of AME are more complicated because it also interacts with histone acetyltransferases p300/CBP-associated factor (P/CAF) and general control of amino-acid synthesis 5-like (GCN5), which are generally considered as co-activators of transcription. Both P/CAF and GCN5 efficiently acetylate the central region of AME in vivo, but the function of this modification and its role in oncogenesis are still unknown.

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Similar to many other fusion proteins that are activated by chromosomal translocations in human leukemia, AME is able to oligomerize and displays a complex pattern of self-interaction that involves at least three oligomerization regions, which are the proximal and the distal zinc finger domains and the Runt domain. The distal zinc finger domain is quite important in AME oligomerization because it mediates the interaction with the other two domains and an internal deletion that removes the three zinc finger motifs virtually sufficient to repair (though not completely) the self-renewal and differentiation programs of normal murine bone marrow progenitors in vitro. In vitro, this domain efficiently cooperates with CtBP in disrupting normal hematopoiesis and the internal deletion, and a point mutation that abolishes CtBP binding reestablishes almost completely the hematopoietic differentiation in murine cells. Probably AME belongs to a growing group of chimeric transcription factors which inappropriately maintain high local concentration of corepressors at the specific promoter sites because of their ability to oligomerize, resulting in the deregulation of genes involved in differentiation, ▶ apoptosis, and proliferation. It is highly possible that the aggressiveness of AME as an oncoprotein is in part mediated by AME’s ability to abrogate the growth inhibitory effect of ▶ transforming growth factor-b (TGF-b) that controls cell expansion and inhibits proliferation of different cell types. The repression of TGF-beta signaling depends on the ability of the proximal zinc finger of AME to directly interact with and repress Smad3, an intracellular mediator of TGF-b signaling. It should be noted that in contrast to AME, ME cooperates with TGF-b and increases the sensitivity of hematopoietic cells to its stimulus. AME is also indirectly involved in deregulation of the hematopoietic program. It has been shown that CCAAT/enhancer-binding protein a (C/EBPa), a crucial transcription factor for normal granulopoiesis, is suppressed at translation level by more than 90% in AME-expressing U937 cells. In AML patients harboring t(3;21),

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the C/EBPa level is reduced even more, whereas in AML patients without the t(3;21), C/EBPa is not affected. The mRNA levels remain unchanged in both cases indicating that AME does not affect C/EBPa transcription. Most probably AME acts through an intermediate effector, ▶ calreticulin, a ubiquitous multifunctional calcium-binding protein, whose expression is strongly correlated with both AME expression and C/EBPa suppression. It has been shown in reporter gene assays and in Rat1 fibroblasts that AME stimulates activator protein 1 (▶ AP-1) activity with dependence on the distal zinc finger domain. AP-1 activation may increase cell proliferation potentially contributing to AME oncogenic properties. A ▶ mouse model of AME-positive leukemia, generated by bone marrow transplantation of AME-expressing cells using BALB/c mice, showed that AME induces acute myeloid leukemia with a latency of 5–13 months indicating that additional genetic abnormalities are necessary for leukemogenesis. The disease was clonal in origin and resembled human acute myelomonocytic leukemia (AML FAB-M4). It has been also shown in this model that AME efficiently cooperates with breakpoint cluster region/Abelson tyrosine kinase (▶ BCR/ ABL), a product of t(9;22) frequently seen in CML patients. Both proteins together are able to block myeloid differentiation during the pre-leukemia stage and induce AML within 1–4 months. The second mouse model for AME utilized bone marrow infection and transplantation using C57BL/6 mice. The animals displayed a variety of clinical features that are observed in essential thrombocythemia (ET) that resulted in their death after 8–16 months. The molecular etiology of ET, which is classified as an MPD, is poorly understood. An activating somatic point mutation (V617F) of Janus kinase 2 (JAK2) was identified in MPD patients. Nonetheless, this mutation was not detected in ~50% of ET patients, indicating that some other molecular mechanisms exist and t (3;21) could be one of them. The differences between these two mouse models can be explained by taking into consideration that the BALB/c strain of mice is well known to have a higher tumor incidence as

AME Transcription Factor

compared with C57BL/6 mice (because it has a mutated inhibitor of Cdk4/alternative reading frame (INK4a/ARF) locus that at least partially disables p16Ink4a, a ▶ tumor suppressor protein which is frequently mutated in many cancers). A mouse model of AME knock-in has been also reported. The heterozygous mutant embryos obtained by breeding of AME chimeric male (ICR strain) and wild-type female (C57BL/6 strain) were not viable and died of fetal liver hematopoiesis failure at around day 13.5E. Fetal liver hematopoietic progenitor cells from these mice displayed increased self-renewal capacity and impaired erythropoiesis. In addition, myeloid and megakaryocytic cells appeared dysplastic indicating that AME induces multiple defects in several myeloid lineages. Interestingly, the majority of AME chimeric mice demonstrated sudden death at the age of about 7 months without any significant signs of any disease, whereas one of them developed a disease resembling megakaryoblastic leukemia at 5 months of age. Since 1987, when the t(3;21) was described for the first time, our knowledge about AME has increased vastly; however, the prognosis of patients with this abnormality is still extremely poor. Hopefully, the cumulative efforts of different research groups will provide new approaches for the search of a treatment for this selected group of patients.

Cross-References ▶ Tumor Suppressor Genes

References Nucifora G, Rowley JD (1995) AML1 and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood 86:1–14 Nucifora G, Laricchia-Robbio L, Senyuk V (2006) EVI1 and hematopoietic disorders: history and perspectives. Gene 368:1–11 Rubin CM, Larson RA, Bitter MA et al (1987) Association of a chromosomal 3;21 translocation with the blast phase of chronic myelogenous leukemia. Blood 70:1338–1342 Yin CC, Cortes J, Barkoh B et al (2006) t(3;21)(q26;q22) in myeloid leukemia. Cancer 106:1730–1738

Amine Oxidases

Amine Oxidases Bruno Mondovì1, Paola Pietrangeli1, Lucia Marcocci1 and Antonio Toninello2 1 Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2 Department of Biological Chemistry, University of Padua, Padua, Italy

Definition Amine oxidases (AOs) are a class of enzymes which is heterogeneous in terms of structure, catalytic mechanisms, and substrate specificity. Biogenic amines, a group of naturally occurring, biologically active amines, such as monoamines (norepinephrine, histamine, tyramine, dopamine, and serotonin) and ▶ polyamines (putrescine, spermidine, spermine) are oxidatively deaminated by AOs in a reaction that consumes O2 to produce the corresponding aldehydes, amines with a shorter chain, ammonium ions, and hydrogen peroxide (H2O2).

Characteristics Two classes of AOs can be described, which contain different prosthetic groups: the FAD-dependent AOs (FAD-AOs) containing the flavin adenin dinucleotide (FAD), and the copperdependent AOs (Cu-AOs) containing copper and an organic cofactor produced by the copper selfcatalyzed posttranslational oxidation of a tyrosine residue, i.e., TPQ (trihydroxyphenylalanine quinone), or LTQ characteristic of lysyl oxidase (LXAO). The FAD-AOs are subdivided in monoamine oxidase A and B (MAO A, MAO B), polyamine oxidase (PAO), and the discovered spermine oxidase (SMO). The two latter enzymes are cytosolic, catalyze the oxidation of secondary amino groups, and participate in the interconversion metabolism of polyamines. MAOs are tightly bound to mitochondrial outer membranes.

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Cu-AOs are often also named SSAO (semicarbazide sensitive amine oxidase) because of their inhibition by semicarbazide, which binds the organic cofactor. When strictly necessary, the name of the best substrate is used to characterize the enzymes. For instance, Cu-AOs, which oxidize diamine and histamine are named diamine oxidase (DAO) and histaminase, respectively. Sometimes, a single enzyme, such as the enzyme purified from pig kidney, may display both DAO and histaminase activities, so that the name may not imply a specific enzyme. The X-ray structure is available for several Cu-AOs, PAO, and MAO. Functions A plethora of physiological functions, sometimes in contrast with one another, is ascribed to AOs. Although the exact molecular mechanism of their biological activity is not well-defined, a role of these enzymes in various processes through the action of either substrates or reaction products is postulated. Evidences have accumulated on the physiopathological relevance of polyamines, histamine, hydrogen peroxide, and aldehydes in cell death and differentiation, allergic diseases, and postischemic reperfusion damage. Histamine is considered to be a main factor involved in allergic diseases. A plant Cu-AO, showing high histaminase activity, counteracts acute allergic asthma-like reaction in actively sensitized guinea pigs. The same enzyme modulates cardiac anaphylactic response in guinea pig. Protective effects of the plant enzyme were also observed in heart and gut ischemia and reperfusion injury in in vivo rats. Bovine serum Cu-AO was shown to present an antioxidant effect, in vitro, against electrolytically induced reactive oxygen species (ROS). Among other physiopathological functions ascribed to AOs are, for example, the involvement of MAO in psychiatric diseases like schizophrenia, by regulating the dopamine metabolism, and of Cu-AOs in cataract, by the lens damaging effect of amine oxidation products. An important role of VAP1, vascular adhesion protein with AO activity, in inflammation, diabetes, and cerebrovascular and cardiovascular diseases is also indicated.

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A primary involvement of AOs was demonstrated in cancer growth inhibition and progression, especially by means of aldehydes, H2O2, and other ROS, the AOs-mediated products of biogenic amines oxidation. Aminoaldehydes were shown to interact with nucleotides or with DNA. Microinjection of Cu-AO into chick embryo fibroblast, rat cells, and glioma cells caused the inhibition of DNA damage and protein synthesis. Tumor cells, with higher polyamines content than the normal controls, were more sensitive to the injected AOs. When an immobilized Cu-AO was injected into the peritoneal cavity of Swiss mice, 24 h after viable Ehrlich ascites tumor cells transplantation or into a mouse (melanoma) model, a strong inhibition of tumor growth was observed. An induction of tumors in rat bowels (colon cancer) was observed on inhibition of DAO by aminoguanidine. An induction of tumors in rats was observed after carcinogenic treatment combined with AO inhibition. A possible use of AOs in cancer therapy has been suggested. Both H2O2 and aldehydes contribute to cytotoxicity, as demonstrated by incubation of Chinese hamster ovary cells with purified bovine serum AO in the presence of spermine. Catalase, the enzyme involved in H2O2 elimination, is absent in many tumor cells and thus apoptosis occurs. The direct relationship between AOs, apoptosis, and cancer appears to be related to the regulation of biogenic amines and their metabolic products. H2O2 is considered to be a mediator of apoptotic cell death but the mechanism is unclear. H2O2 produced by MAO-catalyzed monoamines oxidation seems extremely important for apoptosis induction by considering the fact that MAO inhibitors are able to prevent apoptosis in human melanoma cells and that catalase inhibits the apoptosis induced by polyamines or their analogs and cathecolamines. The catalytic products of active amine oxidation are strong inducers of mitochondrial membrane permeability transition (MPT). Taken together, these results indicate that active amines, operating as AO substrates, play a critical role in controlling apoptosis through their effects on MPT and the respiratory chain activity by means of fluctuations in their concentrations. The conclusions of the above results may be that

Amine Oxidases

apoptosis is induced by polyamines through their oxidation products. Other studies exist demonstrating instead the ability of polyamines to protect cells from apoptosis. This discrepancy can be explained by taking into account the protective effect of the same polyamines, probably due to a scavenging action of ROS. A crucial role of AOs in cancer promotion has also to be considered. High levels of DAO activity were occasionally found in rapidly growing tissues, while in some patients, even affected by metastatic tumors, the level of circulating DAO was unaltered. A strong correlation between serum AO activity and the factor responsible for ▶ angiogenesis was found in non–small cell lung cancer patients. DAO activity in the small intestine mucosa was reported to increase in parallel with the degree of cell maturation, being highest in differentiated villus tip cells and lowest in the proliferative compartment. It was also found to increase in regenerating rat liver, with a peak between 16 and 48 h after partial hepatectomy. DAO activity peaks at the outset of growth and falls during the logarithmic growth phase of the cells. An increasing degree of malignancy associated with an increase of MAO A activity and decrease of MAO B and Cu-AOs activities in chemically-induced mammary cancer in the rat has been observed. Elevated activity of AO was found in skeletal metastases of prostatic cancer (▶ prostate cancer clinical oncology). DAO and arginase, an enzyme that catalyses the synthesis of ornithine from arginine, increase in tumor tissues as compared with benign prostatic hyperplasia. A linear correlation between arginase and DAO activities was observed in patients with cancer. A high concentration of PAO and DAO was found in the cervical intraepithelial neoplasia. The rise from normal conditions seems to produce cytological changes and to play a role in the etiology of ▶ cervical cancer. DAO activity is present at high levels both in tumor tissues and in biological fluids of tumor-bearing subjects. A correlation between the degree of tumor malignancy and their levels of AO activity has been observed in astrocytomas, where the activity is proportional to the degree of malignancy. The oxidation products of biogenic amines should

AML1/MTG8

also be carcinogenic. Acrolein, produced from the oxidation of spermine and spermidine by AOs, appears to be both carcinogenic and cytotoxic. This compound is considered to be a component of a universal cell growth regulatory system. It may act as mediator of cell transformation under oxidative stress when cells are pretreated with benzopyrene, a major carcinogenic found in cigarette smoke. The oxidation products of spermine, spermidine, and putrescine should be cofactors in the development of cervical cancer. The balance between the cell content of biogenic amine oxidizing enzymes and antioxidizing enzymes appears to be a crucial point for cancer inhibition or progression. As a general conclusion, the cancer inhibition/promotion effect of AOs might be explained by taking into consideration the full pattern of the enzymes contained in the cells. A long-lasting imbalance of antioxidizing enzymes and AO activity may be carcinogenic, while AOs are rapidly cytotoxic for cancer cells, because of their higher biogenic amines concentration in comparison with normal cells.

Cross-References ▶ Angiogenesis ▶ Cervical Cancers ▶ Polyamines ▶ Prostate Cancer Clinical Oncology

203 (2012) Differentiation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1113. doi: 10.1007/978-3-642-16483-5_1616 (2012) Postischemic Reperfusion. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2965. doi: 10.1007/978-3-642-164835_4692

Amino-Bisphosphonate ▶ Minodronate

4-Amino-1-(2-deoxy-beta-D-erythropentofuranosyl)-1,3,5-triazin-2(1H)one ▶ 5-Aza-20 Deoxycytidine

AML1 ▶ Runx1

AML1/ETO ▶ Chromosomal Translocation t(8;21)

References Bachrach U, Eilon G (1967) Interaction of oxidized polyamine with DNA. I. Evidence of the formation of crosslinks. Biochim Biophys Acta 145:418–4263 Floris G, Mondovì B (eds) (2009) Copper amine oxidases. Structures, catalytic mechanisms, and role in pathophysiology. CRC Press/Taylor & Francis Group, Boca Raton Toninello A, Pietrangeli P, De Marchi U et al (2006) Amine oxidases in apoptosis and cancer. Biochim Biophys Acta 1765:1–13

See Also (2012) Allergic disease. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 137. doi: 10.1007/978-3-642-16483-5_190

AML1/EVI-1 ▶ AME Transcription Factor

AML1/MTG8 ▶ Chromosomal Translocation t(8;21)

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AMN107 ▶ Nilotinib

Amph II ▶ Bin1

Amphibian Gastrin-Releasing Peptide ▶ Gastrin-Releasing Peptide

Amphiphysin II ▶ Bin1

Amphiphysin-Like ▶ Bin1

Amphiregulin Matias A. Avila and Carmen Berasain Division of Hepatology, CIMA, University of Navarra, Pamplona, Spain

Synonyms AREG; Schwannoma-derived growth factor; SDGF

Definition Amphiregulin (AREG) is a growth factor that belongs to the ▶ epidermal growth factor receptor

AMN107

(EGFR) family of ligands. AREG was originally described as a regulator of cell growth present in the conditioned media of MCF-7 breast tumor cells. AREG has been implicated in different physiologic processes including mammary gland and bone development, lung and kidney branching morphogenesis, and trophoblast growth. The expression of AREG is upregulated in a variety of cancerous tissues, and signaling triggered by AREG is believed to be important in tumorigenesis.

Characteristics The AREG human gene spans 10 kb in the genomic DNA and it is composed of six exons; upon transcription it produces a 1.4 kb mRNA. AREG gene shows broad constitutive expression, being more prevalent in human ovary and placenta although it is also expressed in pancreas, cardiac muscle, testis, colon, breast, lung, spleen, and kidney, whereas it is undetectable in liver. Transactivation of AREG promoter and AREG gene expression can be induced by the ▶ Wilms’ tumor suppressor and through the activation of the protein kinase C (PKC), mitogen associated protein kinase (MAPK), Yes-associated protein (YAP/TEAD), b-catenin, and cyclic AMP/protein kinase A (cAMP/PKA) pathways (Fig. 1). AREG is synthesized as a 252-amino acid transmembrane glycoprotein, also known as transmembrane precursor or pro-form (Pro-AREG) (Fig. 1). Pro-AREG consists of a hydrophilic extracellular N-terminus (or ectodomain), a hydrophobic transmembrane domain (TM), and a hydrophilic cytoplasmic C-terminus (CT-tail) (Fig. 1). In the extracellular N-terminus we can distinguish an N-terminal pro-region containing glycosylation sites followed by a heparin-binding domain and an EGF-like region (Fig. 1). The EGF-like region is shared by other members of the EGF family of ligands. At the plasma membrane Pro-AREG undergoes proteolytic cleavage to release the mature soluble factor in a process known as “ectodomain shedding.” Cleavage of Pro-AREG at two N-terminal sites gives rise to two major soluble forms of ~19 and ~21 kDa. Alternatively,

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Amphiregulin, Fig. 1 Transcription of the AREG gene can be activated in response to the WT1 protein and the PKC, cAMP/PKA, b-catenin, YAP/TEAD, or MAPK signaling pathways. AREG is synthesized as a membraneanchored precursor (Pro-AREG) encompassing an EGF-like domain, a heparin-binding domain (HB), a transmembrane region, and a carboxy-terminal cytosolic tail (CT-tail). Upon digestion by the protease TACE/ ADAM17, soluble AREG forms are shed from the cell surface and can interact with the EGFR in an autocrine or paracrine fashion, or bind to heparan-sulfate proteoglycans (HSPG) in the extracellular millieu. Exosome-associated Pro-AREG can be also released from cells and engage in

autocrine/paracrine signaling. Alternatively, juxtacrine interaction of membrane-anchored Pro-AREG with the EGFR is also possible. Pro-AREG and the AREG carboxy terminal fragment (AREG-CTF) produced upon TACE/ ADAM17 digestion can translocate to the nucleus and potentially modulate gene expression. Shedding of AREG by TACE/ADAM17 can be enhanced in response to activation of G-protein coupled receptors (GPCRs), other growth factor tyrosine kinase receptors (TK-R), and inflammatory receptors, such as Toll-like receptors (TLRs). Binding and activation of the EGFR by AREG triggers growth and survival intracellular signals essential for the tumor cell

Pro-AREG cleavage can produce a larger 43-kDa soluble protein corresponding to the entire extracellular domain. Cleavage of Pro-AREG at the cell surface can be mediated by tumor necrosis factor-a converting enzyme (TACE), a member of the disintegrin and metalloproteinase (ADAM) family also known as ▶ ADAM17 (Fig. 1). Shedding of AREG, or exosome-mediated Pro-AREG release from cells, allows the autocrine or paracrine interaction of the mature ligand with its

cognate receptor, the EGFR (also known as ErbB1), a transmembrane protein endowed with tyrosine kinase activity, although juxtacrine interaction between membrane-bound Pro-AREG and the EGFR has also been observed (Fig. 1). Besides changes in AREG gene expression, different stimuli can also influence the availability of this growth factor through the stimulation of Pro-AREG cleavage at the cell membrane. This is achieved by the activation of TACE/ADAM17

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in response to agonists acting through GPCRs, other growth factor receptors, or proinflammatory molecules, in a process termed EGFR transactivation (Fig. 1). Binding of AREG to EGFR triggers key intracellular signaling pathways, such as the mitogenic MAPK and survival PI3K/Akt pathways, as well as the mTOR and STAT pathways, which have been demonstrated to participate in the transduction of AREG effects (Fig. 1). Although all members of the EGF family can bind and activate the EGFR, there are differences in the pattern and intensity of EGFR tyrosine phosphorylation, and EGFR turnover dynamics, elicited by AREG. This, together with the ability of AREG to bind HSPGs at the cell surface, may impart specificity in the cellular effects elicited by AREG versus other EGFR ligands. Amphiregulin Expression and Function AREG was originally identified as a factor capable of inhibiting the growth of certain carcinoma cell lines, while stimulating the proliferation of normal cells, a fact that motivated its denomination. In fact, depending on its concentration and the nature of the target cell, AREG promotes the growth and survival of most cell types, both normal and transformed. AREG gene overexpression has been frequently demonstrated in cancerous tissues like colon, breast, bladder, prostate, pancreas, lung, ovary, squamous cell carcinomas, hepatocarcinoma, and myeloma cells. The existence of EGFR transactivation involving the release of AREG has been demonstrated in a variety of cancer cells. In this context AREG could be an important mediator between diverse stimuli, including inflammatory signals, acting on GPCRs and other cell surface receptors, and the activation of protumorigenic signals conveyed through the EGFR (Fig. 1). Interference with AREG production by means of specific antisense RNAs or ▶ siRNAs, or treatment with AREG neutralizing antibodies, has been shown to revert many of the neoplastic phenotypic traits of cancer cells in vitro, even though the expression of other EGFR ligands was preserved in these cells. This

Amphiregulin

suggests that AREG plays a nonredundant role in carcinogenesis. Observations performed in vivo also lend support to a role for AREG in the initiation and maintenance of the neoplastic properties of tumor cells. For instance, tissue-specific transgenic overexpression of AREG in pancreas results in enhanced cell cycle progression, and in mice older than 1 year it induces dysplastic changes and premalignant alterations. AREG is also emerging as an important regulator in the tumor microenvironment. AREG produced by monocyte-derived dendritic cells has been identified as a potent pro-tumorigenic factor in lung cancer progression. Moreover, AREG released by tumorassociated mast cells significantly enhances the activity of regulatory T cells, contributing to the immune suppressive environment within the tumor and therefore to its progression. Although, so far most of the evidences that support a role for AREG in cancer development and progression have been gathered under experimental conditions, there are also clinical studies that point in the same direction. In this regard, a significant correlation has been established between elevated tumor tissue AREG mRNA levels and poor survival in bladder carcinoma patients, or elevated serum AREG concentrations and increased mortality in non–small cell lung cancer patients. AREG expression has been also linked to the development of drug resistance in cancer cells, including targeted drugs such as sorafenib in hepatocellular carcinoma. In summary, the current knowledge on AR in cancer suggests that increased availability of this growth factor can provide transformed cells with a selective advantage. Targeted inhibition of AR expression or action may therefore represent a useful therapeutic strategy for a wide variety of cancers.

Cross-References ▶ ADAM17 ▶ Akt Signal Transduction Pathway ▶ Epidermal Growth Factor-Like Ligands

Amplification

▶ Epidermal Growth Factor Receptor ▶ PI3K Signaling ▶ SiRNA ▶ Wilms’ Tumor

References Berasain C, Avila MA (2014) Amphiregulin. Semin Cell Dev Biol 28:31–41 Fischer OM, Hart S, Gschiwnd A et al (2003) EGFR signal transactivation in cancer cells. Biochem Soc Trans 31:1203–1208 Lee DC, Hinkle CL, Jackson LF et al (2003) EGF family ligands. In: Thomson AW, Lotze MT (eds) The cytokine handbook. Academic, London, pp 959–987 Normanno N, De Luca A, Bianco C et al (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366:2–16 Sanderson MP, Dempsey PJ, Dunbar AJ (2006) Control of ErbB signaling through metalloprotease mediated ectodomain shedding of EGF-like factors. Growth Factors 24:121–136

See Also (2012) CAMP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 603. doi:10.1007/978-3-642-16483-5_788 (2012) EGFR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1828 (2012) EGFR Transactivation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1829 (2012) G-protein Couple Receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1587. doi:10.1007/978-3-642-164835_2294 (2012) MAPK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532

AMPL ▶ Bin1

Amplaxin ▶ Cortactin

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Amplification Manfred Schwab German Cancer Research Center (DKFZ), Heidelberg, Germany

Definition Amplification is the selective increase of DNA copy number either intracellularly, as a local genomic change, or experimentally, by polymerase chain reaction (PCR). Increase of the level of mRNA or protein alone should not be referred to as amplification.

Characteristics Intracellular amplification results in a selective increase in gene copy number with the consequence of elevated gene expression. Gene amplification has been seen in three different settings • Scheduled amplification as part of a developmental gene expression program, e.g., chorion genes in ovaries of the fruitfly Drosophila melanogaster or actin genes during myogenesis in the chicken. • Unscheduled amplification during acquisition of cellular ▶ drug resistance. For example, amplification of the gene encoding dihydrofolate reductase (DHFR) can result in up to 1,000 gene copies per cell with the consequence of cellular resistance against the ▶ chemotherapy drug methotrexate. • Unscheduled amplification of cellular genes involved in growth control (▶ oncogenes) during tumor progression. Amplification of oncogenes can result in up to several hundred gene copies and enhanced gene expression. Usually large DNA stretches (from 100 Kb up to several Mb) are amplified, and therefore syntenic genes in addition to the particular

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Amplification, Fig. 1 Cytogenetics of MYCN amplification in neuroblastoma cells. Chromosomal fluorescence in situ hybridization (FISH). High-level MYCN amplification appears in human neuroblastoma cells as two alternative cytogenetic manifestations: (a). Double minutes (DMs) (left), this tumor cell has in addition to amplified MYCN (red) amplification of another oncogene MDM2 (green). The two oncogenes are non-syntenic (2p24, and 12q13–14, respectively), and the amplification is the result of two independent genetic events. (b) Homogeneously

staining region (HSR) (right), multiple copies are amplified in an HSR on chromosome 12 (with strong signal), while single copy gene is retained on the two parental chromosomes (arrows). The retention of MYCN at 2p24 indicates that not the original MYCN gene but rather a copy, presumably the result of extra-replication, has been amplified. Note also the strong signal in interphase nuclei which allows detection of amplified MYCN in tumor biopsies when chromosomes cannot be prepared

oncogene can be co-amplified due to their close linkage to the oncogene. Alternatively, different non-syntenic oncogenes can amplify independently in the same cell. The prototypic human cancer with oncogene amplification is ▶ neuroblastoma. Here, the amplified gene, MYCN, is a biomarker for patient management.

about genomic or environmental elements involved in amplification. Unscheduled amplification presumably is a sporadic event that can become stabilized under selective pressures, i.e., cytostatic drugs or if cells acquire a growth advantage within a certain tissue architecture.

Amplified DNA can be visualized cytogenetically as a homogeneously staining region (HSR) within chromosomes, as double minutes (DM), or as C-bandless chromosomes (CM) (Fig. 1). Cellular Regulation Amplification can follow different The “onion skin model” and fusion-bridge” (BFB) cycles (Fig. fit experimental observations. Little

pathways. “breakage 2), both is known

Clinical Relevance Resistance against cytostatic drugs poses a big problem in cancer therapy. Amplified oncogenes contribute to tumor progression, many different oncogenes have been found amplified (e.g., RAS, MYC, MYCN, MYCL, HER-2 (▶ HER-2/Neu), ABL in some tumor types the oncogene status provides information about patient prognosis: Amplified MYCN indicates poor prognosis for stage 1–3 ▶ neuroblastoma; and amplified HER-2 indicates unfavorable outcome in a subgroup of ▶ breast cancer.

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Amplification, Fig. 2 Breakage-fusion-bridge (BFB) cycles in early stages of amplification. (a) BFB cycles start from common ▶ fragile sites, where a DNA break can occur in both sister chromatids. DNA repair systems will be recruited to the break and may join the free DNA ends of the two sister chromatids to form a dicentric chromosome, one that has two centromers. At anaphase, where sister chromatids are moved to the daughter cells, the

6x

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dicentric chromosome at some point will break. Of the two daughter cells, one will carry a deletion, the other an inverted duplication of DNA, which is equivalent to a low-level amplification. By subsequent BFB cycles, the level of amplification can increase. (b) Low level amplification as the result of BFB cycles. FISH image, where each color shows the position and copy-number of a particular DNA sequence

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Cross-References ▶ Breast Cancer ▶ Chemotherapy ▶ Drug Resistance ▶ Fragile Sites ▶ HER-2/neu ▶ MYC Oncogene ▶ Neuroblastoma ▶ Oncogene ▶ RAS Genes

References Savelyeva L, Schwab M (2001) Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett 167:115–123 Schwab M (1998) Amplification of oncogenes in human cancer cells. Bioessays 20:473–479 Schwab M, Westermann F, Hero B et al (2003) Neuroblastoma: biology, and molecular and chromosomal pathology. Lancet Oncol 4:472–480

See Also (2012) ABL. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 14. doi:10.1007/978-3-642-16483-5_15 (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 408– 409. doi:10.1007/978-3-642-16483-5_6601 (2012) C-Bandless chromosome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 684. doi:10.1007/978-3-642-16483-5_896 (2012) Double minute. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1155. doi:10.1007/978-3-642-16483-5_1717 (2012) Homogeneously staining region. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1725. doi:10.1007/978-3-642-164835_2797 (2012) Methotrexate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2274. doi:10.1007/978-3-642-16483-5_3680 (2012) MYCL. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2430. doi:10.1007/978-3-642-16483-5_3924 (2012) MYCN. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2430–2431. doi:10.1007/978-3-642-16483-5_3925 (2012) Non-syntenic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2554. doi:10.1007/978-3-642-16483-5_4120 (2012) PCR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2803. doi:10.1007/978-3-642-16483-5_4417

Amplified in Breast Cancer 1 (2012) Sister-chromatids. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3418. doi:10.1007/978-3-642-16483-5_5329 (2012) Syntenic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3595. doi:10.1007/978-3-642-16483-5_5628 (2012) Tumor progression. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6046

Amplified in Breast Cancer 1 Jianming Xu Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Synonyms AIB1; Coactivator ACTR; NCoA3; Nuclear receptor coactivator 3; p/CIP; p300/CBPinteracting protein; RAC3; Receptor-associated coactivator 3; SRC-3; Steroid receptor coactivator-3; Thyroid hormone receptor activator molecule 1; TRAM-1

Definition AIB1 is a 160-kDa intracellular protein that enhances gene expression through interacting with nuclear hormone receptors and some other transcription factors and serving as a transcriptional coactivator. The AIB1 gene is amplified and overexpressed in some human breast tumors.

Characteristics Molecular Structure and Functional Domains The human AIB1 gene is located in chromosome 20, and it encodes for a 160-kDa intracellular protein with 1402 amino acid residues. AIB1 is a member of the p160 steroid receptor coactivator (SRC) family that also includes SRC-1 and the transcriptional intermediary factor 2 (TIF2). AIB1 contains multiple structural and functional

Amplified in Breast Cancer 1 CR1 bHLH/PAS

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Ac Receptor & AIB1 complex

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Amplified in Breast Cancer 1, Fig. 1 Schematic presentation of the structure and function of AIB1. CR1, CR2, and CR3 conserved regions 1, 2, and 3 in the p160 SRC family, bHLH/PAS the basic helix-loop-helix/Per-Ah receptor nuclear translocator-Sim domain, S/T the serine and threonine-rich domain, L, L, and L the three LXXLL motifs responsible for interaction with nuclear receptors, Q and Q the two glutamine-rich regions, HAT the histone acetyltransferase domain, H hormone, NR nuclear

receptors, CBP the CREB (cAMP response elementbinding protein)-binding protein, p300 the 300-kDa protein homologous to CBP, p/CAF the p300- and CBP-associated factor, CARM1 the coactivator-associated arginine methyltransferase 1, PRMT1 the protein arginine methyltransferase 1, TBP the TATA-binding protein, TAFIIs TBP-associated general transcription factors (GTFs), Pol II RNA polymerase II

domains (Fig. 1). The N-terminal basic helixloop-helix/Per-Ah receptor nuclear translocatorSim (bHLH/PAS) domain is the most conserved region in the molecule with ~70% sequence similarity to the respective regions of SRC-1 and TIF2. The bHLH/PAS domain contains a nuclear localization signal, which is required for AIB1 to get into the cellular nucleus where AIB1regulated gene transcription takes place and where AIB1 degrades in a proteasome-dependent manner. The bHLH/PAS domain also can interact with certain transcription factors such as myogenin to mediate their transcriptional functions. The serine/threonine (S/T)-rich domain contains many serine and threonine residues, and some of these residues are targets of serine/threonine kinases. The phosphorylation status of AIB1 is related to its interaction specificity and affinity with transcription factors and other coactivators. A sequence in the S/T domain is also found to interact with transcription factor E2F1. Through interaction and function with E2F1, AIB1 can play a role in direct regulation of cell cycle. Following the S/T domain is the second conserved region of AIB1 with ~60% sequence similarity to SRC-1 and TIF2. This region contains three

LXXLL (L, leucine; X, any amino acid) a-helix motifs that are responsible for interaction with the ligand-binding domain of nuclear receptors in a hormone binding-dependent manner. The third conserved region located in the C-terminus of AIB1 has ~50% sequence similarity to SRC-1 and TIF2 and contains two poly-glutamine stretches and a weak histone acetyltransferase activity. This domain can steadily interact with CREB (cAMP response element-binding protein)-binding protein (CBP) and p300, which are strong histone acetyltransferases. This domain also can interact with the coactivator-associated arginine methyltransferase (CARM1) and the protein arginine methyltransferase 1 (PRMT1), which are histone methyltransferases. Functional Mechanisms Two transcriptional activation domains of AIB1 have been identified. The first one is located in the region that interacts with CBP or p300, and the second one is located in the region that interacts with CARM1 or PRMT1 (Fig. 1). The transcriptional activation function of AIB1 is mainly executed through these acetyltransferases and methyltransferases, which are chromatin-

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remodeling enzymes. In the case of steroid hormone-regulated gene expression, hormone binding triggers a series of events for steroid receptors, including the dissociation of heat shock proteins, change of receptor conformation, receptor dimerization, and DNA binding. Importantly, the hormone binding also induces the steroid receptors to expose their coactivator-binding motifs in their ligand-binding domains and allows coactivators such as AIB1 to be recruited to the enhancer region of the nuclear receptor target genes. Through the further interaction of AIB1 with CBP, p300, the p300- and CBP-associated factor (p/CAF), CARM1, and PRMT1, a steroid receptor-directed transcriptional activation complex is built up on the hormone response elements of their target gene. This protein complex uses its protein acetyltransferase and methyltransferase activities to remodel the chromatin structure, to facilitate the assembly of general transcription factors on the promoter, and thereby to promote target gene transcription. In addition to steroid receptors and other nuclear receptors, AIB1 also serves as a coactivator for certain other transcription factors such as E2F1, AP-1, and Ets transcription factors. Physiological Function AIB1 mRNA is expressed in many different human tissues and cell lines when examined by Northern blot analysis. Detail analyses with mouse tissues revealed that AIB1 is mainly expressed in the mammary gland epithelial cells, oocytes, vaginal epithelial layer, hepatocytes, smooth muscle cells, endothelial cells, and the hippocampus and olfactory bulbs of the brain. At this time, our knowledge regarding the in vivo physiological function of AIB1 is mainly learned from the AIB1 knockout mice. AIB1-deficienct mice have a much lower level of insulin-like growth factor-I and 17b-▶ estradiol in their circulation. Accordingly, these mice are smaller in size, and they exhibit delayed puberty, retarded mammary gland development, and reduced female reproductive function. In addition, AIB1 plays a beneficial role in estrogen and ▶ estrogen receptor-mediated vascular protection after vessel injury by enhancing estrogen receptor function and contributes to the control of acute

Amplified in Breast Cancer 1

inflammatory responses by inhibiting the production of pro-inflammatory cytokines. Role in Cancer The AIB1 gene is amplified (or increased in the number of gene copies) in about 5–10% human breast tumors. The AIB1 mRNA is overexpressed in about 30–60% breast tumors, depending on the resources of reports. However, some study only found about 10% of breast tumors that have elevated AIB1 protein levels. AIB1 overproduction is observed in breast tumors both positive and negative to the estrogen receptor a. In ▶ tamoxifentreated patients, high levels of AIB1 are associated with the HER-2/neu expression, the tamoxifen resistance, and the lower disease-free survival rates. In the cultured ▶ breast cancer cells, AIB1, together with the estrogen and estrogen receptor, enhances cyclin D1 expression and cell cycle progression. Downregulation of AIB1 in breast cancer cells inhibits cell proliferation, cell motility, and anchorage-independent growth in the culture and tumor formation in the immune-deficient mice. Animal experiments further demonstrate that AIB1-deficient mice are resistant to either transgenic ▶ oncogene- or chemical carcinogeninduced mammary gland tumorigenesis. The transgenic v-Ha-ras oncogene can no longer induce mammary gland tumors in the ovariectomized AIB1 knockout mice, suggesting that inhibition of AIB1 function and removal of ovarian ▶ hormones may be a potential strategy to control breast tumorigenesis. On the other hand, it has been demonstrated that overexpression of AIB1 in the mouse mammary epithelial cells is sufficient to induce a high frequency of mammary gland tumors, indicating that AIB1 is an oncoprotein. Similar to the role of AIB1 in breast cancer, AIB1 is also found to be overexpressed in certain human prostate tumors and to play a detrimental role in prostate epithelial tumorigenesis in mouse models.

References Anzick SL et al (1997) AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968

Amrubicin Kuang SQ et al (2004) AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Cancer Res 64:1875–1885 Torres-Arzayus MI et al (2004) High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene. Cancer Cell 6:263–274 Xu J, Li Q (2003) Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692 Xu J et al (2000) The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development. Proc Natl Acad Sci USA 97:6379–6384

Amrubicin Michiko Yamamoto, Noriyuki Masuda and Tomoya Fukui Department of Respiratory Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan

Synonyms (+)-(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-b-Derythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro6,11-dihydroxy-5,12-naphthacenedione hydrochloride; SM-5887

Definition The anthracyclines that have been tested clinically so far have been limited to those produced by fermentation or semi-synthetic processes. In contrast, 9-aminoanthracycline oramrubicin is a fully synthetic drug. Amrubicin differs from daunosamine in that it contains a 9-amino group and a simple sugar moiety (Fig. 1).

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tumor tissue, through reduction of its C-13 ketone group to a hydroxy group. Despite the similarity of its chemical structure to that of a representative anthracycline such as doxorubicin, amrubicin has a different mode of action that differs from that of doxorubicin. Amrubicin and amrubicinol are inhibitors of DNA topoisomerase II, which exert their cytotoxic effects by stabilizing a topoisomerase II-mediated cleavable complex (▶ topoisomerase enzymes as drug targets), and are approximately only one-tenth as potent as doxorubicin in generating intercalated DNA. Preclinical Studies In in vitro experiments, amrubicin and its metabolite amrubicinol have been found to be active against a broad spectrum of human cell lines established from cancers of the lung, prostate, urinary bladder, colon, kidney, pancreas, and uterus. Amrubicinol has been shown to exhibit a 20- to 220-fold increase in antitumor activity compared to amrubicin in vitro, with amrubicin being as potent as doxorubicin. In addition, amrubicin and amrubicinol have also been shown to demonstrate a degree of noncross resistance with doxorubicin. Amrubicin has been shown to be more effective against five human xenografts including a breast, a lung, and three gastric cancers; equally effective against two gastric cancers; and less effective against a lung and two gastric cancers. Amrubicin caused dose-dependent weight loss, ataxia, myelosuppression, and hair loss in mice after a single intravenous (iv) injection. The maximum tolerated dose (MTD) for amurubicin was estimated to be 25 mg/kg in four mouse strains. Cardiotoxicity is one of the dose-limiting toxicities (DLTs) in case of anthracyclines; however, amrubicin showed little delayed-type cardiotoxicity in rabbit or dog experimental models. Furthermore, amrubicin did not aggravate doxorubicin-induced myocardial injury. Clinical Studies

Characteristics Amrubicin Monotherapy

Amrubicin is converted to its active 13-hydroxy metabolite, amrubicinol, in the liver, kidney, and

On the basis of the finding that amrubicin exhibited enhanced antitumor efficacy, in vitro

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Amrubicin, Fig. 1 Chemical structure of amrubicin and its active metabolite, amrubicinol

phase I trials of amrubicin were carried out in patients with lung cancer for three consecutive days. In the phase I study, four patients with non–small cell lung cancer (NSCLC) were enrolled at dose level 1 (40 mg/m2/day) and four at dose level 2 (45 mg/m2/day). No DLTs was observed at these dose levels. At dose level 3 (50 mg/m2/day), three of five patients experienced DLTs (leukopenia, neutropenia, thrombocytopenia, or gastrointestinal toxicities). The MTD and recommended dose (RD) were determined to be 50 mg/m2/day and 45 mg/m2/day, respectively. Another phase I trial showed the MTD of amrubicin was 40 mg/m2 in previously treated patients with lung cancer [8 NSCLCs and 7 small cell lung cancers (SCLCs)]. The DLTs were grade 4 neutropenia, febrile neutropenia, or grade 3 arrhythmia in the three patients treated at this dose. The RD for patients with refractory lung cancer was 35 mg/m2/day for three consecutive days every three weeks. In a multicenter phase II study, 61 previously untreated patients were enrolled who had been diagnosed with advanced NSCLC (1). Amrubicin

was administered by a single, daily iv injection at a dose of 45 mg/m2 for three consecutive days every three weeks. The overall response rate (ORR) was 27.9%, and the median survival time (MST) was 9.8 months. The major toxicity was myelosuppression. The incidences of grade 3 or 4 toxicity were 72.1% for neutropenia, 52.5% for leukopenia, 23.0% for anemia, and 14.8% for thrombocytopenia. In relapsed SCLC patients who had previously been treated with at least one platinum-based chemotherapeutic regimen, three phase II studies have been conducted in Japan. Amrubicin was administered at a reduced dose of 35–40 mg/m2/ day for three consecutive days every 3–4 weeks, in view of prior chemo- or radiotherapy. The ORR in patients who relapsed after 3 months from completion of the initial therapy (sensitive group) was 52–60% and that in patients who experienced disease progression within 3 months (refractory group) was 17–50%. The median progression-free survival (PFS) and MST were 3.9–4.5 and 9.2–12 months in the sensitive group, and 2.6–4.0 and 5.3–11 months in the

Amrubicin

refractory group, respectively. The major toxicity was hematological, including neutropenia, thrombocytopenia, and anemia with febrile neutropenia, while nonhematological toxicities were mild (2). In randomized phase II studies, amrubicin (A) was evaluated in comparison with topotecan (T) as second-line treatment for patients with SCLC. Sixty patients who had previously been treated with platinum-based chemotherapy were randomly assigned to receive either amrubicin (40 mg/m2 on day1–3, every three weeks) or topotecan (1.0 mg/m2 on day1–5, every three weeks) in Japanese study (3). The ORR was 38% [95% confidence interval (CI), 20–56%] for the A arm, 53% in sensitive group, and 17% in refractory group, while the ORR for the T arm was 13% (95% CI, 1–25%), 21% in sensitive group, and 0% in refractory group. The PFS and OS were 3.5 and 8.1 months on the A arm, respectively, and 2.2 and 8.4 months on the T arm, respectively. Neutropenia was severe in the A arm compared with the T arm (grade 4 neutropenia 79% vs. 43%; febrile neutropenia 14% vs. 3%), while one treatment-related death due to infection occurred in the A arm. In a US randomized phase II study for SCLC patients with sensitive relapse (4), 76 patients were randomly assigned 2:1 to either amrubicin (40 mg/m2 on day1–3, every three weeks) or topotecan (1.5 mg/m2 on day1–5, every three weeks). Treatment with amrubicin also resulted in a significantly higher ORR than topotecan (44% vs. 15%). The PFS and OS were 4.5 and 9.2 months with amrubicin and 3.3 and 7.6 months with topotecan, respectively. Toxicities were similar with both agents, although grade 3 or higher neutropenia and thrombocytopenia seemed to be more frequent in the T arm as compared with the A arm (78% and 61% vs. 61% and 39%, respectively). For SCLC patients with refractory relapse, confirmatory studies of amrubicin were conducted in Japan (5) and the USA/EU (6). The ORR, PFS, and OS were 32.9% (95% CI, 22.9–44.2%), 3.5 months, and 8.9 months in Japan, and 21.3% (95% CI, 12.7–32.3%), 3.2 months and 6.0 months in the USA/EU, respectively. Neutropenia was the most common

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grade 3 or 4 toxicity (93.9% in Japan and 67% in the USA/EU), with febrile neutropenia (26.8% in Japan and 12% in the USA/EU). From these results, it was concluded that amrubicin showed significant activity against both sensitive and refractory relapsed SCLC, as well as predictable and manageable toxicities. Amrubicin Combination Chemotherapy

To determine the MTDs for a combination of amrubicin and a platinum-based therapeutic (cisplatin or carboplatin), the efficacy and safety of these drugs at their RD were assessed in phase I studies (2). Amrubicin was administered daily for 3 consecutive days every three weeks. These studies determined that the RDs of amrubicin and cisplatin were 40 mg/m2 and 60 mg/m2, respectively, for previously untreated patients with ED-SCLC; the RDs of amrubicin and carboplatin were 35 mg/m2 and AUC 5 for patients with ED-SCLC, and 35 mg/m2 and AUC 4 in elderly patients with ED-SCLC. The common toxicities were neutropenia, leucopenia, thrombocytopenia, and febrile neutropenia. For patients with advanced NSCLC, we conducted a phase I trial of the topoisomerase I inhibitor, irinotecan (CPT-11), in combination with amrubicin (7). Eleven patients were treated with amrubicin on days 1–3, combined with 60 mg/m2 of CPT-11 on days 1 and 8, every three weeks. The amrubicin dose of 30 mg/m2 was one dose level above the MTD, since three of the five patients experienced DLT, namely, diarrhea and leukopenia. Amrubicin did not affect the pharmacokinetics of CPT-11, SN-38, or SN-38 glucuronide. The RD for phase II studies was determined to be 60 mg/m2 for CPT-11 (days 1 and 8) and 25 mg/m2 for amrubicin (days 1–3), administered every three weeks. In a first-line setting, a randomized phase III study (8) comparing amrubicin plus cisplatin (AP) with irinotecan plus cisplatin (IP) in patients with ED-SCLC was conducted to confirm the noninferiority of AP in terms of overall survival. A total of 284 patients were assigned to either an AP (40 mg/m2 of amrubicin on day1–3 plus 60 mg/m2 of cisplatin on day1, every three weeks) or IP (60 mg/m2 of irinotecan on day1,

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8, and 15 plus 60 mg/m2 of cisplatin on day1, every four weeks) arm. After 66% (n = 94 of 142) of patients in the AP arm had been enrolled, the protocol was revised to reduce the initial dose of amrubicin to 35 mg/m2 because of the high incidence of severe hematological toxicities. The ORR was 77.9% (AP) vs. 72.3% (IP) while the PFS was 5.1 months (AP) vs. 5.6 months (IP) and the MST was 15.0 months (AP) vs. 17.7 months (IP) (hazard ratio, 1.43; 95% CI, 1.10–1.85). The adverse events observed in both the AP and IP arms were grade 4 neutropenia (79.3% and 22.5%), febrile neutropenia (32.1% and 10.6%), and grade 3 or 4 diarrhea (1.4% and 7.7%). Since AP therapy proved inferior to IP therapy in this study, AP is considered as a salvage therapy for IP in standard treatment regimens for ED-SCLC in Japan. A European group reported a randomized phase II study (9) of single-agent amrubicin or combination therapy with cisplatin versus etoposide plus cisplatin as first-line treatment in patients with ED-SCLC. Ninety-nine patients were randomized, 33 in every arm, and each received 3 weekly cycles of either 45 mg/m2 of amrubicin on day1–3 (A); 40 mg/m2 of amrubicin on day1–3 plus 60 mg/m2 of cisplatin on day1 (AP); or 100 mg/m2 etoposide on day1, iv/oral on day 2–3, plus75 mg/m2 cisplatin on day1 (EP). The ORR was 61%, 77%, and 63% for the A, AP, and EP regimens, respectively. The PFS and OS were 5.2 and 11.1 months (A), 6.9 and 11.1 months (AP), 5.8 and 10 months (EP), respectively. Grade 3 or 4 toxicities in A, AP, and EP were neutropenia (73%, 73%, and 69%, respectively), thrombocytopenia (17%, 15%, and 9.4%, respectively), anemia (10%, 15%, and 3.1%, respectively), and febrile neutropenia (13%, 18%, and 6%, respectively). One treatment-related death occurred in the A arm, three in the AP arm, and a further three in the EP arm. This study suggested that a combination therapy of amrubicin plus cisplatin had the highest response rate, despite higher hematological toxicities. For elderly patients with ED-SCLC, a phase II study (10) of amrubicin vs. carboplatin plus etoposide was conducted in Japan. Thirty-six elderly patients (aged 70 years) received 35 mg/m2 of amrubicin on day1–3 plus AUC 4 of carboplatin on day1 every three weeks. The

Amrubicin

ORR was 89%, the PFS was 5.8 months, and the OS was 18.6 months. Grade 3 or 4 neutropenia was observed in 97% of the patients. Six (17%) experienced grade 3 or 4 febrile neutropenia, while other toxicities were moderate and treatment-related death was not observed. The results suggested that a combination of amrubicin with carboplatin is effective, and the toxicities are acceptable even in elderly patients with SCLC. Conclusion The preclinical studies described here demonstrate that amrubicin has a unique mechanism of action as an anthracycline-derivative and displays a broad spectrum of antitumor activity. The results of clinical studies using amrubicin as a part of combination regimens in treating lung cancer have been promising. Although amrubicin has been investigated over the last decade, many of its characteristics still remain unclear. Further studies exploring the usefulness of amrubicin in treating other malignancies, either as a single or as a combinatorial agent with other cytotoxic agents including novel, molecular-targeting drugs, are therefore warranted.

Cross-References ▶ Topoisomerases

References Ding Qand Zhan J (2013) Amrubicin: potential in combination with cisplatin or carboplatin to treat small-cell lung cancer. Drug Des Devel Ther 7:681–689 Ettinger DS, Jotte R, Lorigan P et al (2010) Phase II study of amrubicin as second-line therapy in patients with platinum-refractory small-cell lung cancer. J Clin Oncol 28:2598–2603 Inoue A, Sugawara S, Yamazaki K et al (2008) Randomized phase II trial comparing amrubicin with topotecan in patients with previously treated small-cell lung cancer: North Japan Lung Cancer Study Group Trial 0402. J Clin Oncol 26:5401–5406 Inoue A, Ishimoto O, Fukumoto S et al (2010) A phase II study of amrubicin combined with carboplatin for elderly patients with small-cell lung cancer: North Japan Lung Cancer Study Group Trial 0405. Ann Oncol 21:800–803

Anaplastic Large Cell Lymphoma Jotte R, Conkling P, Reynolds C et al (2010) Randomized phase II trial of single-agent amrubicin or topotecan as second-line treatment in patients with small-cell lung cancer sensitive to first-line platinum-based chemotherapy. J Clin Oncol 29:287–293 Murakamia H, Yamamotoa N, Shibata T et al (2014) A single-arm confirmatory study of amrubicin therapy in patients with refractory small-cell lung cancer: Japan Clinical Oncology Group Study (JCOG0901). Lung Cancer 84:67–72 O’Brien ME, Konopa K, Lorigan P et al (2011) Randomised phase II study of amrubicin as single agent or in combination with cisplatin versus cisplatin etoposide as first-line treatment in patients with extensive stage small cell lung cancer – EORTC 08062. Eur J Cancer 47:2322–2330 Satouchi M, Kotani Y, Shibata T et al (2014) Phase III study comparing amrubicin plus cisplatin with irinotecan plus cisplatin in the treatment of extensivedisease small-cell lung cancer: JCOG 0509. J Clin Oncol 32:1262–1268 Sawa T, Yana T, Takada M et al (2006) Multicenter phase II study of amrubicin, 9-amino-anthracycline, in patients with advanced non-small-cell lung cancer (Study 1): West Japan Thoracic Oncology Group (WJTOG) trial. Invest New Drugs 24:151–158 Yanaihara T, Yokoba M, Onoda S et al (2007) Phase I and pharmacologic study of irinotecan and amrubicin in advanced non-small cell lung cancer. Cancer Chemother Pharmacol 59:419–427

Anaerobic ▶ Hypoxia

Anaplastic Astrocytoma ▶ Astrocytoma

Anaplastic Large Cell Lymphoma Angelo Rosolen Department of Pediatrics, Hemato-oncology Unit, University of Padua, Padova, Italy

Synonyms Ki1 lymphoma

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Definition Anaplastic large cell lymphoma (ALCL) was originally described in 1985 as a separate entity among the non-Hodgkin lymphomas (NHL); it is characterized by the cohesive proliferation of large cells expressing the CD30/Ki1 antigen on their membrane. The World Health Organization stated that the term ALCL should be applied to tumors with a T-cell or null phenotype, thus further restricting the identification of this specific subtype of lymphoma.

Characteristics Systemic ALCL accounts for 2–8% of all lymphomas (▶ Malignant lymphoma: hallmarks and concept) but represents ~10–15% of NHL of childhood. In addition to the primary systemic disease, a form of ALCL limited to the skin is also recognized. Isolated cutaneous ALCL may spontaneously remit but can also progress to a more aggressive disease. Systemic ALCL has some clinical features that are less common in other NHL subtypes: patients at diagnosis often have B-symptoms (fever, weight loss, sweats) as in Hodgkin lymphoma, mediastinal (Mediastinum) and extranodal involvement, including skin, bone, and soft tissues. Among lymph nodes, the inguinal nodes are often site of disease, particularly in childhood, and rather frequently lymphadenopathy may be painful. Central nervous system and bone marrow are rare sites of disease, although with more sensitive techniques bone marrow may have submicroscopic infiltration of lymphoma cells more often than previously expected. Clinical differences have been reported between ALK (anaplastic lymphoma kinase)-positive and ALK-negative subtypes. Patients with ALK-positive ALCL are significantly younger and have a better prognosis that the negative counterpart, suggesting that ALK-positivity, rather than age, may confer distinct and relevant clinical features to systemic ALCL. Secondary ALCL may arise in the progression of other lymphomas, most commonly during the course of T-cell NHL, mycosis

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Anaplastic Large Cell Lymphoma

Anaplastic Large Cell Lymphoma, Fig. 1 This panel depicts the classic variant of anaplastic large cell lymphoma with numerous large tumor cells that often contain horseshoe- or kidneyshaped nuclei, distinct nucleoli, and abundant cytoplasm

fungoides, Hodgkin lymphoma, or lymphomatoid papulosis, and has a poor outcome. Except for the cases of very aggressive disease, ALCL may show multiple recurrences that respond to therapy, although eventually a considerable number of patients die despite intensive treatment. Diagnosis Diagnosis of ALCL relies on histopathology and immunophenotyping, but for a complete characterization of the tumor, chromosomal analysis and molecular genetic studies are warranted. It is now clear that ALCL includes several variants: classical or common type (corresponding to the original description of the disease) that accounts for ~60–70% of the cases, lymphohistiocytic variant, giant cell-rich, small cell type, and mixed. The large cell-rich type is characterized by multinucleated cells, often with Reed-Sternberglike features that make the differential diagnosis with Hodgkin lymphoma (▶ Hodgkin disease) sometimes difficult. Irrespective of the histotype, neoplastic ALCL cells are characterized by a distinctive phenotypic profile. They express CD30, a cell membrane glycoprotein, found in activated lymphoid cells, the epithelial membrane antigen (EMA), and T-cell antigens including CD3. Perforin and granzyme B are also expressed in the majority of the cases and, together with the absence of CD15 expression, they are useful markers in the differential diagnosis with Hodgkin

disease. The development of antibody against the ALCL kinase (ALK) (▶ ALK protein) has further refined the immunohistochemical analysis of ALCL. Expression of ALK occurs in tumors carrying ▶ chromosomal translocations that involve the corresponding gene. The most common rearrangement between chromosome 2 and 5 causes strong ALK positivity of neoplastic cells in the nucleus and cytoplasm (Figs. 1 and 2), whereas other chromosomal translocations involving ALK produce accumulation of the translocation product at the cytoplasmic level only. ALK reactivity of tumor cells has diagnostic relevance given that ALK is not detected in normal lymphocytes or in Hodgkin lymphoma cells and only few cases of rare forms of lymphoma and nonlymphomatous tumors (inflammatory myofibroblastic tumors, rare neuroblastoma, and rhabdomyosarcoma) show ALK reactivity. Ultimately, combination of morphological, immunophenotypic, and genetic analysis allow the diagnosis of ALCL and the differentiation of this lymphoma from other tumors. A peculiar aspect to consider is the differentiation of cutaneous ALCL from other CD30positive lymphoproliferative disorders including lymphomatoid papulosis. In this case, because the great majority of isolated cutaneous ALCL is ALK-negative, clinical observation and evolution of the disease are of foremost importance. In addition, because skin involvement in the context of a

Anaplastic Large Cell Lymphoma

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A

Anaplastic Large Cell Lymphoma, Fig. 2 This panel immunohistochemical staining of anaplastic large cell lymphoma containing the chromosomal translocation t(2;5) originating the NPM–ALK fusion protein. Nuclear and

cytoplasmic reactivity to an anti-ALK specific monoclonal antibody is detectable in most lymphoma cells (brownish color). Pictures were a courtesy of Dr. E. S. d’Amore, Institute of Pathology, University of Padua, Italy

systemic ALCL may bear prognostic implications, suspect skin involvement must be documented through biopsy. Imaging procedures used in the diagnosis and staging of ALCL are similar to other NHL. Special attention should though be given to the examination of soft tissues and skin, given the relatively high frequency of involvement of those sites. Chest x-ray is usually sufficient to detect a mediastinal mass, but a CT scan of the neck, thorax, and abdomen should always be obtained to define the extent of disease. Ultrasound is routinely used for diagnosis and monitoring of lymph nodes and abdominal organ involvement, including liver, spleen, kidney, and soft tissues. This technique is easy to perform and can give accurate information not only for diagnostic purposes but also to evaluate tumor response to treatment and during follow-up, once treatment is completed. Whole skeletal scintigraphy may be useful, especially in cases with bone pain, and should be complemented with x-ray of positive bones. Brain MRI or CT scan are usually performed, but central nervous system involvement at diagnosis is infrequent in ALCL. ▶ Positron emission tomography (PET) with 18-fluorodeoxyglucose (FDG) has been introduced in the routine evaluation of ALCL patients, mainly for staging.

Lymphoma cells characteristically have a higher uptake of FDG compared with normal cells and this gives high activity features to the vital tumor mass. Because a high reactivity may also be seen in other nonmalignant tissues with high glucose metabolism, including reactive lymph nodes and inflammatory tissues, PET findings have to be interpreted with caution at present, until large prospective clinical studies where this technology is routinely applied are completed. To completely define the extent of the disease, as in other NHL, bone marrow biopsy and bone marrow smear should be performed and analyzed for the presence of tumor cells as well as a lumbar puncture to exclude the presence of lymphoma cells in the central spinal fluid. Genetics The ▶ Chromosomal Translocation t(2;5)(p23; q35) was originally reported in patients with malignant histiocytosis, which indeed represented ALCL cases diagnosed according to old criteria. Break of chromosome 2 and subsequent fusion to chromosome 5 in a reciprocal chromosome rearrangement is the main genetic feature of ALCL. As a result, the ALK gene on chromosome 2 is juxtaposed to the nucleophosmin (NPM) gene on chromosome 5. The NPM–ALK fusion gene

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gives rise to a fusion protein composed of ALK and NPM domains that can be detected by ▶ immunohistochemistry using an anti-ALK monoclonal antibody. While ALK is not usually expressed in normal tissues, except in few neuronal cells, NPM gene encodes a shuttle protein that undergoes dimerization in the cytoplasm and in this conformation can move to the nucleus. In the cytoplasm of NPM–ALK-positive ALCL cells, heterodimers between NPM and NPM–ALK are formed that, while retaining the ability to move to the nucleus, are reactive against the anti-ALK antibody. This explains the cytoplasmic and nuclear reactivity of ALCL cells harboring the t(2;5) translocation. The wide use of antibodies to ALK protein revealed that about 10% of the ALK-positive ALCLs showed an immunohistochemical reactivity confined to the cytoplasm. Molecular genetic studies demonstrated that those cases were associated with a series of different chromosomal translocations, all involving the ALK gene, but with partners other than NPM. The deriving fusion genes all cause hybrid protein overexpression that lack the shuttling properties of NPM-containing fusion proteins, thus preventing them from nuclear localization. From the functional point of view, NPM–ALK can dimerize and as such it possesses constitutive tyrosine kinase activity, mimicking the normal functional activity of the ALK receptor in normal cells upon binding and oligomerization by its specific ligand. A number of experimental data suggest that NPM–ALK has a causative role in tumorigenesis of ALK-positive ALCL, although it may need concomitant events. In fact, NPM–ALK displays transforming activity in both hematopoietic and fibroblastic cell lines in vitro. Overall, ~90% of childhood ALCLs are positive for the ALK-hybrid protein, whereas this percentage is only 50–60% in adult ALCLs. As suggested by clinical studies, ALK-positivity not only is a relevant tool in the diagnosis of ALCL but represents also a prognostic marker in that ALK-positive ALCLs far better than ALK-negative lymphomas in the adult population. Therapy Treatment of ALCL, similarly to other NHL, is almost exclusively based on chemotherapy. The

Anaplastic Large Cell Lymphoma

therapeutic approach has witnessed a variety of chemotherapy regimens, ranging from acute lymphoblastic leukemia-type regimens lasting 24 months to shorter chemotherapy more frequently used in aggressive B-cell lymphomas. Differently from most European studies where ALCL is considered as a separate entity, in North America all large cell lymphomas, regardless of the histologic subgroup and immunophenotype, are treated according to the same chemotherapy scheme. Primary systemic ALCL in adults has been treated mostly with CHOP (cyclophosphamide, vincristine, prednisone), CHOP-derived chemotherapy, and MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, prednisone, and bleomycin) regimens, but occasionally patients have been treated with Hodgkin lymphoma-type chemotherapy (e.g., ABVD regimen). Collaborative trials have been conducted in childhood and adolescence ALCLs. Overall results were comparable with very different treatment strategies, ranging from leukemia-like treatment, such as modified LSA2-L2 protocol, to chemotherapy regimes derived from B-cell NHL as the BFM (Berlin-Frankfurt-Munster) and the French and British Pediatric Hemato-Oncology Society protocols. The latter are based on the administration of short (usually 5-days) courses of rather high-intensive chemotherapy (based on the rotational use of corticosteroid, anthracyclines, cyclophosphamide or ifosfamide, cytarabine, methotrexate, epipodophyllotoxin) administered at intervals of ~3 weeks. With these treatments, 60–75% of patients obtain cure of their disease and do not experience disease recurrences. Similar results were obtained with the APO regimen in the Children’s Oncology Group (USA) that includes higher cumulative doses of ▶ anthracyclines without ▶ alkylating agents (▶ Cyclophosphamide/Ifosfamide) or epipodophyllotoxins (substance class of anticancer drugs that are isolated or derivatives of natural products from Podophyllum peltatum, e.g., ▶ etoposide). Treatment intensity and/or duration have been differentiated based on various risk factors.

Anaplastic Lymphoma Kinase

Although stage of disease has some relevance, in the adult population a high International Prognostic Index (IPI) score, high serum lactate dehydrogenase levels, ALK-negativity, and expression of the surface antigen CD56 have been associated with a significant lower outcome. In children and young adults, possibly because of the very high frequency of ALK-positivity, ALK expression does not seem to be a relevant prognostic indicator, whereas high stage of disease, elevated lactate dehydrogenase levels, and specific site of disease, including liver, spleen, lung, and skin involvement, seem to be associated with a less favorable prognosis. A distinct clinical condition is represented by the isolated cutaneous ALCL. Once other disease localizations have been excluded, given that spontaneous regression of the lesions can occur, a strict monitoring of the patient is often preferred, postponing initiation of chemotherapy when signs of lymph nodes or other organ involvement is demonstrated. A peculiar aspect of ALCL is that most patients who relapse respond well to salvage therapies. Although early disease recurrences can be extremely aggressive, moderate intensity chemotherapy, including single drug treatment with vinblastine, can achieve long-lasting remission. In case of resistant disease or very aggressive relapse, high-intensive chemotherapy with bone marrow transplant has also been used both in children and adults. Further studies are needed to definitively establish the benefit of bone marrow transplant in ALCL and to identify the subpopulations of patients who may benefit from such a treatment approach.

Cross-References ▶ ALK Protein ▶ Alkylating Agents ▶ Anthracyclines ▶ Chromosomal Translocations ▶ Cyclophosphamide ▶ Etoposide ▶ Hodgkin Disease ▶ Immunohistochemistry

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▶ Malignant Lymphoma: Hallmarks Concepts ▶ Positron Emission Tomography

and

References Brugieres L, Deley MC, Pacquement H et al (1998) CD30(+) anaplastic large-cell lymphoma in children: analysis of 82 patients enrolled in two consecutive studies of the French Society of Pediatric Oncology. Blood 92:3591–3598 Falini B (2001) Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol 114:741–760 Seidemann K, Tiemann M, Schrappe M et al (2001) Shortpulse B-non-Hodgkin lymphoma-type chemotherapy is efficacious treatment for pediatric anaplastic large cell lymphoma: a report of the Berlin-Frankfurt-Munster Group Trial NHL-BFM 90. Blood 97:3699–3706

See Also (2012) ALK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 128. doi:10.1007/978-3-642-16483-5_178 (2012) Immunophenotype. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1826. doi:10.1007/978-3-642-16483-5_3000 (2012) Lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2124. doi:10.1007/978-3-642-16483-5_3463 (2012) Mediastinum. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2199. doi:10.1007/978-3-642-16483-5_3597 (2012) NPM. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2565. doi:10.1007/978-3-642-16483-5_4133 (2012) NPM–ALK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2565– 2566. doi:10.1007/978-3-642-16483-5_4134 (2012) Scintigraphy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3341. doi:10.1007/978-3-642-16483-5_5179 (2012) Vinblastine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3907. doi:10.1007/978-3-642-16483-5_6186

Anaplastic Lymphoma Kinase Definition Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) having an extracellular, a

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single transmembrane, and an intracellular domain containing the tyrosine kinase activity. ALK belongs to the insulin receptor subfamily of RTKs, most closely related to leukocyte tyrosine kinase receptor. It localizes mostly in neuronal cells and may play a role in the nervous system development and maintenance. ALK and some of the ALK partners or closely related genes are found implicated both in anaplastic large-cell lymphoma and in inflammatory myofibroblastic tumors.

Cross-References ▶ ALK Protein ▶ Pleiotrophin

Anaplastic Thyroid Carcinomas ▶ Follicular Thyroid Tumors

Anchorage-Independent Definition The ability of cells to survive and multiply in the absence of a protein matrix for adhesion.

Anaplastic Thyroid Carcinomas

Androgen Ablation Therapy Synonyms ADT; Androgen deprivation therapy; Androgen suppression therapy

Definition Androgen ablation therapy is a therapeutic modality with the purpose of removing or reducing (ablating) male androgens – testosterone and dihydrotestosterone produced endogenously. This type of therapy is primarily used in metastatic ▶ prostate cancer, as opposed to localized disease where the tumor mass can be surgically removed. Procedures to reduce/remove endogenous androgens may include combinations of orchiectomy, antiandrogens, or gonadotropin-releasing hormone agonists/antagonists in combinations with antiandrogens. Androgen receptor antagonists include flutamide, bicalutamide, and nilutamide. Although classified as androgen ablaters, 5a-reductase inhibitors are approved primarily for treatment of benign prostatic hyperplasia. 5a-reductase inhibitors also reduce levels of dihydrotestosterone by preventing its conversion from testosterone and include finasteride and dutasteride. Treatment for benign prostatic hyperplasia may include a1-adrenergic receptor antagonists (terazosin, tamsulosin) in combination with 5a-reductase inhibitors. Although a1-adrenergic receptor antagonists are not androgen ablaters, they are used to relax smooth muscle in the prostate facilitating easier urine flows in addition to the relief provided by shrinkage of the prostate with androgen ablaters.

Cross-References ▶ Syk Tyrosine Kinase

Cross-References ▶ Prostate Cancer

See Also (2012) Adhesion molecules. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 66. doi: 10.1007/978-3-642-164835_96

See Also (2012) Testosterone. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3660. doi: 10.1007/978-3-642-16483-5_5741.

Androgen Receptor

Androgen Deprivation Therapy ▶ Androgen Ablation Therapy

Androgen Insensitivity Syndrome (AIS) ▶ Androgen Receptor

Androgen Receptor Kaustubh Datta and Donald J. Tindall Department of Urology Research, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA

Synonyms AR; Androgen insensitivity syndrome (AIS); Dihydrotestosterone receptor; HUMARA; Kennedy disease (KD); NR3C4; SMAX1; Spinal and bulber muscular atrophy (SBMA); Testicular feminization (TFM)

Definition The androgen receptor or AR is a member of the steroid/thyroid receptor superfamily, in which all members share basic structural and functional homology. The AR is an intracellular ligand (androgen)-dependent transcription factor, which regulates the expression of genes that control cell proliferation, ▶ apoptosis, ▶ angiogenesis, and differentiation in many hormonally regulated tissues including the prostate. It is an important regulator of male sexual differentiation and maturation.

Characteristics The biological function of androgen is mediated through the AR. Except for the spleen and bone

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marrow, the AR is ubiquitously expressed in human organs. The human AR gene is more than 90-kb long and is present as a single allele located at chromosome Xq11.2–12. It has eight exons and possesses a coding region of 2,757 bp. This region encodes a 110 KDa protein (919 amino acids) with four distinct functional domains: a conserved Zn-finger DNA-binding domain (exons 2 and 3), a hinge region (exon 4), a COOH-terminal ligandbinding domain (exons 4–8), and a less conserved and structurally flexible amino (NH2)-terminal transactivation domain (exon 1). Structure–Function Relationships Between Different Domains of Androgen Receptor that Are Required for its Transcriptional Activity Regulation of Unliganded AR

Unbound AR in the cytoplasm remains in an inactive but androgen responsive state as part of a large dynamic heterocomplex composed of heat shock proteins, cochaperones, and tetratricopeptide repeat containing proteins. This large complex helps to modulate the ligand-binding domain (LBD) of AR into a relatively stable, partially unfolded, and inactive intermediate which has a high affinity for the potent biologically active androgen, dihydrotestosterone. The androgen-free LBD and the associated chaperone proteins inactivate the function of the transactivation domain of the AR. This transactivation domain becomes constitutively active in the mutant AR lacking a ligand-binding domain (LBD). As such, this molecular chaperone complex and the LBD of AR prevent unwanted activation of the AR in the absence of androgen. Regulation of Ligand-Bound AR

Because of their lipophilic nature, androgens cross the cell membrane, both passively, and actively via the transport protein, megalin. Once within the cell, the androgen binds to the AR, which stabilizes it and translocates into the nucleus. Within the nucleus, the AR homodimerizes and recruits transcriptional cofactors to the promoters and enhancers of AR-targeted genes, thus facilitating their transcription (Fig. 1). Androgen binding to the LBD

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224 Androgen Receptor, Fig. 1 A schematic diagram of AR structure and its functions

Androgen Receptor

LBD

Lipid raft

Ligand N

C

N

C

NTD Hinge

Non-genomic function

AR

DBD

Differentation Proliferation

Nucleus CC

AF

1

Gene transcription N

AF 5

N

Survival

AF2

Cytoplasm

Synthesis of secreted proteins (e.g. PSA, hK2, PAP) Lipogenesis

DNA

ARE

of the AR induces an overall change in the AR structure leading into an active conformation which is characterized by dissociation of the receptor–chaperone complex. However, molecular chaperones also play important roles in the events downstream of the AR activation such as translocation to the nucleus, transcriptional activation, and transcription complex disassembly and degradation. LBD relieves its inhibitory function upon androgen binding. The AR LBD configuration is highly structured and resembles other steroid receptors’ ligand-binding domains. 1. AR-DNA Binding Domain and Hinge Region: Androgen binding to the LBD initiates a conformational change of the AR leading to several secondary effects important for the AR transcriptional activity. One such effect is the unmasking of the bipartite nuclear-localization signal (NLS) that overlaps the DNA-binding domain and hinge regions (amino acid 625–671). Another weak NLS (amino acid 722–805) is present in the LBD of AR, which is also exposed upon androgen binding. The NLS is recognized by an import protein that mediates translocation of the AR through the

nuclear-pore complex. The AR homodimerization occurs in the nucleus upon treatment with ligand. Both the DNA and ligand-binding domains of the AR are involved in subsequent receptor dimerization. The stronger hydrophobic interaction occurs between the ligand-binding domains. The binding of the AR dimer to the specific androgen response elements (ARE) of a gene occurs in a cooperative manner. The DNA binding domains of the AR, like other nuclear receptor superfamily members, consist of two zinc fingers that provide the structural basis required for ARE recognition in the promoter region of a gene. The consensus ARE is a 6 bp palindromic core sequence (50 -AGEACA-30 ) separated by a three nucleotide spacer. Sequences outside the DNA binding domain also play a role in AR-DNA binding. 2. AR AF-2 Domain: A hydrophobic proteinprotein interaction surface known as activation function-2, or AF2, is present at the carboxy terminal, and becomes accessible after androgen binding to the AR. AF2 is the potential binding site for the AR coactivators such as the p160 family (TIF2, SRC1, and AIB1). These

Androgen Receptor

coactivators enhance the AR transcriptional activity by modulating the AR conformation and recruiting cofactors to the promoter. Ligand binding also induces interaction of AF2 with a specific motif (FXXLF) (F = phenylalanine, L = leucine, and X = any aminoacid) in the NH2-terminal activation domain of the AR. The interaction of NTD and LBD or N/C interaction is important for the AR transcriptional activity. Although the mechanistic details of how this interaction leads to the AR activity are not precisely known, it appears that ligand binding induces intramolecular folding of the AR, leading to the interaction between NTD and LBD. This influences receptor dimerization in the nucleus and reduces ligand degradation from LBD, AR protein dissociation, AR chromatin binding ability and receptor transactivation. 3. AR N-Terminal Transactivation Domain: The N-terminal transactivation domain (NTD) of the AR is longer and structurally different as compared to other steroid receptors. The highly flexible and disordered domain containing NTD is largely globular in nature. Association with coactivators and transcription factors helps to induce folding in the domains of the NTD to optimize efficient binding. Due to its allosteric nature of binding, the AR NTD is able to bind a broad spectrum of transcriptional coactivators, corepressors, and other factors. Therefore, the AR NTD may be responsible for mediating androgen-regulated expression of genes whose functions consist of protein folding, trafficking and secretion, metabolism, cytoskeletal rearrangement, cell cycle regulation, and signal transduction. AF1 and AF5 Domain of NTD: There are two major overlapping activation functions present in the AR NTD, AF1 (amino acids 142–485) and AF5 (amino acids 351–528). These regions contain microsatellite repeats, protein-protein interaction surfaces, and phosphorylation and sumoylation sites. AF1 is considered the major transactivation domain that binds to basal transcription factors, coregulators, cell cycle regulatory proteins, heat shock proteins, etc. The interaction with molecules like TIFIIF

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increases folding in the AF1 domain and facilitates further protein-protein interactions for the formation of a transcriptionally competent receptor complex. Unlike AF1, the activation of AF5 is ligand independent. An inhibitory domain within AF5 inhibits the DNA-binding domain of AR from binding to AREs. Glutamine and Glycine Repeats: AR NTD contains two polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts. The first trinucleotide repeat sequence, CAG, spans amino acids ~58–78 and encodes for the amino acid glutamine. The second stretch consists of GGN repeats that encode glycines and span amino acids ~449–472. Shortened trinucleotide repeat stretches result in an increased AR activity and are often associated with prostate cancer predisposition. On the other hand, overexpansions of the CAG repeat (more than 40) correlate with a significant decrease in the AR activity and are associated with diseases such as X-linked Spinal and Bulbar Muscular Atrophy (SBMA), also known as Kennedy’s disease. Both the polyglutamine and polyglycine repeat size appear to regulate the N/C interaction and thereby influence the AR activity. FXXLF and WXXLF Motifs: The AR NTD also contains two short, highly conserved peptide motifs that mediate the ligand-dependent N/C interaction as discussed previously. These motifs are FXXLF (in humans FQNLF), ranging from amino acids 23–27 and WXXLF (in humans WHTLF), ranging from amino acids 434–438. AR Coregulators: AR functions as a tripartite receptor system involving AR itself, the androgens and AR coregulators. The AR interaction with either coactivators or corepressors enhances or represses its transcriptional activity, respectively, without affecting the basal levels of transcription. Possible mechanisms of coregulator function include modulation of chromatin structure, promotion of AR post-translational modifications, control of androgen/AR binding affinity, AR expression, AR stability, AR nuclear translocation, and AR recruitment of transcription machinery.

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4. Post-translational Modification of AR: Posttranslational modification of the AR is one of the key mechanisms regulating its function. One of the major post-translational modifications of the AR is phosphorylation. These phosphorylation sites may be the sites for possible cross-talk between peptide growth factors and the AR signaling axis, which is important for normal prostate epithelial cell growth and function as well as for the progression of cancer. Most of the phosphorylation sites reside in the NTD at different serine residues. However, findings suggest that Src kinase mediates phosphorylation at tyrosine 534 of AR, and therefore acts as a potential regulator of AR transcriptional activity. Another post-translational modification is acetylation of AR at the hinge domain (at lysine residues 630, 632, and 633) by histone acetyl transferases such as p300, p/CAF, and TIP60. This modification regulates recruitment of coregulators as well as the growth properties of the AR. Acetylation is also required for regulation of the AR by the AKT, PKA, and JNK signaling pathways. Sumoylation of the AR at two specific domains (NRM1 and NRM2 at NTD) also appears to be important for the AR activation, localization, and degradation. Nongenomic Function of the AR

A nongenomic mechanism for the AR function independent of its transcriptional activity has been postulated. This nongenomic event is very rapid (2–15 min) and activates signaling events such as the Src/Raf1/ERK, PI3K-AKT, and IL-6-STAT3 pathways. Interestingly, membrane lipid rafts are considered to be the privileged site for this AR-mediated nongenomic signaling. Upon androgen binding, most of the ARs translocate to the nucleus and act as transcription factors. The few remaining cytosolic ARs may then enter into ▶ caveolin positive or negative rafts to initiate different signaling pathways. Androgen Receptor in Human Physiology and Pathology Androgen and the AR are not only necessary for the initiation of prostate development; they are also important factors for the survival,

Androgen Receptor

proliferation, secretary function, morphology, angiogenesis, and differentiation of the adult prostate gland. AR is also important for Wolffian duct development and spermatogenesis in males. Various clinical disorders due to functional abnormalities of the AR have been reported, suggesting that a wide range of physiological responses and developmental processes are mediated by the AR. Androgen Receptor and Prostate Cancer: Approximately 80–90% of prostate tumors are dependent on androgen at the initial diagnosis, suggesting the importance of the AR signaling in all stages of prostate carcinogenesis. A developed bioinformatics approach known as Cancer Outlier Profile Analysis (COPA) together with standard genomic techniques has identified recurrent gene fusions of the 50 untranslated region of the TMPRSS2 gene to ▶ ETS transcription factors (ERG or ETV1) in human prostate cancer tissues. Interestingly, this gene fusion enables expression of the fused product under the control of the AR, as the TMPRSS2 promoter contains an ARE. This TMPRSS2-ETS fusion appears to be frequent in prostate cancer and might be an initiating event for prostate cancer, underscoring the importance of the AR-mediated signaling in prostate cancer. Both the tumor epithelia and adjacent stroma express AR. Retardation of tumor growth occurs in response to androgen ablation therapy. Until now, the primary therapy for advanced (locally extensive or metastatic) prostate cancer consists of androgen ablation by pharmaco-therapeutic or surgical means. Eventually, the tumor recurs due to a transition from androgen-dependence to a highly aggressive and androgen-depletion-independent (refractory) phenotype. The detailed molecular mechanism underlying the development of the androgen refractory phenotype of prostate cancer is poorly understood. As such, it has been difficult to develop effective treatments for this stage of the disease. Disruption of androgen receptor function inhibits the proliferation of androgen refractory prostate cancer, demonstrating the importance of the AR even at subnormal physiological levels of androgen. Mechanisms for ligand-independent AR reactivation include AR mutation, gene amplification, increased stability and nuclear localization, coregulators and cross talk between different

Aneuploidy

signal transduction pathways. Studies also suggest an acquired capacity of recurrent prostate tumors to biosynthesize testicular androgens from adrenal androgens or cholesterol, thereby reactivating the AR. Targeting AR for Therapy: Androgen ablation therapy, which is achieved by pharmacotherapeutic (steroidal and non-steroidal antiandrogens) or surgical (subcapsular or subepididymal bilateral orchiectomy) means, is the standard initial systemic therapy for locally advanced or metastatic prostate cancer. This therapy is based on either inhibiting the synthesis of active androgen or inhibiting the physiological androgen from binding to AR. Novel therapeutic strategies for the androgen-depletion-independent stage of prostate cancer will focus on inhibiting expression of the AR, blocking the binding of AR coactivators, and enhancing the binding of AR corepressors. Using intelligent high-throughput screening and structural and computational chemistry, it may be possible to develop peptide antagonists, small molecules or antisense oligonucleotides that can target AR-coactivator binding surfaces. Potential drugs targeting the N-terminal domain of AR may also prevent or delay the progression of both hormonaldependent and -independent prostate cancer.

References Debes JD, Tindall DJ (2004) Mechanisms of androgenrefractory prostate cancer. N Engl J Med 351:1488–1490 Dehm SM, Tindall DJ (2006) Molecular regulation of androgen action in prostate cancer. J Cell Biochem 99:333–344 Heinlein CA, Chang C (2004) Androgen receptor in prostate cancer. Endocr Rev 25:276–308 MacLean HE, Warne GL, Zajac JD (1997) Localization of functional domains in the androgen receptor. J Steroid Biochem Mol Biol 62:233–242 Shen HC, Coetzee GA (2005) The androgen receptor: unlocking the secrets of its unique transactivation domain. Vitam Horm 71:301–319

Androgen Suppression Therapy ▶ Androgen Ablation Therapy

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Androgen-Independent Prostate Cancer (AIPC) Synonyms Hormone-refractory prostate cancer

Definition AIPC is a prostate cancer that does not require androgen to progress.

Cross-References ▶ Hormone-Refractory Prostate Cancer ▶ Hormone Replacement Therapy ▶ Prostate Cancer Hormonal Therapy

Aneuploidy Subrata Sen Department of Molecular Pathology (Unit 951), The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Definition Aneuploidy refers to the presence of either less than or more than the normal diploid number of chromosomes in a cell. Such losses or gains (aneusomy) of chromosomes may involve segments of chromosomes or complete chromosomes leading to major imbalances in the genetic makeup of the affected cells. Instead of the two copies of structurally intact homologous chromosomes (disomy) present in a normal cell, aneuploidy may result in the loss of one chromosome (monosomy) or both homologous chromosomes (nullisomy) as well as gain of one chromosome or

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more to each homologous pair of chromosomes (trisomy, tetrasomy, etc.) in a cell. Occasionally, gain of one or more copies of the entire haploid set of chromosomes (triploidy, tetrapolidy, etc.) or loss of one haploid set of chromosomes (haploidy) have also been reported.

Characteristics ▶ Chromosomal instability in the form of aneuploidy is one of the most common genetic anomalies detected in human cancer cells. While von Hansemann had, at the end of the nineteenth century, first discussed the biological consequences of chromosome segregation errors based on observations of abnormal mitoses in human cells, possible contribution of aneuploidy in tumorigenic transformation of cells resulting from multipolar mitosis was later proposed by Theodore Boveri at the beginning of the twentieth century. Germline inheritance of aneuploid condition often leads to developmental defects, and somatic origins of similar anomalies are frequently associated with malignant phenotypes. It has been convincingly demonstrated that aneuploidy arises due to underlying genetically determined chromosomal instability in malignant cells. Discovery and functional characterization of genes regulating faithful segregation of chromosomes required for the maintenance of chromosomal stability in somatic cells have helped elucidate the genetic pathways involved in the process. Findings of tumor suppressor genes and DNA mismatch repair genes as upstream regulators of these pathways have provided compelling evidence in favor of aneuploidy being a genetically determined phenomenon associated with cancer. Whether aneuploidy is a cause or consequence of cancer has long been debated. Although the physiological relevance of aneuploidy in cancer still remains a subject of investigation, increasing number of observations, over the years, have revealed frequent changes in gene copy number in conjunction with changes in chromosome copy number in cancer cells during cancer initiation and/or progression processes. These observations made in primary tumors with in situ hybridization

Aneuploidy

(ISH) and comparative genomic hybridization (CGH) techniques have revealed that specific chromosome aneusomies sometimes correlate with distinct tumor phenotypes. In addition, aneuploid tumor cell lines and in vitro transformed rodent cells have been reported to display an elevated rate of chromosome instability, thus indicating that aneuploidy is a dynamic chromosome mutation event associated with transformation of cells. Finally and most importantly, a number of genes regulating chromosome segregation have been found aberrantly expressed in human cancer cells, and genetically engineered mice expressing abnormal levels of some of these genes have been reported to display higher incidence of aneuploidy associated with tumor development. These findings provide a new direction towards understanding the molecular mechanisms responsible for the origin of aneuploidy in cancer, and the knowledge is expected to help design novel cancer therapeutic strategies in the future. Aneuploidy in Cancer Aneuploidy involving one or more chromosomes has been commonly reported in human tumors. It is estimated that aneuploidy is the most prevalent genetic change recorded among over 20,000 solid tumors analyzed thus far. These observations were originally made using classical cytogenetic techniques late in a tumor’s evolution and were difficult to correlate with cancer progression. Studies have reported an association of specific nonrandom chromosome aneuploidy with different biological properties such as loss of hormone dependence and metastatic potential. Classical cytogenetic studies of analyzing metaphase chromosomes from tumor cells had serious limitations in scope since these were applicable only to those cases in which mitotic chromosomes could be obtained. Because of low spontaneous rates of cell division in primary tumors, analyses depended on cells either derived selectively from advanced metastases or those grown in vitro for varying periods of time. In both instances, the metaphase chromosomes analyzed represented only a subset of the tumor cell population. The two major advances in analytical cytogenetic techniques, in situ hybridization

Aneuploidy

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A

Aneuploidy, Fig. 1 Fluorescence in situ hybridization (FISH) analyses of normal ovarian epithelial cells (a) and human ovarian cancer cells (b) performed with chromosome 20 short arm (labeled with red fluorescent dye) and

long arm probes (labeled with green dye). Note the presence of two copies of chromosome 20 in normal epithelial cells in (a) but five to seven copies of chromosome 20 as evidence for aneuploidy in the cancer cells (b)

(ISH) and comparative genomic hybridization (CGH), have allowed better resolution of chromosomal aberrations in freshly isolated tumor cells. ISH analyses with chromosome-specific DNA probes can be performed on interphase nuclei and allow assessment of numeric chromosomal anomalies within tumor cell populations in the contexts of whole nuclear architecture and tissue organization (Fig. 1). CGH allows genome-wide screening of chromosomal anomalies without the use of specific probes, even in the absence of knowledge of the chromosomes involved. While both techniques have certain limitations in terms of their resolution power, they nonetheless provide a better approximation of chromosomal changes occurring among tumors of various histology, grade, and stage compared with what was possible with the classical cytogenetic techniques. DNA ploidy measurements have also been performed with flow cytometry and cytofluorometric methods. Although these assays underestimate chromosome ploidy due to the possibility of a gain occasionally masking a loss in the same cell, several studies employing these methods have concluded that DNA copy number changes or DNA aneuploidy correlates with poor prognosis in different cancers. A few published examples of aneuploidy in cancer are mentioned in the following discussion that deal with DNA ploidy measurements as well. Most of these

observations are correlative without a direct proof of specific involvement of genes on the respective chromosomes. However, Findings on the role of specific genes in regulating chromosome segregation and maintenance of chromosomal stability together with the development of mouse models genetically manipulated to either upregulate or downregulate some of these genes displaying increased incidence of tumors associated with aneuploidy provide strong evidence in favor of aneuploidy playing a critical role in the tumorigenesis process. In renal cancer, either segmental or whole chromosome aneuploidy appears to be uniquely associated with specific histologic subtype. Tumors from patients with hereditary papillary renal carcinomas (HPRC) commonly show trisomy of chromosome 7, when analyzed by CGH. Germline mutations of a putative oncogene MET have been detected among patients with HPRC. It was demonstrated that an extra copy of chromosome 7 results in nonrandom duplication of the mutant ▶ MET allele in HPRC, thus implicating this trisomy in tumorigenesis. The study suggested that mutation of MET may render the cells more susceptible to errors in chromosome replication, and clonal expansion of cells harboring duplicated chromosome 7 reflects their proliferative advantage. In addition to chromosome 7, trisomy of chromosome 17 in papillary tumors

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and also of chromosome 8 in mesoblastic nephroma are commonly seen. Association of specific chromosome imbalances with benign and malignant forms of papillary renal tumors not only contributes to understanding of tumor origins and evolution but also implicate aneuploidy of the respective chromosomes in the tumorigenic transformation process. In ▶ colorectal cancer, aneuploidy is common occurrence. Molecular allelotyping studies have suggested that the limited karyotyping data available from these tumors actually underestimate the true extent of these changes. Losses of heterozygosity, reflecting loss of the maternal or paternal allele in tumors, are widespread and often accompanied by a gain of the opposite allele. Thus, for example, a tumor could lose a maternal chromosome while duplicating the homologous paternal chromosome leaving the tumor cell with a normal karyotype and ploidy but an aberrant allelotype. It has been estimated that on an average, cancer of the colon, breast, pancreas, and prostate may lose 25% of the alleles and it is not unusual for a tumor to have lost over half of all its parental alleles. In clinical settings, DNA ploidy changes indicate high risk of developing premalignant changes among patients with ulcerative colitis and also lymph node metastasis among patients with gastric carcinoma. Similarly, chromosome copy number alterations or aneuploidy has been detected in precancerous lesions of colon, cervix, head and neck, esophagus, and bone marrow. Between 60% and 80% of colorectal polyps from individuals with adenomatous polyposis syndrome, predisposed to develop colorectal cancer, have been reported to show aneuploid changes. Comparative analysis of genomic alterations in AdAPC driven mouse intestinal tumors have identified loci syntenic with human chromosomes 1, 12, 9, and 22 that are frequently gained or lost in familial adenomas and sporadic colorectal cancers suggesting that genetic mechanisms manifested in the form of aneuploidy are conserved across species. The molecular karyotype of amplified chromosomal segments (amplotype) generated from colorectal cancer was reported to indicate that over representation of loci on chromosomes 8 and 13 may be critical for metastatic colorectal cancer.

Aneuploidy

Incidence of chromosome aneuploidy has also been evaluated as a marker of risk assessment and prognosis in several other cancers. Analyzing aneuploidy in nonsurgically obtained squamous epithelial cells offers a promising noninvasive tool to identify individuals at high risk of developing head and neck cancer. Interphase FISH studies have revealed extensive aneuploidy in tumors from patients with head and neck squamous cell carcinomas (HNSCC) and also in clinically normal distant oral regions from the same individuals. It has been suggested that a panel of chromosome probes for FISH analyses may serve as an important tool to detect subclinical tumorigenesis and for diagnosis of residual disease. The presence of aneuploid or tetraploid populations is commonly seen in 90–95% of esophageal adenocarcinomas, and when detected in ▶ Barrett esophagus, a premalignant condition, predicts progression of disease. Aneuploidy in most solid tumors coexists with structural chromosomal aberrations giving rise to complex karyotypes. Such karyotypic complexities could be reflective of similar underlying mechanisms responsible for the origin of both kinds of chromosomal aberrations as well as their selective value for the evolution of malignant cells during carcinogenesis. These possibilities appear credible in view of the findings that tetraploid p53 null mouse mammary epithelial cells show an increased frequency of whole chromosome missegregation and chromosomal rearrangements together with increased propensity to give rise to malignant mammary epithelial cancers. Despite complex karyotypes, different cancers also show shared minimal regions of gains and losses of specific chromosomes. By analyzing such regions of genomic imbalances in various solid tumors, karyotypic pathways of evolution of cancers involving specific chromosomal aneusomies have been described. For pancreatic cancer, the recurrent early imbalances included loss of chromosomes 1, 5, 7, 8, 15, 17, and 18, while the late recurrent imbalances were identified as gain of chromosomes 2, 6, 7, and 11 and loss of chromosome 19. Besides clinical correlative observations, role of aneuploidy in oncogenesis has also been

Aneuploidy

supported by in vitro and in vivo transformation experiments performed with human and rodent cells. These studies revealed that aneuploidy is induced at early stages of transformation. Transgenic mouse models with chromosome segmentspecific duplications and deletions have been generated to investigate the effect of chromosome ploidy alterations during development. Three duplications for a portion of mouse chromosome 11 syntenic with human chromosome 17 were established in the mouse germline. Mice with duplication of 1 Mb chromosomal DNA developed corneal hyperplasia and thymic tumors. The findings document a direct role of chromosome aneusomy in tumorigenesis. Developments of mouse models with targeted upregulation or downregulation of genes regulating chromosome segregation giving rise to increased incidence of aneuploidy and cancer have further strengthened the idea of aneuploidy being a cause driving tumorigenesis rather than a consequence of cancer. Aneuploidy as a “Driving Force” and Not a “Consequence” in Cancer The presence of numerical chromosomal alterations in a tumor does not mean that the change arose as a dynamic mutation due to genomic instability. While aneuploidy as a dynamic mutation due to genomic instability in tumor cells would occur at a certain measurable rate per cell generation, a consequential state of aneuploidy is expected to be fixed in similar tumors at an unpredictable random rate possibly decided by differences in environmental factors such as humoral, cell substratum, and cell-cell interaction differences of the tumor and normal cell microenvironments. It could be argued that despite similar rates of spontaneous aneuploidy induction in normal and tumor cells, the latter are selected to proliferate due to altered selective pressure in the tumor cell microenvironment while the normal cells are eliminated through activation of apoptosis. Alternatively, it could be postulated that selective expression or over expression of antiapoptotic proteins or inactivation of proapoptotic proteins in tumor cells may counteract default induction of apoptosis in G2/M phase

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cells undergoing missegregation of chromosomes. To investigate if aneuploidy is a dynamic mutational event, different human tumor cell lines and transformed rodent cell lines have been analyzed for the rate of aneuploidy induction. When grown under controlled in vitro conditions, such conditions ensure that environmental factors do not influence selective proliferation of cells with chromosome instability. In one study, Lengauer and colleagues provided evidence by FISH analyses that losses or gains of multiple chromosomes occurred in excess of 102 per chromosome per generation in aneuploid colorectal cancer cell lines. The study further concluded that such chromosomal instability appeared to be a dominant trait. Utilizing another in vitro model system of Chinese hamster embryo (CHE) cells, Duesberg and colleagues have also obtained similar results. With clonal cultures of CHE cells, transformed with nongenotoxic chemicals and a mitotic inhibitor, these authors demonstrated that the majority of the transformed colonies contained more than 50% aneuploid cells, indicating that aneuploidy would have originated from the same cells that underwent transformation. All the transformed colonies tested were tumorigenic. It was further documented that the ploidy factor, representing the quotient of modal chromosome number divided by the normal diploid number, in each clone correlated directly with the degree of chromosomal instability. Thus chromosomal instability was found proportional to the degree of aneuploidy in the transformed cells, and the authors hypothesized that aneuploidy is an effective mechanism of destabilizing the genome and changing normal cellular phenotypes. Genetic Mechanisms of Aneuploidy in Cancer Numerical chromosomal aberrations giving rise to aneuploidy result when chromosomes are missegregated unequally to the daughter cells during mitotic cell division process. Failure to correct misattachments of kinetochores with spindle microtubules through mitosis is the major cause of such chromosome missegregation. The cell cycle control mechanism that ensures faithful equal segregation of chromosomes during mitosis

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Aneuploidy

(a) Amphitelic

Checkpoint complex inactive APC/C active Diploid

“wait anaphase signal”

APC/C inactive

(b) Syntelic

Checkpoint complex active

Aneuploid

(C) Monotelic Aneuploid Prometaphase (d) Merotelic Aneuploid with structural chromosome aberration

Metaphase

Anaphase

Daughter cells

Aneuploidy, Fig. 2 Mitotic checkpoint regulation of chromosome segregation

is referred to as the mitotic checkpoint or the spindle assembly checkpoint (Fig. 2). The mitotic checkpoint prevents chromosome missegregation and aneuploidy by inhibiting metaphase to anaphase transition in cells until the sister kinetochores of all the replicated chromosomes attach appropriately to the spindle microtubules from the two opposing poles in the cell. This form of attachment is known as amphitelic attachment and until such time as this attachment is achieved, mitotic checkpoint proteins recruited to the unattached kinetochores generate a diffusible signal (wait anaphase signal) that inhibits the anaphase promoting complex/ cyclosome (APC/C) from facilitating the degradation of the substrates necessary for transition from metaphase to anaphase and mitotic exit. Thus with an active mitotic checkpoint, inappropriately attached sister kinetochores, such as those with both kinetochores attached to the same pole known as syntelic attachment or others with only one kinetochore attached to one pole known as monotelic attachment or to the two opposing poles known as merotelic attachment, are

prevented from proceeding to anaphase with the likely outcome of giving rise to aneuploidy. Aberrant expression of the checkpoint proteins leading to weakening of the mitotic checkpoint, however, allows missegregation of inappropriately attached sister chromatids to proceed to anaphase leading to the generation of aneuploid daughter cells. Chromosome segregation errors may also result in cells with centrosome anomalies giving rise to multipolar spindles. Among the mitotic processes implicated in cancer, defects in centrosome function have been frequently suggested to be involved in a wide variety of malignant human tumors. Centrosomes play a central role in organizing the microtubule network in interphase cells and the mitotic spindle during cell division. Multipolar mitotic spindles have been observed in human cancers in situ and abnormalities in the form of supernumerary centrosomes, centrosomes of aberrant size and shape, as well as aberrant phosphorylation of centrosome proteins have been reported in prostate, colon, brain, and breast tumors. It is conceivable that cells with abnormal centrosomes may missegregate chromosomes

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Aneuploidy, Table 1 Genes-proteins regulating chromosome ploidy in cancer Gene name Cenp-A Bub 1

Function Kinetochore assembly Mitotic checkpoint

Bub R1

Mitotic checkpoint

Bub 3 Mad1 Mad 2

Mitotic checkpoint Mitotic checkpoint Mitotic checkpoint

Cenp E

Motor protein/mitotic checkpoint Motor protein Chromosome segregation Sister chromatid cohesion

KIF 4 Aurora-B PTTG (Securin) Survivin Aurora-A PLK 1 Nek 2 Brca1 Brca2 AdAPC Msh2

Chromosome segregation Chromosome segregation Chromosome segregation Chromosome segregation Tumor suppressor Tumor suppressor Tumor suppressor DNA mismatch repair

Mutation/altered expression Upregulated Mutated/upregulated/ downregulated Mutated/upregulated/ downregulated Upregulated/downregulated Upregulated/downregulated Mutated/upregulated/ downregulated

Human cancer Yes Yes

Animal models of cancer

Yes Yes Yes

Yes Yes Yes

Upregulated Upregulated

Yes Yes

Upregulated Upregulated Upregulated Upregulated Mutated/downregulated Mutated Mutated/downregulated Mutated/upregulated/ downregulated

Yes Yes Yes Yes Yes Yes Yes Yes

producing aneuploid cells. The molecular genetic mechanism(s) regulating centrosome structure/ function that are aberrant in cancer cells remain to be elucidated. The presence of supernumerary centrosomes in aneuploid p53-deficient fibroblasts and over expression of the centrosome associated kinase Aurora-A/STK15 and PLK1 in human cancers have further validated the possibility that aberrant centrosome function is involved in aneuploidy and oncogenesis. A number of genes involved in the mitotic checkpoint pathway and those regulating chromosome segregation have been found to be aberrantly expressed in human cancer cells raising the possibility that aberrant expression of the respective mitotic checkpoint and chromosome segregation regulatory proteins contribute to the origin of aneuploidy in cancer (Table 1). In addition to the genes with known functions in mitotic checkpoint and chromosome segregation, mutant alleles of tumor suppressor genes,

Yes

Yes Yes Yes Yes

AdAPC, BRCA1, and BRCA2, have also been shown to induce aneuploidy in murine fibroblasts derived from mice expressing mutated forms of these proteins. Similarly, murine fibroblasts lacking the mismatch repair gene Msh2 also reveal widespread aneuploidy indicating that mutations in this gene may be contributing to tumorigenesis by inducing DNA mismatch repair defects and aneuploidy. Complementing these findings on the likely involvement of aneuploidy inducing genes in the tumorigenesis process, two publications on genetically engineered mice aberrantly expressing genes involved in the regulation of chromosome segregation further advance the case for aneuploidy being a cause of cancer with some caveats. In one of these studies, mice heterozygous for Cenp-E gene, involved in the alignment of chromosomes on mitotic spindle, were reported to develop cancer accompanied by an increase in age dependent whole chromosome aneuploidy

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although Cenp-E heterozygosity inhibited tumorigenesis in animals lacking the tumor suppressor gene p19/ARF. In the second study, mice over expressing the mitotic checkpoint protein Mad2 developed a wide range of tumors with extensive chromosomal rearrangements. However, silencing of Mad2 after tumor formation had no effect on tumor growth, suggesting that Mad2 over expression acts early to promote tumorigenesis. Together, these studies indicate that, like other types of genetic instability, aneuploidy promotes susceptibility to cancer rather than make it obligatory. The concept gains further credence from observations in the human genetic disease mosaic variegated aneuploidy, associated with inactivated mitotic checkpoint gene Bub1b, which reveal constitutional aneuploidy and predisposition to develop cancer. Conclusions The role of aneuploidy as a cancer causing mutation event helps resolve the paradox that with known mutation rate in somatic cells (~107 per gene per cell generation), tumor cell lineages cannot accumulate enough mutant genes during a human life time. Evidence from human tumor cytogenetic and molecular genetic studies provide compelling evidence in favor of aneuploidy being directly involved in the development of tumor phenotypes. Results from clinical findings support a correlation between origin of aneuploidy and tumorigenic transformation of cells. Molecular genetic analyses of tumor cells suggest that mutations/aberrant expression of genes involved in controlling mitotic checkpoint and chromosome segregation play critical roles in causing chromosome instability leading to aneuploidy in cancer.

Cross-References ▶ Renal Cancer Clinical Oncology

References Mitelman F, Johansson B, Mertens F (eds) (2006) Mitelman database of chromosome aberrations and gene fusions in cancer. http://cgap.nci.nih.gov/ Chromosomes/Mitelman

Angiogenesis Pellman D (2007) Aneuploidy and cancer. Nature 446:38–39 Rajagopalan H, Lengauer C (2004) Aneuploidy and cancer. Nature 432:338–341 Sen S (2000) Aneuploidy and cancer. In: Lengauer C (ed) Current opinion in oncology, vol 12. Lippincott Williams & Wilkins, Philadelphia, pp 82–88 Weaver BAA, Cleveland DW (2006) Does aneuploidy cause cancer? Curr Opin Cell Biol 18:658–667. Elsevier Ltd

Angiogenesis Arjan W. Griffioen Angiogenesis Laboratory, Department of Pathology, Maastricht University, Maastricht, The Netherlands

Synonyms Formation of Neovascularization

new

blood

vessels;

Definition Angiogenesis is the formation of new capillary vasculature out of pre-existing blood vessels under the regulation of growth factors and inhibitors. It occurs in physiological (e.g., wound healing, ovulation, placental growth) and pathological (e.g., ▶ cancer, arthritis, ▶ inflammation) conditions.

Characteristics The formation of new blood vessels out of pre-existing capillaries, the process that is called angiogenesis, is a sequence of events that is of key importance in a broad array of physiologic and pathologic processes. Normal tissue growth such as in embryonic development, wound healing, and the menstrual cycle is characterized by dependence on new vessel formation for the supply of

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Angiogenesis, Fig. 1 The angiogenesis cascade of endothelial cell activation, degradation of the extracellular matrix and the basement membrane, migration, and

proliferation. EC, endothelial cells; BM, basement membrane; AS, angiogenic stimulus

oxygen and nutrients as well as for removal of waste products. Also, in a large number of different and non-related diseases, formation of new vasculature is involved in abnormal physiology. Among these pathologies are diseases such as tissue damage after reperfusion of ischemic tissue or cardiac failure, where angiogenesis is low and should be enhanced to improve disease conditions. In a larger number of diseases, excessive angiogenesis is part of the pathology. These diseases include cancer (both solid tumors and ▶ hematological malignancies), cardiovascular diseases (atherosclerosis), chronic inflammation (rheumatoid arthritis, ▶ Crohn disease), diabetes (diabetic retinopathy), psoriasis, endometriosis, and adiposity. These diseases may benefit from therapeutic inhibition of angiogenesis. The initial recognition of angiogenesis being a therapeutically interesting process began in the oncological arena in the early 1970s, when the hypothesis was put forward that tumors are highly vascularized and therefore most vulnerable at the level of their blood supply (Carmeliet 2005). The endothelial cells that line the blood vessels play a pivotal regulatory role in the execution of angiogenesis. The sequence of events in endothelial cells that follow the initiation of angiogenesis by exposure to (e.g., tumor derived) angiogenic stimulation consists of:

• Synthesis of proteases that degrade the ▶ extracellular matrix • ▶ Migration toward the stimulus • Proliferation to increase the number of endothelial cells • Differentiation in order to form a functional vessel (Fig. 1) Negative interference in the different steps of the angiogenesis cascade enables different approaches for treatment of cancer: • Neutralization of angiogenic factors – antigrowth factor antibodies (Avastin) and dominant negative growth factor receptors • Inhibition of growth factor receptors – antigrowth factor receptor antibodies • Desensitization of growth factor-mediated intracellular signaling pathways – ▶ Receptor tyrosine kinase inhibitors • Inhibition of ▶ matrix metalloproteinases • Inhibition of endothelial cells adhesion • Inhibition of endothelial cell ▶ migration • Inhibition of endothelial cell growth/ proliferation Clinical Aspects Although the field of angiogenesis research is rather new, the first compounds with angiostatic activity (Anti-Angiogenic Drug) have been

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approved by the US Food and Drug Administration (Folkman 2006). Most of these compounds are based on interference with growth factors produced by the tumor. Avastin (▶ Bevacizumab) is a monoclonal antibody that blocks ▶ vascular endothelial growth factor. Other currently approved compounds act through inhibition of signaling (kinase inhibitor function) of growth factor receptors. Other angiogenesis inhibitors that directly act on endothelial cells are currently in development. One of the advantages of antiangiogenic therapy is believed to be the lack of induction of resistance to the therapy. This is explained by the fact that endothelial cells are genetically stable cells that are considered not to mutate into drug resistant variants. Although this is a beneficial feature of the anti-angiogenic approach, it is expected that inhibitors of angiogenesis will mainly be used in the future in combination with other anticancer modalities such as chemotherapy, irradiation, and/or ▶ immunotherapy.

Cross-References ▶ Extracellular Matrix Remodeling ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Trefoil Factors

References Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438:932–936 Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285:1182–1186 Folkman J (2006) Angiogenesis. Annu Rev Med 57:1–18 Griffioen AW, Molema G (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 52:237–268

Angiogenesis-Inhibiting Agents ▶ Vascular Targeting Agents

Angiogenesis-Inhibiting Agents

Angiogenin Zhengping Xu Zhejiang University School of Medicine, Hangzhou, China

Synonyms Ribonuclease 5; RNase A family 5

Definition Angiogenin (ANG), originally isolated and characterized as a tumor angiogenic factor, is a member of the vertebrate secreted ribonuclease superfamily. Besides its angiogenic activity, the biological functions of ANG have been extended to tumorigenesis, neuroprotection, and host defense. The mechanism of action of ANG is mainly related to its ribonucleolytic activity toward ribosomal RNA (rRNA) and transfer RNA (tRNA). It has been developed as a clinical therapy target for treatment of cancer, angiogenesis-related diseases, and neurodegenerative diseases.

Characteristics Angiogenin is the Fifth Member of the Human Ribonuclease A Superfamily The human ANG gene is located on chromosome 14q11.2, within the RNase genes cluster of ~400 kb length. The ANG and RNASE4 share the same promoters and 50 -untranslated region (50 -UTR) followed by two distinct exons encoding the two proteins, respectively. In this unique gene arrangement, the transcription of ANG and RNase4 is controlled by a universally promoter and a liver-specific promoter. The reasons for this unique gene arrangement of ANG and RNASE4 are thought to ensure coexpression of the two proteins that act in concert to regulate important biological events. The entire open reading frame (ORF) of human ANG gene encodes a single-chain protein

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consisting of 147 amino acid residues (the first 24 amino acid residues of signal peptide is cleaved before secretion). The human ANG is a 14.4 kDa basic protein (pI 10.1) that has 33% sequence identity and 65% homology with bovine pancreatic ribonuclease A (RNase A). The threedimensional structure of ANG from NMR spectroscopy and X-ray crystallography confirms its structural similarity to RNase A. ANG is designated as the fifth member of human ribonuclease A superfamily after RNASE1, RNASE2 (END, liver, eosinophil-derived neurotoxin), RNASE3 (ECP, eosinophil cationic protein), and RNASE4. The special structure of ANG is important for its function different to other family members. ANG has all the three main catalytic residues of RNase A (His-13, Lys-40, and His-114). However, its ribonucleolytic activity, which is necessary for its angiogenic activity, is 105–106 lower than that of RNase A. On the one hand, this weak enzymatic activity is because the pyrimidine basebinding site in ANG is occluded by the side chain of Gln-117 compared with the structure of RNase A. On the other hand, the fourth disulphide bond in other members of the RNase A superfamily is replaced by two cysteine residues in ANG. The missing of fourth disulphide bond results a sequence of residues (loop region from Lys-60 to Lys-68), which interacts with cell-surface receptor. ANG also has a nuclear localization sequence (NLS) consisting of 30-Met-Arg-Arg-Arg-Gly34, which is required for its angiogenic activity. The known physiological substrate of ANG includes the rRNA and tRNA. ANG prefers to cleave the 30 side of pyrimidine by a transphosphorylation/hydrolysis mechanism. It is reported that ANG is more active than RNase A when compared by their activity on the 28 s and 18 s rRNA. ANG can degrade 28 s and 18 s rRNA to the major products of 100 ~ 500 nucleotides in length. Later, ANG was demonstrated to be responsible for the first cleavage site (A0) of the 47 s pre-rRNA. tRNA was first used as a quantitative enzymatic assay of ANG. A series of publications have highlighted that ANG can cleave the tRNA anticodon loop to form exact tiRNA (tRNA-derived, stress induced small RNA). The tiRNA inhibits protein translation in a phosphorylation-eIF2a-independent manner

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under stress, including heat shock, hypothermia, hypoxia, and radiation. ANG takes key role in tiRNA-mediated protein translation inhibition. In addition, ANG binds to the placental ribonuclease inhibitor (RI), which is one of the most abundant proteins in cytosol. The ANG-RI binding interaction with an extremely low Kd of ~7.1  1016 M is more potent than other family members. The X-ray crystallographic analysis of ANG-RI complex reveals that ANG is located inside the central cavity of RI and the complex pair crystallizes as a dimer, in contrast to the other RNases/RI, which forms a monomeric complex. The tight binding of RI to ANG not only inhibits ANG ribonucleolytic activity but also its tumor angiogenic activity. Angiogenin Is an Angiogenic Factor Angiogenin, characterized by Professor Valle and his colleague at Harvard in 1985, is the first identified human tumor-derived protein that stimulates the growth of blood vessels. It provides the first direct proof for Professor Folkman’s hypothesis that tumor growth depends on neovascularization. ANG is very potent in inducing angiogenesis comparing with most other angiogenic factors. It can induce new blood vessel formation in the chicken chorioallantoic membrane and rabbit cornea only in a femtomole amounts. Until now, the actions of ANG and its mechanisms in angiogenesis have been well documented. ANG is one of the secreted proteins by tumor cells and acts on endothelial cells. In the tumor microenvironment, when ANG reaches to the cell surface of endothelial cells, it binds to the actin and dissociates as a complex. This complex stimulates tissue-type plasminogen activator (tPA)catalyzed generation of plasmin from plasminogen. Degradation of basement membrane and extracellular matrix may thus allow endothelial cells to penetrate and migrate into the tumor. ANG binds to a potential receptor, a 170-kDa transmembrane protein which is not yet fully characterized. On one hand, it triggers a number of downstream signaling pathways, including extracellular signal-related kinase 1/2 (ERK1/2) and protein kinase B/Akt. Activation of these pathways by ANG is considered to produce more

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ribosomal proteins that enhance cell growth. On the other hand, ANG undergoes a receptormediated endocytosis from the cell surface to the nucleus and accumulates in the nucleolus. This process is very important for its angiogenic activity. Either its nuclear localization signal variants or receptor binding site variants lose the angiogenic activity. The ribonucleolytic activity of ANG is also essential for its angiogenisis function. It is clear that the role of ANG in nucleolus is promoting ribosomal transcription by binding the promoter region of ribosomal DNA, which is called angiogenin binding element (ABE), and might act as the enzyme to cleave the first cleavage site (A0) of 47 s pre-rRNA. ANG has been proposed as a permissive factor for angiogenesis induced by other angiogenic factor including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), and epidermal growth factor (EGF). Combined with its signaling pathways, ANG induced ribosome biogenesis is generally required for tumor angiogenesis. ANG circulates in human plasma at a normal concentration of 200–400 ng/ml. However, it fails to cause new blood vessel formation compared its ability in chicken embryo chorioallantoic membrane assay as little as 0.5 ng from the same source. It looks like a paradox. However, ANG does not trigger the ribosome biogenesis in normal endothelial cells that constitute the blood vessel. Now it is known that ANG receptor presents only on the sparsely cultured endothelial cells, but not in confluent cells that exist in blood vessels. This suggests that ANG promotes wound healing at the loss of vascular integrity. When injured clot disrupts endothelial cell confluence, the high concentration of ANG in blood vessel could facilitate rapid blood vessel growth and tissue repair. Besides endothelial cell, smooth muscle cell is another ANG target cell. ANG has been reported to enhance human arterial smooth muscle cell proliferation and bind to a-actinin-2, a cytoskeletal protein. The binding of ANG to a-actinin-2 may result in the phosphorylation of stress-associated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK), which is not affected by the binding of ANG to the receptor in endothelial cells.

Angiogenin

Taken together, we propose the hypothesis of ANG in angiogenic process (1) tumor or tissue damage results in the release of ANG; (2) ANG binds to the endothelial cell-surface actin to activate the protease system and the dissolution of basement membrane; (3) the sparsely endothelial cells express ANG receptor, which activates signal transduction and mediates ANG nuclear translocation to stimulate the ribosome biogenesis; (4) proliferation of endothelial cells penetrate through the basement membrane to form new blood tube; and finally (5) the maturation of the new blood vessel wall by smooth muscle cell migration and proliferation, which also is activated by ANG. Angiogenin Is a Tumorigenic Factor ANG is closely related to tumor growth and progression, and even its aggressiveness. Clinical studies have been found that the protein and mRNA levels of ANG are universally upregulated in the plasma and tissue of patients with various types of cancers. For example, ANG is significantly and progressively upregulated in prostatic epithelial cells while evolving from a benign to an invasive phenotype in the same patients. ANG was once thought to promote cancer progression by its ability to induce neovascularization. ANG was reported to play a direct role on cancer cells themselves. ANG plays a double role in cancer cells by stimulating ribosome biogenesis and sustaining survival under adverse conditions. It can constantly translocate into the nucleus of tumor cells in a cell density-independent manner. This progress is different from the endothelial cells which only occurs under sparse cell density. Besides, ANG can activate AKT, which enhances ribosomal protein production. ANG and AKT pathway have fulfilled the ribosomal biogenesis required for cancer cells growth. ANG’s inhibitors (including an anti-human monoclonal antibody 26-2F, small chemical compound neomycin and neamine, siRNA, antisense, soluble binding proteins, and enzymatic inhibitors) would therefore have a profound effect on cancer cells rRNA transcription, ribosome biogenesis, proliferation, and tumorigenesis.

Angiogenin

ANG also shows cell protection ability. It has been shown to be responsible for stress-induced cleavage of tiRNA. ANG-mediated production of tiRNA in response to stress results in reprogramming of the protein translation thereby promoting damage repairs and cells survival. ANG can also protect cell apoptosis through its capacity to inactivate p53 function and upregulate antiapoptotic genes expression, including Bag1, Bcl-2, Hells, Nf-jb, and Ripk1, and downregulate proapoptotic genes, such as Bak1, Tnf, Tnfr, Traf1, and Trp63. However, its mechanism is still need to be clarified. Angiogenin Is a Neuroprotective Factor Since 2006, a total of 29 unique, nonsynonymous variants of ANG gene have been identified in 6,471 amyotrophic lateral sclerosis (ALS) patients (0.46%) and 3,146 Parkinson’s disease (PD) patients (0.45%) compared with 7,668 control subjects. Several mutations have been characterized to impair the ribonucleolytic activity, nuclear translocation capacity, or angiogenic activity of ANG. ANG is shown to be the first “loss of function” gene so far identified in ALS and PD patients. ANG is the second angiogenic factor associated with ALS pathogenesis. Mice with a homozygous deletion in the hypoxia responsive element of VEGF gene result in an ALS-like phenotype. Subsequently, VEGF exerts neuroprotective on motor neurons not only by increasing neurovascular perfusion but also via directly effects on the neuron cells themselves. Since ANG-mediated rRNA transcription is essential for VEGF to stimulate angiogenesis, it is possible that a deficiency in ANG function may also impair the physiological role of VEGF toward motor neurons. ANG concentration is abnormally reduced in the plasma and cerebrospinal fluid of some ALS patients. ANG protects motor neurons under excitotoxic insults and serum starvation in vitro assays. Data show that stressed motor neuron secretes ANG, then astrocytes endocytose ANG to cleave RNA (unknown group of RNA). In ALS mice model, recombinant ANG delays the death. However, the precise mechanisms of ANG in

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neuron protective response remain determined.

to be

Angiogenin Acts in Other Diseases ANG may also play roles in a variety of nonmalignant angiogenesis-dependent diseases such as endometriosis, peripheral vascular disease, inflammatory bowel disease (IBD), rheumatoid arthritis, diabetes, and so on. In these disorders, ANG expression levels increase and may contribute to the local pathological angiogenesis conditions. Summary ANG is a vertebrate-specific secreted ribonuclease with angiogenic, tumorigenic, and neuroprotective activity. It was first isolated and identified solely by its ability to induce new blood vessel formation in chick embryo chorioallantoic membrane. Subsequently, it was soon discovered to be a 14-kDa basic protein that has 33% sequence identity to bovine pancreatic ribonuclease A (named as the fifth member of ribonuclease family, RNASE5). The role of ANG in angiogenesis is dependent on stimulating rRNA transcription and processing. ANG expression level is upregulated in a various cancer types. It can sustain tumor cells growth by enhancing ribosomal biogenesis and promote cell survival by cleaving the tRNA to form tiRNA. ANG “loss-of-function” has been associated with ALS and PD. It protects motor neuron and delays the death of the ALS mice. As ANG has multiple functions in physiological and pathological processes, it would be a potential therapeutic target.

Acknowledgments We apologize to colleagues whose work has not been cited due to the space limitation.

References Gao X, Xu Z (2008) Mechanisms of action of angiogenin. Acta Biochim Biophys Sin (Shanghai) 40(7):619–624 Li S, Hu GF (2012) Emerging role of angiogenin in stress response and cell survival under adverse conditions. J Cell Physiol 227(7):2822–2826

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240 Riordan JF (2001) Angiogenin. Methods Enzymol 341:263–273 Tello-Montoliu A, Patel JV, Lip GY (2006) Angiogenin: a review of the pathophysiology and potential clinical applications. J Thromb Haemost 4(9):1864–1874

Angiopoietins Harprit Singh De Montfort University, Leicester, UK

Definition Angiopoietins are a group of secreted glycoproteins that play a vital role in vascular development. These growth factors are important in maintaining blood vessel maturation, vascular integrity, and vascular remodeling during adulthood.

Characteristics The angiopoietin family of growth factors consists of four members, Ang1–4. Angiopoietins 1–4 act as ligands for the membrane receptor tyrosine kinase Tie2. The Tie2 receptor is predominately expressed on the vasculature endothelium. Different members of the angiopoietin family act as agonist and antagonist toward the Tie2 receptor. The best characterized angiopoietins are angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). Angiopoietin 1 acts as an agonist and hence activates the Tie2 receptor by phosphorylating several key tyrosine residues present at the carboxyl terminus. On the other hand, Ang2 has the ability to antagonize or partially phosphorylate tyrosine residues. Structure of Angiopoietins The angiopoietins share similar structure, each containing an amino-terminal superclustering angiopoietin-specific domain, which is followed

The entry “Angiopoietins” appears under the copyright Her Majesty the Queen in Right of United Kingdom both in the print and the online version of this Encyclopedia.

Angiopoietins

by a coiled-coil domain as illustrated in Fig. 1. A linker peptide and a carboxyl-terminal fibrinogen homology domain then follow. The C-terminal fibrinogen homology domain (FRED) is further made up of three regions, A, B, and P. The P domain is responsible for the binding of ligand to the Tie2 receptor. The coiled-coil domain is responsible for oligomerization of monomer angiopoietins, while the superclustering domain allows formation of higher-order multimers. Ang1 exists as trimeric, tetrameric, and pentameric homo-oligomers which cluster into multimers. This multimerization of a tetrameric or high-order structure is essential for Ang1 to activate Tie2 receptors in endothelial cells. The ability of Ang2 to act as an antagonist is that it exists only as a homodimer and has no capability of forming higher-order multimers which are essential in activating Tie2 receptors. The linker allows secreted Ang1 to bind to extracellular matrix. Angiopoietin 1: A Protective Ligand Angiopoietin 1 is distributed throughout the normal adult vascular system and is constantly released by smooth muscle cells and pericytes that surround the endothelial layer. In addition, other cells including neutrophils and monocytes also generate Ang1. Genetic studies in mice lacking Ang1 ligand have shown them to die by embryonic day 12.5 with similar vascular defective phenotypes as mice lacking the Tie2 receptor. The main role of Ang1 is that it maintains vessel quiescent, suppresses vascular leakage, inhibits vascular inflammation, and maintains endothelial survival. It exerts its protective effects by binding and activation of the Tie2 kinase domain causing auto- and transphosphorylation of specific tyrosine residues, which act as docking sites for secondary messengers for downstream signaling pathways. Tie2 triggers several cell signaling cascades and downstream targets as illustrated in Fig. 2. Ang1-induced survival and migration of endothelial cells are aided by activation of Tie2 and downstream signaling pathways including phosphatidylinositol 3-kinase (PI3 Kinase), Extracellular signal-regulating kinases 1 and 2 (Erk1/2), and Dok-R/PAK pathways. Dok-R binds to Nck and p21-activated kinase (PAK)

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Angiopoietins, Fig. 1 Schematic representative of the structure of Ang1 and Ang2

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Angiopoietins, Fig. 2 Key downstream Ang1/Tie2 signaling pathways

and has a migratory effect. Activation of PI3 Kinase by recruitment of p85 subunit to specific Tie2 tyrosine-phosphorylated residues further activates the serine-threonine kinase AKT signal transduction pathway. This PI3-K/Akt pathway mediates antiapoptotic/survival effect of Ang1. Ang1 also regulates the MAPK signaling cascade by phosphorylating ERK1/2 which again is involved in migration and survival. Ang1-induced Tie2 activation also facilitates the interaction with ABIN2, a regulatory protein for the transcription

factor NF-kB, and has an anti-inflammatory effect. In addition, Ang1-stimulated Tie2 activation also plays an important role in the recruitment of pericytes to the vessels. The protective effects of Ang1 make this ligand an attractive therapeutic target for manipulation. Vascular regression contributes to various diseases including sepsis and diabetic retinopathy, and so the antiapoptotic effects of Ang1 would have therapeutic usage in counteracting such regression. Also inflammatory conditions such

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as asthma and sepsis could also be regulated by anti-inflammatory effects of Ang1. A potent Ang1 variant, COMP-Ang1, has been developed that shows therapeutic effects in various vascular pathology models including stroke, diabetic nephropathy, and asthma. Further work in understanding the mechanism of Ang1 action will allow development of potent mimetic of Ang1 for clinical use. Angiopoietin 2 Promotes Vascular Destabilization Angiopoietin 2 is stored in Weibel-Palade bodies in the cytoplasm of endothelial cells and hence has an autocrine action. In contrast to the constant expression and secretion of Ang1, expression of Ang2 is predominantly at sites of vascular remodeling including wound healing, female reproductive tract, and tumors. Levels of Ang2 are also elevated in various pathologies including sepsis, diabetic retinopathy, and cardiac allograft vasculopathy. Evidence that Ang2 binds to Tie2 and acts as antagonist comes from early transgenic studies that show overexpression of Ang2 displays similar phenotypes of mice that lack Ang1 or Tie2. At sites of vascular remodeling, the Ang1/Ang2 ratio is dramatically decreased allowing more Ang2 to accommodate Tie2 receptors and hence block the protective and stabilization effects of Ang1. The consequence of Ang2-induced destabilization effect in tumors allows certain angiogenic cytokines such as Vascular endothelial growth factor (VEGF) to act on the vasculature promoting tumor angiogenesis. Ang2 also aids in the recruitment of tumor-associated monocytes which are capable of promoting angiogenesis within the tumor. Therapeutic Target for Tumor Angiogenesis Over the years, a huge interest has been drawn in the development of therapeutic agents to block the activity of Ang2 to inhibit tumor angiogenesis and growth. Ang2 monoclonal antibody inhibitors are common agents used for such models. Some preclinical models have shown that these inhibitors are quite potent in inhibiting tumor growth. One example is MEDI3617. This Ang2-specific

Angiopoietins

monoclonal antibody inhibitor has shown to suppress lung metastasis and lung lymph node metastasis from non-small cell carcinoma of the lung by blocking the Ang2 destabilization effect. Other studies have shown that combining selective Ang2 inhibitors with anti-VEGF antibodies in tumor models significantly reduces tumor growth compared to using Ang2 inhibitors on their own. Hence work on the effects of combined inhibitors of Ang2, VEGF, and other angiogenic cytokines including bFGF and PDGF is currently being investigated to maximize therapeutic potential. In conclusion, angiopoietins are involved in vascular stability and remodeling. The level of Ang1 and Ang2 determines the fate of the vasculature. Increased levels of Ang2 or a fall in the Ang1/Ang2 ratio is linked to several pathologies including cancer making the angiopoietin-Tie2 axis an attractive target in the treatment in tumor therapy.

Cross-References ▶ AKT Signal Transduction Pathway ▶ Angiogenesis ▶ Cytokine ▶ Extracellular Signal-Regulated Kinases 1 and 2 ▶ Fibrinogen ▶ Inflammation ▶ Metastasis ▶ Monoclonal Antibodies for Cancer Therapy ▶ Nuclear Factor-κB ▶ Pathology ▶ PI3K Signaling ▶ Receptors ▶ Receptor Tyrosine Kinases ▶ Vascular Endothelial Growth Factor

References Brindle NPJ, Saharinen P, Alitalo K (2006) Signaling and functions of angiopoietin-1 in vascular protection. Circ Res 98:1014–1023 Hashizume H, Falcon BL, Kuroda T, Baluk P, Coxon A, Yu D, Bready JV, Oliner JD, Mcdonald DM (2010) Complementary actions of inhibitors of

Angiotensin II Signaling angiopoietin-2 and VEGF on tumor angiogenesis and growth. Cancer Res 70:2213–2223 Kim KT, Choi HH, Steinmetz MO, Maco B, Kammerer RA, Ahn SY (2005) Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2. J Biochem 280:20126–20131 Moss A (2013) The angiopoietin: Tie2 interaction: a potential target for future therapies in human vascular disease. Cytokine Growth Factor Rev 24:579–592 Yuan HT, Khankin EV, Karumanchi SA, Parikh SM (2009) Angiopoietin 2 is a partial agonist/antagonist of Tie2 signaling in the endothelium. Mol Cell Biol 29:2011–2022

Angiotensin ▶ Angiotensin II Signaling

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homeostasis. There are two well-defined receptors of angiotensin II (subtype 1 (AT1) and subtype 2 (AT2)), both of which have seven transmembrane, ▶ G-protein coupled receptors and are encoded by different genes (AT1 (agtr1), 3q21–25; AT2 (agtr2), Xq22–23). The major isoform, AT1 receptor, is expressed in a wide variety of tissues. The AT2 receptor, the second major isoform, is expressed abundantly in fetal mesenchymal tissues, but its expression decreases significantly immediately after birth. The AT2 receptor expression level is low in adult tissues but is inducible and functional under pathophysiological conditions. In addition to these angiotensin II receptors, leucyl/cystinyl aminopeptidase and Mas-related G-protein-coupled receptor member F have been identified as receptors for angiotensin IV and angiotensin-(1–7), respectively.

Angiotensin II Signaling Characteristics Masaaki Tamura and Takaya Matsuzuka Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA

Synonyms Angiotensin

Definition The angiotensin peptides (angiotensins I, II, III, IV, and -(1–7)) are derived from the precursor angiotensinogen by sequential processing proteases such as renin, angiotensin I-converting enzyme (ACE), chymase, and other peptidases. Among these peptides, angiotensin II has been well studied and is shown to be the most biologically active peptide. This peptide hormone production system is called the renin-angiotensin system and is one of the phylogenetically oldest hormone systems that has been conserved throughout evolution. The renin-angiotensin system plays a key role in the maintenance of arterial blood pressure and fluid and electrolyte

Angiotensin II Signaling in Carcinogenesis The renin-angiotensin system plays a key role in fluid homeostasis and in blood pressure control. Circulating renin, produced by the juxtaglomerular apparatus of the kidney, and other tissue renin cleaves angiotensinogen to angiotensin I. Angiotensin I-converting enzyme (ACE) catalyzes the subsequent production of the active peptide angiotensin II. Angiotensin II stimulates a variety of biologically important actions, such as vasoconstriction, aldosterone release, and cell proliferation. A large portion of these biological actions are executed by locally generated angiotensin II in an autocrine and paracrine manner. The diversity of angiotensin II-induced biological reactions is determined through the expression of two receptors and their coupling with various ▶ G-proteins. The AT1 receptor is expressed in a wide variety of tissues and is mainly responsible for most angiotensin II-dependent actions in cardiovascular/renal tissues. The AT1-mediated angiotensin II signaling stimulates an increase in vasoconstriction (Gq), cardiac hypertrophy (Gq), cell mortality (G12/13), nitric oxide (Gi), and ▶ prostaglandin (Gi)

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Angiotensin II AT 1 receptor

AT 2 receptor NH2

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HOOC

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Ca2+ EGFR kinase

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Angiotensin II Signaling, Fig. 1 Schematic illustration for diverse angiotensin II signaling

formation (G-proteins in the parenthesis indicate their specific roles, Fig. 1). AT1-mediated signaling also stimulates production of various growth factors such as EGF, basic-FGF, TGF-b, and ▶ VEGF. AT2 receptor-mediated angiotensin II actions are also diverse, and this diversity is also determined through Gi and Gs protein coupling. Protein tyrosine and serine/threonine phosphatase activation (Gs), nitric oxide/cGMP, and arachidonic acid/prostaglandin production (Gi) are involved in the mechanism of AT2 receptor-mediated biological reactions (Fig. 1). The AT2 receptor can function to counteract AT1 receptor-mediated angiotensin II bioreactions. However, the AT1 and AT2 receptors can also unidirectionally mediate the angiotensin II signal. Angiotensin II also stimulates FGF-2 expression through both the AT1 and AT2 receptors. In addition, the AT2 receptor mediates ▶ apoptosis in a few types of cells derived from cardiovascular and neuronal tissues in vitro. The stimulation of cell proliferation by angiotensin II-AT1 signaling has been studied in various cancer cell lines such as ▶ breast cancer, pancreatic cancer, ▶ ovarian cancer, and prostate cancer. The activation of AT1 stimulates growth factor pathways such as tyrosine kinase phosphorylation and induces phospholipase C, leading to

activation of downstream proteins such as MAPK, JNK, and STAT pathways. Furthermore, AT1 signaling also stimulates ERK1/2 via ▶ epidermal growth factor receptor (EGFR) transactivation. The AT1 signaling-induced shedding of heparin-binding EGF by stimulation of metalloproteinases causes the transactivation of EGFR. However, since it is implied that the involvement of transactivation of EGFR by AT1 signaling is dependent on cell type, pathophysiological significance of angiotensin II-AT1dependent EGFR transactivation in carcinogenesis is not yet clear. Clinical Aspects Angiotensin II induces the expression of protooncogenes, such as c-fos and c-myc, and promotes cell proliferation and growth through the AT1 receptor. AT1 receptor signaling also stimulates the expression of hypoxia-inducible factor (HIF) 1a and VEGF, which causes resultant neovascularization, a requirement for solid tumor growth. Accordingly, angiotensin II is a mitogenic and pro-angiogenic factor. The AT1 receptor expression has been shown in the tissues of breast cancer, ovarian cancer, pancreatic cancer, melanoma, prostate cancer, and bladder cancer. There is a strong positive relationship

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Angiotensin II Signaling, Fig. 2 The schematic model of the angiogenic effect of angiotensin II in tumorigenesis

Cancer cells

A VEGF

Angiotensin II

Angiogenesis VEGF COX-2

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between the expression level of the AT1 receptor and ovarian cancer malignancy, and the survival rate of AT1 positive ovarian cancer patients is significantly lower than the AT1 negative patients. ACE is also detected in tumor stroma of several types of cancers. These observations suggest that local renin-angiotensin system exists in these various cancer tissues, and the AT1 receptormediated angiotensin II signaling may play a significant role in tumor growth. Subcutaneous tumor xenografts in AT1a-KO mice demonstrated that AT1 signaling in host stromal fibroblasts is also an important regulator of tumor-associated ▶ angiogenesis. Angiogenesis is an important support mechanism in tumor development. Angiotensin II can directly stimulate capillary network formation by upregulation of VEGF production in endothelial cells and vascular smooth muscle cells. VEGF is known as a strong angiogenic factor in a variety of cancers. VEGF promotes endothelial cell proliferation, migration, and survival. An ACE inhibitor attenuates VEGF-mediated tumor growth, accompanied with the suppression of neovascularization in the tumor and VEGF-induced endothelial cell migration. VEGF expression is upregulated by AT1 signaling not only in cancer cells but also in tumor-associated stromal cells including fibroblasts and infiltrated macrophages. Angiotensin II-AT1 signaling also induces tumor-associated macrophage infiltration. Angiotensin II significantly induced cyclooxygenase-2 (▶ COX-2) expression in the mouse lung stromal fibroblasts

through AT1 signaling. Prostaglandin E2, the main product of COX-2, is known to have a pro-angiogenic effect as well. In fact, the COX-2 inhibitors reduced tumor growth accompanied by an antiangiogenic effect on tumor tissue. The expression levels of COX-2 and VEGF appears to be tightly associated since VEGF stimulates COX-2 mRNA expression and prostaglandin E2 increases VEGF mRNA expression in vascular endothelial cells. The COX-2-specific inhibitor suppresses tumor angiogenesis by decreasing VEGF expression in a rat colon cancer model. Furthermore, the selective COX-2 inhibitor celecoxib and ACE inhibitors or AT1 receptor antagonists synergistically inhibited colon cancer growth. Accordingly, angiotensin II-AT1 signaling promotes tumor growth by upregulation of both COX-2 and VEGF expression in cancer cells and stromal cells (Fig. 2). Attenuation of the AT1 receptor function by a clinically employed AT1-specific receptor antagonist has been shown to block lung metastasis of renal cell carcinoma in mice. A potential mechanism underlying the AT2 receptor-dependent modification of carcinogen susceptibility appears to be in part due to a modulation of cytochrome P450 expression and stromal fibroblast-dependent support of tumor growth. In addition to angiotensin II receptor blockers, ACE inhibitors retard the growth of cancer cells in vitro. ACE inhibitors also inhibit angiogenesis and the growth of tumor xenografts in rats. Therefore, the renin-angiotensin system is

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an important component in both cancer and cardiovascular diseases. Epidemiological Study of the Effect of ACE Inhibitors on Cancer Risk Although the ACE inhibitors (e.g., captopril, lisinopril, enalapril, or perindopril) have demonstrated significant antitumor effects in in vitro studies or animal studies, results of epidemiological studies are not consistent with these studies. In 1998, Lever et al. reported that ACE inhibitors decreased the risks of cancer, particularly breast and lung cancer for the first time. However, most of the other epidemiological studies did not find any clear association between ACE inhibitors and risk of cancer. Although the reason for these controversial results remains unclear, the variety of conditions among studies (the use of different ACE inhibitors, populations, the dose, and duration of treatment) might cause these different results. Since angiotensin II is produced not only by ACE but also by other enzymes such as chymase, ACE inhibitors cannot completely block the effect of angiotensin II. Therefore, an epidemiological study to determine the association between AT1 receptor inhibitors and risk of cancer will also be required. Perhaps the most critical issue is that there is no ACE inhibitor or AT1 receptor blocker case-controlled study. There is a strong negative correlation between the expression levels of AT1 in ovarian cancer tissue and the 5-year survival ratio of patients. Although the sample number is small, this study indicates that angiotensin II signaling has a crucial impact on some types of cancer prognosis. Taken together, angiotensin II signaling is an important component in carcinogenesis and is a potential target for Chemotherapy for various cancers.

ANLL Egami K, Murohara T, Shimada T et al (2003) Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth. J Clin Invest 112:67–75 Ino K, Shibata K, Kajiyama H et al (2006) Manipulating the angiotensin system – new approaches to the treatment of solid tumours. Expert Opin Biol Ther 6:243–255 Kanehira T, Tani T, Takagi T et al (2005) Angiotensin II type 2 receptor gene deficiency attenuates susceptibility to tobacco-specific nitrosamine-induced lung tumorigenesis: involvement of transforming growth factorbeta-dependent cell growth attenuation. Cancer Res 65:7660–7665 Lever AF, Hole DJ, Gillis CR et al (1998) Do inhibitors of angiotensin-I-converting enzyme protect against risk of cancer? Lancet 352:179–184

ANLL ▶ Acute Myeloid Leukemia

Anoikis Steven M. Frisch Mary Babb Randolph Cancer Center and Department of Biochemistry, West Virginia University, Morgantown, WV, USA

Synonyms Detachment-induced mediated death

cell

death;

Integrin-

Definition Apoptosis that is suppressed by extracellular matrix.

References Berry C, Touyz R, Dominiczak AF et al (2001) Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am J Physiol Heart Circ Physiol 281:H2337–H2365

Characteristics Cells that are released from extracellular matrix attachment or cells that are attached to an

Anoikis

inappropriate type of matrix are normally programmed to undergo apoptosis. This phenomenon prevents the reattachment and possible mis-localized colonization of epithelial cells shed during normal turnover, for example, in the gastrointestinal tract. Metastatic tumor cells have undergone genetic or epigenetic changes that invariably render them resistant to anoikis, permitting them to survive during metastasis and underscoring the cancer relevance of this phenomenon. Anoikis is primarily a property of epithelial and endothelial cells, and the epithelial-to-mesenchymal transition (EMT) of tumor cells is accompanied by resistance to anoikis. Accordingly, many activated oncogenes confer anoikis resistance.

Mechanisms Anoikis occurs when survival signaling by ligated integrins is interrupted or when unligated integrins actively recruit and activate caspases. Survival signaling by integrins is complex. Two pivotal effectors are the ERK subfamily of MAP kinases and the kinase known as Akt/PKB. ERKs can promote cell survival through several different effects, including: (i) phosphorylation and inactivation of the pro-apoptotic action of the Bcl-2 family member BAD, (ii) downregulation of the pro-apoptotic Bcl-2 family member Bim and upregulation of Bcl-xl, and (iii) phosphorylation and inactivation of caspase-9. Akt activates several other survival pathways, through other effects: a. inactivation of glycogen synthase kinase-3, which regulates both Wnt/APC/beta-catenin/LEF-1 signaling and certain pro-apoptotic transcription factors; b. activation of the pro-survival transcription factor complex NF-kB; c. inactivation of the p73-associated cofactor, YAP; and d. phosphorylation and inactivation of caspase-9. Upstream of these kinases, Focal Adhesion Kinase (FAK) and Integrin Linked Kinase (ILK) contribute to integrin-mediated cell survival. Epithelial cells containing a constitutively active FAK or ILK are resistant to anoikis, and many

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human tumors overexpress FAK protein. Thus, FAK is considered a potential anticancer drug target. FAK, usually in a complex with c-src, can activate ERKs through several pathways: (i) by binding paxillin and augmenting signaling through p21-activated kinase (PAK), the p130cas/crk complex, and an exchange factor (PIX) for rac-related GTPases, which are, in turn, important factors in determining anoikis sensitivity, and (ii) by activating the Ras/Raf/MEK/ ERK pathway through Grb2/sos1 interaction. FAK can also activate PI3-kinase, activating Akt. FAK can also rescue cells from anoikis by inactivating the pro-apoptotic activity of RIP1, a death receptor adaptor protein. Certain Bcl-2 family members now have an established role in anoikis. Although the translocation of Bax to mitochondria occurs in detached cells, a report shows that it is the conformational change of Bax rather than its translocation per se that is rate-limiting. Mitochondrial permeabilization by Bax is regulated by several factors, including the “BH3 domain-only” Bcl-2 family members, Bim and Bmf. Both of these latter factors play an important role in anoikis, and they are both regulated transcriptionally and posttranslationally. This may occur, for example, by the loss of active EGFR-mediated ERK signaling in detached cells, facilitating pro-apoptotic activity of Bim at mitochondria, or by the association/ dissociation of the BH3 factors with respect to the actin cytoskeleton. Several highly cancer relevant genes have been implicated in regulating anoikis, two of which are non-integrin proteins involved in cell adhesion and one is a receptor. First, E-cadherin is a major invasion suppressor protein involved in epithelial cell-cell adhesion that is frequently downregulated in carcinoma cells. Interestingly, mouse genetics data show that cells lacking E-cadherin (in a p53-null background) are resistant to anoikis, indicating that epithelial cells are normally sensitized to anoikis through E-cadherin-mediated cell interactions. This has important implications for the mechanism by

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ASPP

EMT

SCRIB P LATS

YAP/TAZ LATS

P

Dsh

WNT

CK1 GSK3β P P

CtBP ZEB snail twist NF| B FAK ILK TrkB

βP catenin

x CK1

DSH

Akt

WNT

Smad 3

TGF-β/TGFβR

x PI3K

CRB

x

GRHL2

Epithelial specific inhibitory complex

GSK3 β x TGF-β/TGFβR

YAP/TAZ Smad3 P YAP, TAZ, SMAD3, and β-catenin target genes silent

ANOIKIS-SENSITIVE EPITHELIAL CELL

βcatenin

YAP, TAZ, SMAD3, and β-catenin target genes acve

ANOIKIS-RESISTANT MESENCHYMAL CELL

Anoikis, Fig. 1 The epithelial specific cell polarity proteins maintain anoikis sensitivity by regulating the Hippo, Wnt and TGF-b pathways. In normal, interacting epithelial cells (left panel), the cell polarity complexes, crumbs (crb) and scribble (srb) stimulate the phosphorylation of YAP and TAZ through the LATS kinase. This maintains YAP and TAZ in the cytoplasm, sensitizing cells to anoikis. In addition, cytoplasmic YAP and TAZ interact with Smad3 and prevent its nuclear translocation, even in the presence of active TGF-β receptors. Furthermore, cytoplasmic TAZ

interacts with disheveled (Dsh), inhibiting canonical Wnt signaling. Expression or activation of the factors listed in the figure induce EMT (right panel), compromising cell polarity complexes, promoting YAP, TAZ and Smad3 nuclear translocation and inducing cell survival genes. The absence of cytoplasmic TAZ allows Dsh to be activated by casein kinase-1 (CK1), inhibiting GSK3β, and thus allowing b-catenin to transactivate pro-survival genes in the nucleus. Alternatively, the Akt-GSK-3-b-catenin axis could be stimulated by activation of TrkB, as shown

which EMT allows tumor cells to resist anoikis. An E-cadherin-associated cytoskeletal protein, ankyrin-G, sensitizes normal epithelial cells to anoikis by sequestering a transcription factor, NRAGE, that represses the p14ARF tumor suppressor gene. Second, the neurotrophin receptor protein, trkB, that is over-expressed in pancreatic and prostate tumors, is a potent activator of the PI3-kinase/Akt pathway and thus renders these tumor cells resistant to anoikis, providing an opportunity for trkB-based therapy. The third is carcinoembryonic antigen (CEA), which is over-expressed on the surface of a variety of tumor cells, appears to program tumor cells to resist anoikis by causing integrin clustering and ensuing survival signaling.

Metabolic pathways of metastatic tumor cells (after EMT) may favor anoikis-resistance by maintaining high oxidative phosphorylation while at the same time suppressing the levels of reactive oxygen species (ROS). This can be achieved by increased glumate dehydrogenasemediated glutaminolysis and/or over-expression of anti-oxidant enzymes. Cell-matrix detachment (acting, in part, through ROS) also may engage autophagic pathways that protect against anoikis by generating biosynthetic precursors, reducing potential and energy charge. Several mechanisms have been implicated in the acquisition of anoikis-resistance that accompanies EMT, including altered cell polarity complexes, as depicted in Fig. 1.

Anoxia

Anoxia Yerem Yeghiazarians1, Adrian L. Harris2 and Kurosh Ameri1 1 Department of Medicine, Division of Cardiology, Translational Cardiac Stem Cell Program, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Cardiovascular Research Institute, University of California San Francisco (UCSF), San Francisco, CA, USA 2 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Cancer Research UK, Headington, Oxford, UK

Synonyms Extreme hypoxia; Hypoxia; Severe hypoxia

Definition Literal Definition Anoxia literally means the complete absence of oxygen (O2) and has been described as the state where no O2 (0% O2) is detected in the tissue. This definition contrasts the definition of hypoxia, which means low levels of oxygen as opposed to complete absence. Conceptual Definition The major function of the vasculature is to deliver oxygen and nutrients to cells and remove carbon dioxide and other metabolic by-products from them. Oxygenated blood is distributed in each tissue according to the functions and needs of that tissue, which differs from one tissue to another. Therefore, when studying different tissues and cell types, there are significant variations in cellular response(s) based on oxygen level and/or corresponding nutrient level. Hence, oxygen tension has to be viewed with respect to a particular tissue/organ and is therefore essentially a functional definition, because there are marked normal differences in oxygen tension in the body. For example, normal physiological oxygen level

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in the superficial skin region is 1.1%, whereas in subpapillary plexus it is 4.6% and in intestinal tissue it is 7.6% oxygen. Thus, a decrease in the normal O2 for a tissue or organ, sufficient to induce a molecular or physiological response, would be an operational definition with respect to a specific tissue/organ. Several studies have shown that when cancer cells are exposed to hypoxia (defined as 1%, 0.5%, or 0.1% oxygen in those studies) versus anoxia (defined as O2 < 0.5%, 0.1%, or 0.001% in various studies), distinct pathways are switched on in anoxia that are either absent or switched on in much lower levels in hypoxia. Hence, anoxia has been referred to lack of oxygen that triggers cellular and molecular responses that differ to the response in hypoxia. These differences in response of cells to hypoxia versus anoxia have been correlated to cell-fate differences, respectively. Cellular fates during oxygen deprivation are diverse, including death, survival, continued proliferation, quiescence (or hibernation/dormancy), senescence, and differentiation. Such diverse fates depend on the severity and/or duration of oxygen/nutrient deprivation and the genetic background of the cell type. Therefore, the distinction of terminology between hypoxia and anoxia is important because in hypoxia, cells have a much better chance to adapt and survive compared to anoxia. Therefore, anoxia can also be defined with respect to cell fates that differ to the fates observed in hypoxia. Whereas cells can continue to grow in hypoxia for some time, anoxia on the other hand can redirect cell fate toward hibernation/dormancy or death. These key cell-fate differences in hypoxia versus anoxia are due to the key pathways induced, epigenetic changes, and metabolic switches. Indeed, experiments performed with Caenorhabditis elegans have demonstrated that sensing anoxia is a separate pathway to sensing and adapting to hypoxia, where organisms survive anoxia via undergoing suspended animation.

Characteristics Oxygen is absolutely essential for life, so the molecular mechanisms underlying responses to

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low levels of oxygen are central to the cell. The cell has to be able to sense and interpret the level of oxygen present in its environment, and based on this interpretation, the cell will make a decision (termed cellular decision-making) for a particular fate such as death versus survival. For example, when cells are exposed to hypoxia, such as during intensive exercise, information flow within the cell interprets the oxygen level as being “hypoxia” (low), which in turn results in anaerobic metabolism that enables the cell to produce energy and survive under anaerobic conditions. This adaptive response can be viewed as a normal physiological process, which is primarily modulated via the hypoxia-inducible factor 1 (HIF1) pathway [see entry on “▶ Hypoxia”]. In contrast, fate of cells in a pathological setting will be different than the aforementioned physiological condition of exercise. Several diseases such as ischemic heart disease, stroke, and cancer are associated with oxygen and nutrient deprivation. In ischemic conditions such as in the heart or brain, cells initially respond by adapting and surviving via switching on anaerobic metabolism. As ischemic conditions become severe, cells receive no oxygen or nutrients (notably glucose) and eventually die but can also hibernate and survive. This scenario is similar in solid tumors, which are known to contain regions of hypoxia and anoxia. Tumor cells may survive anoxia due to diminished apoptotic pathways, genetic mutations, protein mislocalization, as well as via dormancy-mediated survival. In Vitro Creation of Hypoxia and Anoxia Several units have been used to describe the amount of oxygen present in the experimental atmosphere. It has been proposed that the partial pressure of oxygen should be given in the SI unit kilopascal (kPa, 1000 N per m2) in line with international agreements. 1 kPa equals 10 bar or 7.5 torr (or mmHg where 760 mmHg equals 100% O2). In gas mixtures containing 10,000 ppm (parts per million) of oxygen, the partial pressure is 1 kPa. Most reports have used the unit mmHg or % O2 to refer to the amount of oxygen present in experimental atmosphere. The use of ambient air has been referred to the “normal oxygen tension” (normal levels of oxygen) often termed normoxia

Anoxia

or 21% oxygen. Normoxia is used as means of experimental control, to which hypoxia is compared to. Typically, experiments testing the effects of hypoxia tend to culture cells in incubators with a gas mix of 5% CO2 and 95% N2 until the desired level of hypoxia is reached. The hypoxic cells are then compared to cells cultured in normoxia, which consist of ambient air and 5% CO2. Anoxia has been achieved in vitro by using incubators with an atmosphere of 5% CO2, 90% N2, and 5% H2 and a palladium catalyst to scavenge traces of oxygen. Alternatively, a continuous flow of 95% N2 and 5% CO2 has been used. Such conditions have achieved O2 levels lower than 0.1% and even 0.001% O2 in tissue cultures of moderate to low cell density, and therefore anoxia has been addressed as O2 levels 10 mmHg or 1% O2) tumors are mostly hypoxic compared to in vitro conditions of 21% O2 and express HIF1, indicating that many tumors live under hypoxia. The in vitro conditions of hypoxia and anoxia coincide well with oxygen measurements performed with polarographic O2 electrode needles on patient tumors, which have demonstrated extremely low levels of oxygen such as 2 mm (Tot 2007). One of the problems in multifocal cases is that seemingly clear surgical margins may be created during breast-conserving surgery if the surgeon cuts the breast tissue in between two distant individual foci, which can result in the false impression that the surgical margins are clear. This may lead to local recurrence of the cancer in a considerable number of cases, more often than in patients with unifocal tumors. Both the increased metastatic capacity of the multifocal tumors and their increased local recurrence rates lead to decreased disease-free survival of these patients. Several studies have demonstrated that multifocality has a negative impact on diseasespecific and overall survival of patients. This negative impact seems to be independent of the applied oncological therapy (Pekar et al. 2014; Vera-Badillo et al. 2014). Diffuse Cases Diffuse growth of both the invasive and in situ components of tumors is a distinct morphological pattern with a substantial impact on prognosis. Diffuse in situ carcinomas (including the in situ component of invasive tumors) can be observed in

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approximately one-quarter of breast cancer cases. In these cases, the tumor involves mainly the large ducts and often the main duct of the sick lobe. Half of such cases are detected by mammography findings of typical long and branching calcifications; other such tumors may cause architectural distortion on the mammogram without calcifications. Invasive carcinomas associated with an in situ component such as this have a much poorer prognosis compared to other invasive tumors. In contrast with diffuse in situ carcinomas, invasive carcinomas with a diffuse growth pattern are rare and comprise approximately 5% of all breast carcinoma cases. Most of these tumors are of the lobular subtype and comprise small dispersed tumor cells that infiltrate the surrounding tissue without producing any reaction from it. These tumors are most often of intermediate histological grade and are estrogen receptor positive; however, despite their favorable molecular characteristics, these are the most aggressive breast carcinomas with the worst outcome in modern breast healthcare.

References

Conclusions Multifocality and a diffuse growth pattern of both the in situ and invasive tumor components are frequent in breast carcinoma and are powerful negative prognostic parameters independent of other characteristics of the tumors. The prognosis in unifocal invasive breast carcinomas is relatively favorable, while the prognosis is intermediate in multifocal cases and worst in diffuse cases. Modern multimodal breast radiology and special large-format histopathological slides can enable detection of multiple tumor foci in most multifocal cases and provide valuable guidance in therapeutic decision-making.

Definition

Cross-References ▶ Breast Cancer ▶ Breast Cancer Prognostic and Predictive Biomarkers ▶ Breast Cancer Stem Cells ▶ Ductal Carcinoma In Situ

Pekar G, Hofmeyer S, Tabar L et al (2014) Multifocal breast cancers documented in large-format histology sections: long-term follow-up results by molecular phenotypes. Cancer 119:1132–1139 Tot T (2007) Clinical relevance of the distribution of the lesions in 500 consecutive breast cancer cases documented in large-format histologic sections. Cancer 110:2551–2560 Vera-Badillo FE, Napoleone M, Ocana A et al (2014) Effect of multifocality and multicentricity on outcome in early stage breast cancer: a systematic review and meta-analysis. Breast Cancer Res Treat 146:235–244

Breast Cancer Multistep Development Dihua Yu and Jing Lu Departments of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

A ▶ multistep development of breast cancer involves increasingly abnormal stages during ▶ breast cancer ▶ progression as illustrated in Fig. 1.

Characteristics Breast cancer is well recognized as a heterogeneous disease. It can be categorized as five subtypes based on gene expression profiles as determined by ▶ multigene arrays: luminal A, luminal B, HER2+/▶ estrogen receptor (ER)-, basal-like, and normal breast-like (Sorlie et al. 2001). Based on epidemiological and histological observations of mostly the luminal A and luminal B subtypes, these steps can be defined as a series of morphological changes beginning with ▶ hyperplasia; followed by atypical hyperplasia, ▶ carcinoma in situ, and invasive carcinoma; and ending with metastatic breast cancer, the major

B

672 Normal Mammary duct

Breast Cancer Multistep Development

Hyperplasia

Atypical Hyperplasia

Carcinoma in situ

Invasive Carcinoma

Metastasis

Genetic, epigenetic, and microenviroment alterations

• Gain of functions in oncogenes, such as ER, HER2, ras and c-myc; • Loss of functions in suppressor genes, such as BRCA1/2, P53, PTEN and Rb; • Epigenetic alterations such hypermethylation of DNA and hypoacetylation of histones; • Microenviroment alterations such as loss of myoepithelial cells and inflammatory responses;

Breast Cancer Multistep Development, Fig. 1 Schematic representation of the multistep model of breast cancer development. These steps can be defined as a series of

increasingly abnormal stages including hyperplasia, atypical hyperplasia, carcinoma in situ, invasive carcinoma, and metastatic breast cancer

cause of most breast cancer-related deaths. This seemingly continuous but nonobligatory progression can occur over long periods of time, decades in many cases, and many patients can live with the early stage noninvasive lesions through a normal life span, without being diagnosed or treated. Multistep of Breast Cancer Progression

size, shape, number, or growth pattern. It is found in approximately 15% of breast biopsies following the identification of suspicious microcalcification. According to the location of these abnormal cells within the breast tissue, the lobules, or the ducts, AH can be further divided into atypical lobular hyperplasia (ALH) or atypical ductal hyperplasia (ADH).

Hyperplasia

Carcinoma In Situ

Hyperplasia refers to the increased proliferation of normal-looking mammary epithelial cells within the breast. As a benign, noncancerous disease, hyperplasia can be caused by delayed differentiation rather than the essential alterations that will obligatorily lead to breast cancer. However, statistical studies have indicated that women with hyperplasia have a twofold increase in the risk of developing breast cancer.

▶ Carcinoma in situ is the first malignant step in the progression of breast cancer. It is defined by the clonal proliferation of malignant cells that are restrained within the lumen of mammary ducts (termed ▶ ductal carcinoma in situ, or DCIS) or lobules (termed lobular carcinoma in situ, or LCIS). DCIS and LCIS have been indicated to evolve from ADH and ALH, respectively. In both cases, there is no invasion into the surrounding stroma. DCIS is the most common type of noninvasive breast cancer in women, accounting for 25% of all breast cancer diagnoses. As an intermediate stage

Atypical Hyperplasia

Atypical hyperplasia (AH) is characterized as a condition when breast cells appear abnormal in

Breast Cancer Multistep Development

in breast cancer progression between ADH and invasive cancer, DCIS represents a spectrum of heterogeneous breast diseases which vary both morphologically and biologically and therefore remain a challenging task for its classification and clinical management. Traditionally, DCIS classification has been mainly based on architectural growth pattern and thus divided into comedo, solid, cribriform, papillary, micropapillary, clinging, hypersecretory, and apocrine variants. However, this classification does not allow prediction of the clinical behavior of DCIS, particularly its potential for progression into life-threatening invasive disease. To generate better correlation with the clinical outcome of DCIS, several new criteria have been proposed, and most of them are based primarily on nuclear grade (high, intermediate, and low) and secondarily on cell polarization (architectural differentiation) and absence or presence of ▶ Necrosis. These classifications are more predictive of disease recurrence after surgical resection. LCIS is relatively rare compared to DCIS and usually shows a low proliferation rate. In many cases, LCIS is diagnosed in patients before menopause, and the lesions are usually multifocal and bilateral (▶ contralateral breast cancer). Invasive Carcinoma

Invasive carcinoma is defined as cancerous cells having spread beyond the mammary ducts or lobules and invaded into the surrounding stroma. There are many subtypes of invasive carcinoma in the breast, with the invasive ductal carcinoma (i.e., malignant cells have penetrated through the basement membrane of the mammary duct and invaded the fatty tissue of the breast) as the most common type, accounting for three-quarter of all cases. The second most common subtype is invasive lobular carcinoma, which is characterized as cancerous cell invading through the lobules of the breast. Other rare forms of invasive breast cancer include inflammatory carcinoma, medullary breast cancer, and adenocystic breast cancer. Pathologic/clinical and molecular studies have strongly supported the in situ carcinoma as the precursor lesion of invasive carcinoma.

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Metastasis

The end stage of breast cancer as a progressive disease is ▶ metastasis, when breast cancer cells gain the capability to escape the restrain of primary site, metastasize, and colonize a secondary site. Metastasis is extremely devastating to patients because the vast majority of breast cancer mortality is due to metastasis, not the primary tumor. Metastasis is a multistep cascade involving at least the following crucial events: dissemination from the original tissue architecture; increased ▶ matrix metalloproteinase expression to degrade extracellular matrix barrier; elevated ▶ motility and ▶ invasion; intravasation into the blood or lymphatic vessels and survival in the circulation; and extravasation and adaptation to a foreign microenvironment of distant organs for metastatic growth. The most common sites of breast cancer metastasis are the bones, brain, liver, and lungs. Mechanisms that Drive Multistep Development of Breast Cancer It is commonly accepted that the multistep development of breast cancer is driven by progressively accumulated genetic, epigenetic, and microenvironmental alterations (Figs. 1 and 2). Numerous studies have confirmed the essential role of genetic abnormalities in breast cancer progression. Two categories of genetic abnormities are the gain-of-function mutation in proto-oncogenes and the loss-of-function mutation in ▶ tumor suppressor genes. Some well-known ▶ oncogenes involved in breast cancer include HER2 (or ErbB2), RAS, ▶ MYC, and many others, and many others. The activation of proto-oncogenes can occur through gene ▶ amplification, rearrangement by chromosomal translocation, and mutation. Critical tumor suppressor genes in breast cancer include BRCA1 and BRCA2, p53, PTEN, the ▶ retinoblastoma gene RB1, and others. Inactivation is frequently caused by mutation, deletion, or allelic loss. These diverse genetic events contribute to the disruption of normal cellular physiology in various perspectives, such as uncontrolled proliferation, insensitivity to stimuli to undergo ▶ apoptosis, and increased potential for ▶ migration, and eventually lead to the

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Breast Cancer Multistep Development

Increasingly accumulated alterations drive the multiple steps of breast cancer progression

Normal cell

Non-invasive stage

Invasive stage

Metastasis stage

Breast Cancer Multistep Development, Fig. 2 Linear multistep model of breast cancer development. The increasingly accumulated alterations at genetic, epigenetic,

and microenvironment levels gradually drive the progression from normal breast tissue to noninvasive stage, to invasive stage, and ultimately to metastatic breast cancer

ultimate transition to a malignant mammary epithelial cell. The contribution of ▶ epigenetic changes to breast cancer development and progression is increasingly recognized over the past decade. Different from genetic alterations, epigenetic events, such as dysregulated DNA methylation, histone acetylation and methylation, can substantially alter gene expression by modifying chromatin structures (▶ histone modification). Genes affected by epigenetic alterations in breast cancers include HOXA5, MGMT, MLH1, CDH1, and others. The essential role of the microenvironment along breast cancer progression has also been gradually established. The fact that malignant breast cancer cells could dwell in a ▶ dormancy state over a long period of time clinically, and numerous elegant experimental models demonstrating the failure of many tumor cells to thrive in a new environment in spite of high rates of arriving at the secondary organs, effectively reveals the protective role of normal microenvironment in preventing breast cancer development and progression. The suppressive role of the microenvironment during breast cancer progression is perhaps best reflected at its last stage – metastasis. It has been demonstrated that the distinct organ pattern of breast cancer metastasis is highly dependent on the intricate interactions between breast tumor cells and the microenvironment of particular target organs, not

a random process. Therefore, it is increasingly accepted that the progression of breast cancer through the multiple steps is accompanied by tumor cells gradually acquiring capability to convert an oppressive microenvironment to a permissive microenvironment. A distinct example of the microenvironment components in regulating breast tumor progression is the suppressive role of myoepithelial cells in preventing the transition of ductal carcinoma in situ (DCIS) to invasive breast cancer. Emerging data strongly suggested that the layer of myoepithelial cells surrounding mammary ducts functions as a barrier to inhibit the escape of malignant breast tumor cells to other tissues or organs. Unlike the noninvasive breast lesions, which have favorable prognosis if diagnosed and intervened clinically, invasive carcinoma and metastasis significantly contribute to the morbidity and mortality of breast cancer patients. Therefore, extensive efforts in both clinical and basic research have been attributed to better understand the transition from noninvasive carcinoma in situ to invasive carcinoma. Various alterations at the genetic, ▶ epigenetic, and microenvironment levels collaborate to increase the intrinsic cell ▶ migration ability and decrease the rigid intracellular restrains exerted on by both cell-cell and cell-matrix ▶ adhesion, to ultimately convert the noninvasive breast tumor to life-threatening invasive/metastatic breast tumor.

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Alterations occur at any stages of breast cancer

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Non-invasive stage

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Breast Cancer Multistep Development, Fig. 3 Selection model of breast cancer development. Diverse genetic, epigenetic, and microenvironment alterations can occur at any stage of breast cancer development. The successfully

established tumor is the result of selection pressures from the environment and/or clinical treatments, but does not necessarily go through all the steps

Alternative Model of Breast Cancer Development As the central paradigm of breast cancer development, the linear multistep model reflects both the pathological observations and the genetic/epigenetic alterations found in patients and experimental models. However, due to the heterogeneous nature of breast cancer and the enormous number of factors involved in the breast cancer progression, this model mainly applies to the luminal A and luminal B subtypes of this disease, but cannot summarize all subtypes of breast cancer. The massive diversity in both phenotype and genotype of a certain stage of breast tumor formulates an alternative model of breast cancer development and progression: the diversity selection model (Fig. 3). This model proposes that the various subtypes of breast cancer are the results of selective expansion of altered stem or progenitor cells in the breast. And the tumor does not necessarily go through all the linear stages. These two models are not intrinsically incompatible. Multiple genetic/epigenetic alterations can also gradually accumulate in stem cell or progenitor cells (▶ breast cancer stem cells), which may contribute to the intratumoral heterogeneity. Also, somatic breast epithelial cells can acquire

genetic/epigenetic alterations to obtain stem cell or progenitor cell properties. Conclusion In summary, breast cancer multistep progression has been significantly elucidated over the past decade. However, more in-depth investigations are imperative to identify key players in this process. The goal is to develop strategies to detect the early events of breast cancer multistep progression and to intervene effectively this dreadful process.

Cross-References ▶ Adhesion ▶ Amplification ▶ Apoptosis ▶ Breast Cancer ▶ Breast Cancer Epidemiology ▶ Breast Cancer Stem Cells ▶ Carcinoma in Situ ▶ Contralateral Breast Cancer ▶ Dormancy ▶ Ductal Carcinoma In Situ ▶ Epigenetic

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▶ Estrogen Receptor ▶ HER-2/neu ▶ Histone Modification ▶ Hyperplasia ▶ Inflammatory Breast Cancer ▶ Invasion ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Migration ▶ Motility ▶ Multigene Array ▶ Multistep Development ▶ MYC Oncogene ▶ Necrosis ▶ Oncogene ▶ Progression ▶ RAS Genes ▶ Retinoblastoma ▶ Tumor Suppressor Genes

References Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 Polyak K (2001) On the birth of breast cancer. Biochim Biophys Acta 1552:1–13 Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98:10869–10874 Tang P, Hajdu SI, Lyman GH (2007) Ductal carcinoma in situ: a review of recent advances. Curr Opin Obstet Gynecol 19:63–67 Tsikitis VL, Chung MA (2006) Biology of ductal carcinoma in situ classification based on biologic potential. Am J Clin Oncol 29:305–310 Xu J, Acharya S, Sahin O et al. (2015) 14–3-3z turns TGFb's function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2. Cancer Cell 27:177–192.

See Also (2012) Acetylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 17. doi:10.1007/978-3-642-16483-5_24 (2012) Allelic loss. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 137. doi:10.1007/978-3-642-16483-5_186 (2012) Basement membrane. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 349. doi:10.1007/978-3-642-16483-5_537

Breast Cancer Multistep Development (2012) Carcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 657. doi:10.1007/978-3-642-16483-5_848 (2012) Chromatin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 825. doi:10.1007/978-3-642-16483-5_1125 (2012) Deletion. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1080. doi:10.1007/978-3-642-16483-5_1553 (2012) Differentiation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1113. doi:10.1007/978-3-642-16483-5_1616 (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1291–1292. doi:10.1007/978-3-64216483-5_1958 (2012) Gain-of-function mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1489. doi:10.1007/978-3-642-164835_2303 (2012) Histones. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1706. doi:10.1007/978-3-642-16483-5_2762 (2012) HER2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1678. doi:10.1007/978-3-642-16483-5_2676 (2012) HOX. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1739. doi:10.1007/978-3-642-16483-5_2819 (2012) Intravasation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1901. doi:10.1007/978-3-642-16483-5_3125 (2012) Loss-of-function mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2076. doi:10.1007/978-3-642-164835_3414 (2012) Mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Myoepithelial cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2440. doi:10.1007/978-3-642-16483-5_3943 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Proliferation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4766 (2012) Proto-oncogenes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3107–3108. doi:10.1007/978-3-64216483-5_6656 (2012) Stroma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3541. doi:10.1007/978-3-642-16483-5_5532 (2012) Translocation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5942

Breast Cancer New Therapies: HER2, VEGF, and PARP as Targets

Breast Cancer New Therapies: HER2, VEGF, and PARP as Targets Shaheenah Dawood1 and Massimo Cristofanilli2 1 Department of Medical Oncology, Dubai Hospital, Dubai, United Arab Emirates 2 Division of Hematology and Oncology, Robert H Lurie Comprehensive Cancer Center, Chicago, IL, USA

Definition The last two decades have seen an explosion of information in the treatment of both early- and advanced-stage ▶ breast cancer. The Early Breast Trialists’ Collaborative Group 15-year update clearly demonstrates that 6 months of adjuvant ▶ anthracycline-based polychemotherapy reduces the annual breast cancer death rate by 38% and 20% for women younger than 50 years and those aged 50–69 years, respectively. The recognition and understanding of the biological subtypes of breast tumors have helped move its management towards a more personalized approach, further improving these figures. Gene expression profiling has identified at least six subtypes of breast tumors including luminal subtypes (hormone receptor positive), HER2 subtype, and a basal-like subtype. In parallel have been the development and implementation of specific targeted therapies that have not only allowed for more treatment options to be available but have altered the natural history of the disease, positively impacting survival outcomes.

Characteristics Anti-HER2 Therapy HER2 protein overexpression or gene ▶ amplification occurs in approximately 20–25% of breast cancers and is a biomarker of a more aggressive disease associated with an adverse prognostic outcome. Several agents have been developed that abrogate HER2-mediated signaling pathways

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with two agents currently approved for clinical use. ▶ Trastuzumab, a humanized monoclonal antibody, targeting the extracellular component of the HER2 receptor, is approved for use in both the adjuvant and metastatic setting. In the pivotal phase III ▶ clinical trial by Slamon and colleagues that randomized 469 patients with HER2-positive metastatic breast cancer to receive first-line treatment with either ▶ chemotherapy alone or chemotherapy and trastuzumab, the investigators reported a significant improvement in median overall survival from 20.3 to 25.1 months. Four large randomized clinical trials evaluated the role of trastuzumab in the adjuvant setting among women with node-positive or highrisk node-negative breast cancer. A combined analysis of the NSABP B-31 and the NCCTG N9831 studies, in which women with early-stage HER2-positive breast cancer were treated with adjuvant doxorubicin and followed by ▶ paclitaxel with or without 1 year of trastuzumab, demonstrated a 52% increase in disease-free survival and 35% increase in overall survival with the addition of trastuzumab. The HERA study randomized a similar cohort of 5,102 women with HER2-positive early-stage breast cancer who had completed standard chemotherapy up to 1 or 2 years of trastuzumab versus observation. At a median follow-up of 3 years, the investigators reported a significant increase in disease-free survival by 36% and overall survival by 34% among women who had received 1 year of trastuzumab compared to observation. In the BCIRG 006 study 3,222 women with early-stage HER2-positive breast cancer were randomized to receive either anthracycline-based regimen (adriamycin and ▶ cyclophosphamide followed by ▶ docetaxel), a non-anthracycline-based regimen with 1 year of trastuzumab (trastuzumab, docetaxel, and carboplatin), or an anthracycline-based regimen with 1 year of trastuzumab (▶ adriamycin and ▶ cyclophosphamide followed by docetaxel and trastuzumab). The investigators reported an improvement in disease-free survival with the addition of trastuzumab by 39% and 33% in the anthracycline- and non-anthracycline-containing arms of the study, respectively, compared to the

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group of women who did not receive trastuzumab. In contrast to the these large-scale trials that evaluated 1 year of trastuzumab, the FinHer study assessed the efficacy of 9 weeks of adjuvant trastuzumab in a group of node-positive or highrisk node-negative women with HER2-positive early-stage breast cancer. At a median follow-up of 62 months, the authors reported that the addition of trastuzumab resulted in a reduced risk of distant recurrence or death compared to the group who did not receive trastuzumab (hazard ratio with adjustment for presence of axillary nodal metastases was 0.57; p = 0.047). There is currently an ongoing phase III trial evaluating 1 year of trastuzumab compared to 9 weeks of trastuzumab in the adjuvant setting. The main side effect of the use of trastuzumab is cardiotoxicity. In a pooled analysis of the four large adjuvant studies, grade III or IV cardiotoxicity was reported for 4.5% of patients receiving trastuzumab compared to 1.8% of patients. In a separate meta-analysis of over 11,000 patients, the relative risk of cardiotoxicity associated with the adjuvant use of trastuzumab versus no trastuzumab was 5.59 (95% CI 1.99–15.7; p = 0.011). Similar observations were noted in the metastatic setting as well with an important observation that the rate of cardiotoxicity substantially increased with the combination of trastuzumab and anthracyclines. Based on such observations guidelines are now available for cardiac monitoring of patients receiving trastuzumab in either the adjuvant or metastatic setting. The second anti-HER2 agent approved for clinical use is the reversible ▶ tyrosine kinase inhibitor lapatinib that targets the intracellular tyrosine kinase component of both the HER2 receptor and the ▶ epidermal growth factor receptor (EGFR). In phase III randomized clinical trial, over three hundred women with metastatic HER2positive breast cancer who had progressed after receiving ▶ anthracycline, taxanes, and ▶ trastuzumab-based regimens were randomized to receive either capecitabine alone or capecitabine and lapatinib. The investigators reported a significantly improved median time to progression in the combination arm versus the

monotherapy arm of the study (8.4 months vs. 4.4 months). A randomized phase III trial has also evaluated the combination of lapatinib and trastuzumab compared to lapatinib alone in a cohort of heavily pretreated women with HER2positive breast cancer demonstrating significantly improved progression-free survival in the combination arm. Pertuzumab, like trastuzumab, is a monoclonal antibody that binds HER2. However in contrast to trastuzumab it binds to a different epitope, disrupting HER2 dimerization. Phase I and phase II trials have demonstrated good tolerance and clinical benefit in a heavily pretreated population. It is currently being evaluated in the phase III CLEOPATRA trial. ▶ Trastuzumab–DM 1 is trastuzumab that is bound to an inhibitor of tubular polymerization. In the phase II setting trastuzumab–DM1 when administered to a cohort of women with HER2-positive metastatic breast cancer who had progressed on prior anti-HER2 therapy resulted in an overall response rate of 38.2%. This agent is currently being tested in the phase III setting. Anti-VEGF Therapy Tumor ▶ angiogenesis is an important step in the development of breast tumors and is regulated by a number of proangiogenic factors including ▶ vascular endothelial growth factor (VEGF). ▶ Antiangiogenesis agents abrogate signaling pathways promoted by these receptors. ▶ Bevacizumab is a humanized anti-VEGF antibody that is approved for use in the treatment of women with HER2-negative metastatic breast cancer. In the phase III ECOG 2,100 trial, 722 women with HER2-negative metastatic breast cancer were randomized to receive firstline treatment with either ▶ paclitaxel alone or paclitaxel and bevacizumab. The investigators reported a significant improvement in median time to progression (11.8 months vs. 5.9 months, p < 0.001) and overall response rate (36.9% vs. 21.2%, p < 0.001) in the combination arm compared to the group of patients who received paclitaxel alone. Overall survival however was similar between the two groups (26.7 months vs. 25.2 months, p = 0.16). In the phase III

Breast Cancer New Therapies: HER2, VEGF, and PARP as Targets

AVADO trial, a similar cohort of women was randomized to receive first-line treatment with docetaxel alone or in combination with bevacizumab. A significant improvement in progression-free survival and overall survival was observed. Results from the RIBBON-1 and RIBBON-2 studies have also demonstrated the efficacy of bevacizumab in combination with a variety of chemotherapeutic agents in both firstand second-line setting, respectively. A number of phase II and phase III clinical trials are exploring novel combinations with bevacizumab. The CALGB is conducting a phase III clinical trial of the combination of an aromatase inhibitor with bevacizumab in an attempt to overcome or delay endocrine resistance. The combination of bevacizumab with anti-HER2 agents is also being explored. Other anti-VEGF agents such as tyrosine kinase inhibitors ▶ sorafenib and sunitinib are also being evaluated in patients with HER2negative metastatic breast cancer. PARP Inhibitors Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that plays a critical role in cell proliferation and DNA repair, and therefore inhibition of PARP has been explored in a number of phase I and phase II trials. The PARP inhibitor BSI-201 has been evaluated among women with ▶ triple-negative breast cancer in a randomized phase II setting in combination with ▶ gemcitabine and carboplatin where a significant improvement in clinical benefit rate, progression-free survival, and overall survival was observed compared to chemotherapy alone. The oral PARP inhibitor olaparib has in the phase II setting demonstrated 38% response rate as a single agent in a cohort of women with chemotherapy refractory BRAC1- or BRCA2-mutated metastatic breast cancer. Future Directions The use of targeted therapies in the treatment paradigm of patients with breast cancer has been revolutionary in the management of this disease. Current adjuvant and metastatic trials are focused on incorporating these novel agents. Other novel agents such as the ▶ mammalian target of rapamycin (mTOR) inhibitor ▶ rapamycin and

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the heat shock protein 90 (▶ Hsp90) inhibitor tanespimycin (that interacts with HER2 through its kinase domain and has a stabilizing effect on it) are also currently being investigated. Ultimately the goal is to improve prognostic outcomes with minimal toxicity by individualizing treatment using targeted therapies based on the breast tumor subtype presentation.

Cross-References ▶ Adriamycin ▶ Amplification ▶ Angiogenesis ▶ Anthracyclines ▶ Antiangiogenesis ▶ Aromatase and its Inhibitors ▶ Bevacizumab ▶ Breast Cancer ▶ Breast Cancer Antiestrogen Resistance ▶ Chemotherapy ▶ Clinical Trial ▶ Cyclophosphamide ▶ Docetaxel ▶ Epidermal Growth Factor Receptor ▶ Gemcitabine ▶ HER-2/neu ▶ Hsp90 ▶ Mammalian Target of Rapamycin ▶ Microtubule-Associated Proteins ▶ Paclitaxel ▶ Rapamycin ▶ Repair of DNA ▶ Sorafenib ▶ Trastuzumab ▶ Triple-Negative Breast Cancer ▶ Tyrosine Kinase Inhibitors ▶ Vascular Endothelial Growth Factor

References Early Breast Cancer Trialists’ Collaborative Group (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year

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680 survival: an overview of the randomized trials. Lancet 365:1687–1717 Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) (2012) Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100 000 women in 123 randomised trials. Lancet 379(9814):432-444. doi:10.1016/S0140-6736(11) 61625–5 Guarneri V, Conte P (2009) Metastatic breast cancer: therapeutic options according to molecular subtypes and prior adjuvant therapy. Oncologist 14(7):645–656 Martín M, Esteva FJ, Alba E, Khandheria B, Pérez-Isla L, García-Sáenz JA, Márquez A, Sengupta P, Zamorano J (2009) Minimizing cardiotoxicity while optimizing treatment efficacy with trastuzumab: review and expert recommendations. Oncologist 14(1):1–11 Spector NL, Blackwell KL (2009) Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 27(34):5838–5847 Traina TA (2009) Bevacizumab in the treatment of metastatic breast cancer. Oncology (Williston Park) 23(4):327–332

See Also (2012) Adjuvant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 75. doi:10.1007/978-3-642-16483-5_107 (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 408– 409. doi:10.1007/978-3-642-16483-5_6601 (2012) Carboplatin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 641. doi:10.1007/978-3-642-16483-5_833 (2012) Doxorubicin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1159. doi:10.1007/978-3-642-16483-5_1722 (2012) Epitope. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1297. doi:10.1007/978-3-642-16483-5_1966 (2012) Gene Expression Profiling. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1522. doi:10.1007/978-3-642-164835_2368 (2012) HER2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1678. doi:10.1007/978-3-642-16483-5_2676 (2012) Lapatinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1980. doi:10.1007/978-3-642-16483-5_3277 (2012) Monoclonal Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Monoclonal Antibody Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2367–2368. doi:10.1007/978-3-64216483-5_3823

Breast Cancer Prognostic and Predictive Biomarkers (2012) Olaparib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2598. doi:10.1007/978-3-642-16483-5_6689 (2012) Poly(ADP-Ribose) Polymerase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2935. doi:10.1007/978-3-642-16483-5_4655 (2012) Proliferation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4766 (2012) Sunitinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3562. doi:10.1007/978-3-642-16483-5_5575 (2012) Targeted Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3610. doi:10.1007/978-3-642-16483-5_5677 (2012) Taxanes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3614– 3615. doi:10.1007/978-3-642-16483-5_6648 (2012) Tyrosine Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3822. doi:10.1007/978-3-642-16483-5_6079

Breast Cancer Prognostic and Predictive Biomarkers Michael Z. Gilcrease Department of Pathology, Breast Section, MD Anderson Cancer Center, Houston, TX, USA

Definition A biomarker is a body substance or component that can be objectively measured to indicate the status of a biological (usually pathological) process. Normal genes and gene products can serve as biomarkers, as well as alterations in or modifications of normal genes and gene products. Combinations of substances that together indicate a particular biological function can also serve as biomarkers, as well as entire cells. Biomarkers that indicate how a disease will progress in an individual patient are referred to as prognostic biomarkers, whereas those that predict how a disease will respond to a particular therapy are termed predictive biomarkers. A number of prognostic and predictive biomarkers are currently used clinically or are under investigation to guide therapy for ▶ breast cancer patients.

Breast Cancer Prognostic and Predictive Biomarkers

Characteristics Established Prognostic Biomarkers in Breast Cancer Well-established prognostic factors for invasive breast carcinoma include the histologic type, tumor grade, presence or absence of lymphovascular invasion, tumor size, and lymph node status. These traditional prognostic markers, although based on the microscopic assessment of the tumor or regional lymph nodes, are sometimes not regarded as biomarkers per se, as they do not entail the quantitative measurement of a single biological substance. Nevertheless, they are biomarkers in a broad sense, and they have wellestablished prognostic utility. Other prognostic biomarkers are useful only if they provide additional information about disease outcome that is independent of that provided by these wellestablished prognostic factors. Favorable histologic types of invasive breast carcinoma include tubular carcinoma, mucinous carcinoma, medullary carcinoma, low-grade adenoid cystic carcinoma, low-grade adenosquamous carcinoma, and fibromatosis-like metaplastic tumor. Unfavorable histologic types of invasive breast carcinoma include invasive micropapillary carcinoma, some forms of metaplastic breast carcinoma, centrally-necrotizing breast carcinoma, and invasive breast carcinoma with a “large central acellular zone.” Invasive micropapillary carcinoma tends to be high stage at presentation but does not clearly have a worse prognosis than stage-matched invasive ductal carcinomas. Some metaplastic breast carcinomas, particularly those with a predominant sarcomatoid morphology, are aggressive tumors that behave like true sarcomas. Carcinosarcomas are similarly clinically aggressive tumors but have a greater likelihood of axillary lymph node involvement than predominantly sarcomatoid carcinomas. Both centrallynecrotizing carcinomas and those with large central acellular zones have a tendency to metastasize to lungs and brain and have a particularly poor prognosis. The grade of breast cancer is a measure of potential aggressive behavior based on the histologic appearance of well-defined cytological

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parameters. The Nottingham combined histologic grading system is recommended by the College of American Pathologists for grading invasive breast carcinomas. This grading system takes into account the degree of nuclear pleomorphism of invasive tumor cells, the mitotic rate of the invasive tumor, and the degree of tubule formation by the invasive tumor cells. Tumor grade is reported as grade 1 (low grade), grade 2 (intermediate grade), or grade 2 (high grade). Tumor grade is an independent prognostic factor. High-grade tumors have a worse prognosis than low and intermediate grade tumors. Lymphovascular invasion also portends a worse prognosis. The College of American Pathologists recommends using the terminology “vascular invasion” when tumor cells are identified within either lymphatic or blood vascular channels. (It is not necessary to distinguish between the two.) Lymphovascular invasion should be evaluated in the peritumoral breast tissue. It is present in approximately 20% of primary invasive breast carcinomas, and its presence is an adverse prognostic factor, independent of other prognostic factors. Lymphovascular invasion is independently associated with local tumor recurrence and patient survival. The size of an invasive breast carcinoma should be reported at least for the greatest single dimension. The prognostic significance is based on the size of the invasive component only. Associated carcinoma in situ (carcinoma that has not invaded beyond the basement membrane of the normal breast duct system) is not included in the size of the invasive breast carcinoma. Only 10–20 % of patients with invasive breast carcinomas measuring less than 1 cm have axillary lymph node metastases. The recurrence-free survival at 10 years for patients with negative axillary nodes is approximately 90% when the tumor size is less than 1 cm. The lymph node status has long been regarded as the single most important prognostic factor in breast cancer. Only 20–30% of patients with negative lymph nodes develop tumor recurrence within 10 years, compared to almost 70% of patients with positive lymph nodes. Patients with four or more positive lymph nodes have a worse

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prognosis than those with three positive nodes or less. The prognostic significance of micrometastases is not clearly established but appears to be worse than complete absence of metastasis. The significance of isolated tumor cells in the axillary lymph nodes, now staged separately from micrometastases, is even less clear. Prognostic Markers Following Breast Conservation Surgery and Neoadjuvant Chemotherapy With increasing use of breast conservative surgery and neoadjuvant chemotherapy (chemotherapy before surgical excision of the primary tumor), additional important prognostic markers include margin status and pathologic response to neoadjuvant chemotherapy (▶ Neoadjuvant Therapy). A positive margin (invasive tumor at the surgical margin of the excised breast tissue) has been shown to be an independent predictor of decreased survival (RR = 3.9, P = 0.011). Breast conservative surgery, therefore, requires negative margins. Subsequent radiation therapy is also required, even when negative margins are achieved, to reduce the risk of tumor recurrence following breast conservative surgery. A pathologic complete response (pCR) to neoadjuvant chemotherapy is a favorable prognostic factor. It is defined as a complete eradication of invasive carcinoma cells following chemotherapy. In a study of 1,731 patients treated with neoadjuvant chemotherapy, a pCR was observed in 13%. Eight percent of hormone receptor-positive patients had a pCR, while 24% of hormone receptor-negative patients had a pCR. In hormone receptor-positive patients, 5-year survival was 96.4% versus 65.3% with and without a pCR, respectively. In hormone receptor-negative patients, 5-year survival was 83.4% versus 67.4% with and without a pCR, respectively. Because a pCR is an important prognostic factor for patients treated with neoadjuvant chemotherapy, it is important that the tumor site be sampled correctly by the pathologist. It is useful to place a metallic marker in the tumor if a response is observed after initiating chemotherapy to facilitate identification and correct sampling of the tumor site in the surgical

Breast Cancer Prognostic and Predictive Biomarkers

excision specimen in the event of a pCR or nearcomplete response. Established Predictive Biomarkers in Breast Cancer Clinically useful prognostic and predictive biomarkers should have biologic relevance and well-defined scoring criteria. They should be reproducible in different laboratories, confirmed independently by multiple investigators, and validated in large prospective studies. Most reported markers for breast cancer do not yet meet these criteria. As a result, only a few are currently recommended for routine practice. Hormone receptor staining is routinely performed more for its utility in predicting response to hormonal therapy than for its prognostic significance. (There are mixed data on the prognostic significance of hormone receptor expression in invasive breast carcinoma.) A quantitative value for ▶ estrogen receptor (ER) and progesterone receptor (PR) expression is routinely reported for all invasive breast carcinomas, as response to ▶ endocrine therapy has been shown to be proportional to the degree of hormone receptor positivity. Completely negative staining or weak staining in less than 1% of invasive carcinoma cells is regarded as a negative test for estrogen or progesterone receptor. Any degree of staining greater than this is now regarded as a positive test, and the likelihood of response to ▶ hormonal therapy appears to be directly related to the amount of nuclear staining for ER and PR in the invasive tumor cells (Fig. 1). HER2 (c-erbB-2) is a member of the ▶ epidermal growth factor receptor (EGFR) family of growth factor receptors. Overexpression of the protein and/or ▶ amplification of the HER2 gene has been shown to be an adverse prognostic factor in node-positive breast cancer patients, but evaluation of HER2 status is routinely performed on all invasive breast carcinomas more for its utility in predicting response to anti-HER2 therapy, such as ▶ trastuzumab (▶ Herceptin) or lapatinib. In experienced labs, 3+ HER2 staining by ▶ immunohistochemistry correlates well with HER2 gene ▶ amplification as determined by ▶ fluorescence in situ hybridization (FISH). Tumors with 3+

Breast Cancer Prognostic and Predictive Biomarkers

HER2 expression or HER2 gene amplification show the greatest response to trastuzumab therapy, and they are also more sensitive to ▶ anthracyclinecontaining ▶ chemotherapy (Fig. 2).

Breast Cancer Prognostic and Predictive Biomarkers, Fig. 1 Nuclear expression of estrogen receptor in invasive breast carcinoma

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Proposed Biomarkers in Breast Cancer A variety of tumor markers have been proposed, most of which are analyzed by ▶ immunohistochemistry assays. A few of these show promise as potentially useful prognostic markers but have not yet been adopted in routine practice. Several new molecular tests are also reported to have both prognostic and predictive utility. The Ki-67 antigen is expressed in late G1, S, and early G2/M phases of the cell cycle. Immunohistochemical staining for Ki-67 is more sensitive than S-phase analysis or mitotic figure counting for assessing proliferation. Ki-67 analyses, however, lack standardization. The College of American Pathologists recommends reporting mitotic figure counts for every invasive breast carcinoma and designates the use of MIB-1 immunohistochemistry (for detection of Ki-67) as optional.

Breast Cancer Prognostic and Predictive Biomarkers, Fig. 2 Membranous expression of HER2 in invasive breast carcinoma. Scores of 0 and 1+ are negative, 2+ is equivocal, and 3+ is positive for HER2 overexpression

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▶ Urokinase-type plasminogen activator (uPA), a serine protease, is a promising prognostic marker for breast cancer. uPA and its inhibitor, plasminogen activator inhibitor 1 (PAI-1; ▶ Plasminogen-Activating System), stimulate the ▶ adhesion, migration, and proliferation of cells and the degradation of matrix proteins. Elevated levels of uPA and/or PAI-1 consistently correlate with tumor recurrence and decreased patient survival. Some studies also show that elevated levels of these markers predict response to chemotherapy. In a study of more than 3,400 patients with invasive breast carcinoma, uPA/ PAI-1 levels correlated with response to chemotherapy. In a subsequent pooled analysis of 8,377 patients with invasive breast carcinoma, except for lymph node status, a high level of uPA or PAI-1 was the strongest prognostic factor identified. High levels of uPA or PAI-1 correlated with reduced survival in both lymph nodepositive and lymph node-negative subgroups. In particular, uPA or PAI-1 levels had prognostic significance in lymph node-negative patients that received no adjuvant systemic therapy. Unfortunately, uPA and PAI-1 levels are currently evaluated by ELISA, and reliable immunohistochemical assays for uPA and PAI-1 for clinical use are still lacking. This has hindered acceptance of uPA and PAI-1 as routine prognostic markers in the USA. Bcl-2 belongs to a family of proteins that regulate cell survival. Bcl-2 inhibits apoptosis in vitro. Some reports show a correlation between bcl-2 and ER expression and response to tamoxifen. Some data also show that bcl-2 expression appears to be a favorable prognostic factor in lymph node-negative patients. The College of American Pathologists currently does not recommend use of bcl-2 expression as a prognostic factor because of insufficient data. However, in a study published after the latest CAP recommendations, multiple tumor markers were evaluated on tissue microarrays from 930 invasive breast carcinomas, and the most powerful marker to predict survival at 10 years was bcl-2. Moreover, its prognostic significance was independent of the Nottingham Prognostic Index. A large prospective study is needed to confirm the prognostic

Breast Cancer Prognostic and Predictive Biomarkers

utility of bcl-2, but it may prove to be a useful marker for routine practice in the future. A new and controversial putative prognostic marker for breast cancer is cyclin E. Cyclin E exists as multiple functional low molecular weight isoforms in addition to its complete form. The low molecular weight isoforms of cyclin E induce genetic instability and produce increased resistance to hormonal treatment in vitro. These low molecular weight isoforms have also been reported to have adverse prognostic significance. In a paper from the New England Journal of Medicine, overexpression of cyclin E was reported to be “. . .the most powerful prognostic marker for breast cancer that has been identified to date.” Among 114 patients with stage I breast cancer, none of the 102 patients with low cyclin E isoform levels died of breast cancer during the 5 years following the date of diagnosis. In contrast, all of the 12 patients with a high level of cyclin E isoforms died of breast cancer during this time period. These results need to be confirmed, preferably in prospective studies, and verified by independent investigators before cyclin E is adopted as a routine prognostic marker. If the low molecular weight isoforms are more important than the complete protein, Western blotting may be necessary for their identification. Multiple additional prognostic markers, including DNA ploidy/S-phase, p53, cyclin D, cathepsin D, EGFR, and E-cadherin have been reported to have clinical utility, but each has problems with reproducibility and/or assay standardization, and none of these is currently recommended by the College of American Pathologists for routine use as a prognostic marker for breast cancer. Multigene Predictors in Breast Cancer Gene expression profiling is a method of evaluating hundreds or thousands of genes in tumor cells by extracting the RNA and quantifying the expression of genes relative to so-called housekeeping genes that are expressed at a relatively constant level regardless of experimental conditions. Gene expression profiling studies have identified a so-called basal-like subgroup of invasive breast carcinomas, in addition to a subgroup

Breast Cancer Prognostic Biomarkers

that overexpresses genes related to HER2. Both of these subgroups have been reported to have adverse prognostic significance. Another subgroup expressing a so-called 70-gene prognosis signature is reported to have adverse prognostic significance. A commercial assay to detect this signature (Mammaprint) is being tested in lymph node-negative patients in a prospective randomized study in Europe. The study is comparing the 70-gene signature with common clinical-pathological criteria for selecting patients to receive adjuvant chemotherapy. The assay currently requires fresh frozen tumor tissue. Another commercially available molecular test that is becoming more popular in the USA is the Oncytoype Dx assay, which involves quantitation of 21 genes by real-time PCR. This assay provides a so-called recurrence score that correlates inversely with the likelihood of response to tamoxifen in lymph node-negative breast cancer patients. This assay can be performed on paraffin tumor tissue. It is currently being evaluated in a large clinical trial involving over 10,000 patients at 900 sites in the USA and Canada. Both keratin-positive tumor cells in bone marrow and circulating tumor cells in the blood are also reported to be associated with patient outcome. The independent prognostic and predictive value of these tests is still being evaluated. Conclusion Traditional prognostic markers in breast cancer are based on the histologic assessment of the primary tumor and regional lymph nodes. These include histologic type, tumor grade, presence or absence of lymphovascular invasion, tumor size, and lymph node status. Clinically useful biomarkers should provide additional independent prognostic or predictive information. Tumor margin status and pathologic complete response are important prognostic markers following breast conservative surgery and neoadjuvant chemotherapy. Assays for hormone receptors and HER2 are routinely performed as predictive markers for response to endocrine therapy and anti-HER2 therapy, respectively. A variety of additional prognostic markers have been proposed but require

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further validation. These include assays based on gene expression profiling and RT-PCR, as well as the detection of keratin-positive cells in bone marrow and circulating tumor cells.

B References Harvey JM, Clark GM, Osborne CK et al (1999) Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 17(5):1474–1481 Paik S, Shak S, Tang G et al (2004) A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351(27):2817–2826 Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98(19):10869–10874 van de Vijver MJ, He YD, van’t Veer LJ et al (2002) A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347(25):1999–2009 Wolff AC, Hammond ME, Schwartz JN et al (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 131(1):18

Breast Cancer Prognostic Biomarkers Boon-Huat Bay and George Wai-Cheong Yip Department of Anatomy, National University of Singapore, Singapore, Singapore

Definition Biomarkers are distinctive and relatively specific biological indicators (in the form of altered gene, protein, carbohydrate, or lipid expression) of physiological or disease processes. ▶ Clinical cancer biomarkers have been broadly categorized into prognostic biomarkers which aid in determining the disease outcome (prognosis) or predictive markers which predict response to therapy. Identification of prognostic and predictive biomarkers would enhance the management of ▶ breast cancer patients by helping clinicians make better decisions with regard to the mode of treatment

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for each patient, such as which group of patients would benefit from chemotherapy after surgical excision of the tumor. Prognostic biomarkers also form the basis for the development of effective targeted therapy against ▶ breast cancer.

Characteristics Clinical Prognostic Indicators Standard prognostic factors for breast malignancy take into account clinical and pathological criteria such as a patient’s age and the morphological features of the cancer, such as its stage and histological grade. Tumor stage involves measuring the size of the tumor and determining if the tumor has invaded into surrounding structures and draining lymph nodes as well as spread distally to other organs (metastasis). There are two main commonly used systems for staging of tumors: the TNM system (T, tumor; N, lymph node status; M, metastasis) and the American Joint Committee on Cancer (AJCC) staging. Histological grade is assessed by morphological examination of the tissues under a light microscope. Tumors are classified as histological Grade 1 (low grade where the tissue has more resemblance to normal tissue in terms of parameters such as variability of the size of the nucleus and mitosis), Grade 2 (moderately differentiated), and Grade 3 (poorly differentiated) tumors. These parameters provide the basis for prognostic algorithms, such as the Nottingham Prognostic Indicator which is a reliable predictor of long-term survival of breast cancer patients. However, there are limitations in the use of conventional prognostic tools for predicting patient outcome. Herein lies the importance of the continuous search for clinically useful biomarkers that can provide additional prognostic information. Traditional Prognostic Markers Well-established traditional prognostic markers include ▶ estrogen receptor (ER) status, progesterone receptor (PR) status, ▶ HER-2/neu (synonym neu or cerbB2) positivity, and Ki-67 cell proliferation marker.

Breast Cancer Prognostic Biomarkers

Hormone Receptors

Estrogen receptor (ER) is a 65 kDa nuclear molecule and binds to 17b-▶ estradiol as its principal ligand. Two ER subtypes, ERa and ERb, have been described, with the former being present in approximately 70% of breast cancers. Binding of estrogen to ER leads to either homo- or heterodimerization of the receptor, which then interacts with hormone response elements to induce transcription of genes which regulate cellular activity (Fig. 1). This process can be deactivated by blocking the activity of the receptor or depriving the receptor of the estrogen hormone. Patients with ER-negative breast tumors are more likely to have a higher histological grade and decreased overall survival, whereas the prognosis in ER-positive tumors is relatively better. The presence of ER has been used to guide the use of ▶ endocrine therapy. Drugs such as ▶ tamoxifen target and block the ER receptor and therefore possess anticarcinogenic properties. They are able to reduce tumor cell proliferation and significantly reduce the risk of recurrence within 5 years by 40% and overall breast specific mortality by 31%. ▶ Aromatase inhibitors like anastrozole and letrozole inhibit the conversion of precursor molecules to estradiol. Patients need to be assessed of their tumor status for the ER marker (endocrine responsiveness) to qualify for either of the treatments. Furthermore, the presence of ER receptor is associated with fewer benefits from ▶ chemotherapy. Like estrogen, progesterone is a steroid hormone and expression of the progesterone receptor (PR) is known to be strongly dependent on ER activity. Therefore, PR-positive breast cancers have a more favorable prognosis than PR-negative tumors. The ER and PR status of breast cancer tissues is determined by ▶ immunohistochemistry (IHC), a technique which uses an antibody to detect the receptors (Fig. 2). Human Epidermal Growth Factor Receptor-2 (HER-2)

Human ▶ epidermal growth factor receptor-2 (HER-2 or ERBB2) is a member of the family of

Breast Cancer Prognostic Biomarkers Breast Cancer Prognostic Biomarkers, Fig. 1 Diagrammatic representation of ER, PR, and HER-2 pathways (Courtesy of S.L. Bay, National University of Singapore)

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Growth factors

Estrogen Progesteore

B HER2

ER

Tyrosine kinase

PR

Signalling molecule

Nucleus

Breast Cancer Prognostic Biomarkers, Fig. 2 Positive estrogen receptor-immunostaining (immunohistochemistry) in breast cancer tissue with strong reactivity present in the cell nuclei which are stained brown (Courtesy of P.H. Tan, Singapore General Hospital, Singapore)

epidermal growth factor receptors. The HER-2 gene is located on chromosome 17q21 and encodes a 185 kDa tyrosine kinase glycoprotein (Fig. 1). HER-2 regulates cell differentiation, ▶ adhesion, and ▶ motility. The status of HER-2

can be determined by immunohistochemistry or more sophisticated fluorescence in situ hybridization techniques. HER2 expression is estimated to be amplified in approximately 20% of breast tumors. Most clinical studies have shown that

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▶ amplification of the HER-2 gene or overexpression of the HER-2 protein is associated with higher-grade tumors, increased rate of recurrence, lower survival, and poorer prognosis. The identification of HER-2 as a prognostic biomarker has led to the clinical development of ▶ trastuzumab, a humanized monoclonal antibody against HER-2 protein (monoclonal antibody therapy). Targeted therapy using trastuzumab in combination with chemotherapy in either a first-line or adjuvant (▶ adjuvant therapy) setting has demonstrated survival benefits in breast cancer patients with elevated HER-2 expression. Ki-67 Proliferation Marker

The Ki-67 gene, located on chromosome 10q25, codes for a nuclear nonhistone protein. Two protein isoforms (359 and 320 kDa) can be formed by alternative splicing. Ki-67 is found in proliferating cells, where its expression increases during disruption of the nuclear membrane during early mitosis. Elevated expression of Ki-67 is a marker of poor prognosis and increased risk of recurrent disease. Emerging Prognostic Markers Breast cancer is a heterogeneous disease resulting from the accumulation of multiple gene mutations. Numerous studies have been carried out over the years to understand the different molecular mechanisms involved in breast cancer as well as to obtain prognostic markers to improve diagnosis, therapeutic approaches, and patient management. The development of expression profiling technologies has also accelerated the rate of discovery of novel potential markers for breast cancer. Genomic Markers

The BRCA1 gene is located on chromosome 17q21 and encodes a 1,863-amino acid nuclear protein that regulates transcriptional activation, cell cycle checkpoint control, ▶ DNA repair, chromosomal remodeling, and ▶ apoptosis. BRCA1 is a ▶ tumor suppressor gene, mutations of which relate to the progression of familial breast cancer (▶ BRCA1/BRCA2 germline

Breast Cancer Prognostic Biomarkers

mutations and breast cancer risk). Loss of BRCA1 in sporadic tumors results in reduced BRCA1 expression or incorrect subcellular localization of the encoded protein. This well-studied gene is associated with high-grade and largersized tumors, advanced lymph node stages, vascular invasion, negative estrogen receptor, progesterone receptor and ▶ androgen receptor (AR) and ▶ E-cadherin expression, and basallike type of breast carcinoma. Alterations in BRCA1 gene expression result in poor patient survival. Proliferation Markers

Increased proliferative activity is one of the hallmarks of cancer. Besides Ki-67, which is an established proliferation marker, proteins associated with cell proliferation include the cyclins which are involved in regulation of the cell cycle and growth factor receptors such as insulin-like growth factor receptor 1. Other examples of breast cancer proliferation markers are FOXM1 (▶ forkhead box M1; a member of the forkhead box superfamily of transcription factors), metallothionein (a metal-binding protein; metallothionein enzymes), ▶ securin (a regulatory protein), and YB-1 (a member of the cold shock domain DNA- and RNA-binding protein superfamily). Anti-apoptosis Markers

▶ BCL2 is a mitochondrial protein that inhibits chemotherapy-induced ▶ apoptosis (mitochondria apoptosis pathway). Its expression level is inversely correlated with that of oncogenic Ki-67. Patients with BCL2-negative breast cancer are more likely to respond to chemotherapy. However, overexpression of BCL2 is also correlated with increased survival rates, and this may be due to the presence of concurrent estrogen receptor expression. Mutation of the p53 tumor suppressor gene (▶ TP53; ▶ p53 family) has long been implicated in the evasion of apoptosis in human tumors and associated with more aggressive breast cancers. Structural Proteins

Cytokeratins (CKs) are a family of major structural proteins present in the cytoplasm of

Breast Cancer Prognostic Biomarkers

epithelial cells. Their molecular weights range from 40 to 68 kDa. In the human breast, CKs are mainly expressed in basally located myoepithelial cells. Basic CK5 (58 kDa) and acidic CK14 (50 kDa) and CK17 (46 kDa) are associated with high-grade basal-like breast carcinoma, early tumor recurrence, and poor prognosis. Furthermore, expression of these three CKs has been significantly correlated with BRCA1-expressing tumors. Angiogenesis-Associated Markers

Another hallmark of cancer is the formation of new blood vessels (▶ angiogenesis) to help nourish the tumor for its growth. Angiogenic factors include growth factors such as members of the ▶ vascular endothelial growth factor family, ▶ fibroblast growth factor 2, and hepatocyte growth factor (synonym ▶ scatter factor) as well as members of the angiopoietin family. Serum levels of vascular endothelial growth factor (VEGF) may be a useful prognostic factor in breast cancer, as they have been observed to be elevated in malignant breast tumors and predict overall survival and local recurrence. Expression of VEGF has been correlated with estradiol in tumors and may promote cancerous spread by regulating ▶ chemokine receptor CXCR4. However, a report has shown that contrary to expectation, angiogenic factors and receptors were downregulated in primary breast tumors. An intact uterus in postmenopausal women appears to protective females against distal spread of breast cancer by lowering serum VEGF levels.

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Glycosaminoglycans and Proteoglycans

Glycosaminoglycans (GAGs) and proteoglycans (PGs) play vital roles in cancer progression. GAGs are long, unbranched polysaccharides that are formed by repeating disaccharides of an uronic acid residue alternating with an amino sugar. Four major classes of GAGs have been described, namely, heparan sulfate (HS), chondroitin sulfate/dermatan sulfate, keratan sulfate, and hyaluronan. Syndecans are transmembrane HSPGs and consist of four family members. Overexpression of syndecan-1 in breast cancer is linked to poor survival outcome and is predictive of response to neoadjuvant chemotherapy. Through its interactions with heparin-binding growth factors and integrin, syndecan-1 regulates cancer progression and tumor-associated ▶ angiogenesis. Syndecan-4 has also been found to be associated with aggressive ER-negative breast cancer. Glypican-1, an HSPG, has been found overexpressed in high-grade breast cancer tissues. Multigene Arrays

Genomics technologies have led to the development of ▶ multigene arrays known to provide prognostic information such as the Oncotype DX assay, an array of 21 genes (comprising 16 outcome-related genes and five reference genes) and the MammaPrint 70-gene signature. The MammaPrint has been given approval by the US Federal Drug Administration (FDA) for application as a prognostic tool when used in combination with other clinicopathological parameters.

Plasminogen Activators and Inhibitors

MicroRNA

Cancer cells make use of proteolytic enzymes to assist in invading surrounding tissues and distant metastasis. ▶ Urokinase-type plasminogen activator (u-PA) is a serine protease that degrades extracellular matrix thus easing cancer progression. Elevated expression of u-PA and plasminogen activator inhibitor (PAI1; ▶ plasminogen-activating system) is associated with higher recurrence risk and poorer survival in patients with node-negative breast cancer. These patients have also been reported to derive greater benefits from chemotherapy.

▶ MicroRNAs (miRNAs) are small noncoding RNAs that originate from genes transcribed by RNA polymerase II. Studies have demonstrated associations between miRNAs and cancer progression and stimulated interest in identifying the involved miRNAs. By suppressing the translation of mRNA or cleaving mRNA, miRNAs can regulate cellular proliferation, apoptosis, and differentiation. Alterations of miRNA expression have been reported to be associated with breast cancer development, ▶ invasion, and ▶ metastasis.

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Breast Cancer Prognostic Biomarkers

Future Directions Well-established biomarkers of breast cancer are routinely used in medical practice to guide clinical management decisions and to aid in predicting disease outcome. Simultaneous analysis of several molecules has led to the identification of basal-like (also known as triple-negative) breast cancer. This subset of breast cancer is negative for ER, PR, and HER-2 and displays a more aggressive behavior and possesses a poorer prognosis compared with luminal (ER positive) and HER-2like (ER negative, HER-2 positive) breast tumors. Technological advances in genomics, proteomics, and glycomics have led to the discovery of novel predictive and prognostic factors. Efforts are now required to validate the clinical usefulness of these molecules and to determine if they will contribute to personalized breast cancer treatment.

Cross-References ▶ Adhesion ▶ Adjuvant Therapy ▶ Amplification ▶ Androgen Receptor ▶ Angiogenesis ▶ Apoptosis ▶ Aromatase and its Inhibitors ▶ Bcl2 ▶ BRCA1/BRCA2 Germline Mutations Breast Cancer Risk ▶ Breast Cancer ▶ Chemokine Receptor CXCR4 ▶ Chemotherapy ▶ Clinical Cancer Biomarkers ▶ E-Cadherin ▶ Endocrine Therapy ▶ Epidermal Growth Factor Receptor ▶ Estradiol ▶ Estrogen Receptor ▶ Fibroblast Growth Factors ▶ Forkhead Box M1 ▶ HER-2/neu ▶ Immunohistochemistry ▶ Invasion ▶ Metastasis

▶ MicroRNA ▶ Motility ▶ Multigene Array ▶ P53 Family ▶ Plasminogen-Activating System ▶ Repair of DNA ▶ Scatter Factor ▶ Securin ▶ Tamoxifen ▶ TP53 ▶ Trastuzumab ▶ Tumor Suppressor Genes ▶ Urokinase-Type Plasminogen Activator ▶ Vascular Endothelial Growth Factor

References Dowsett M, Dunbier AK (2008) Emerging biomarkers and new understanding of traditional markers in personalized therapy for breast cancer. Clin Cancer Res 14:8019–8026 Duffy MJ, Crown J (2008) A personalized approach to cancer treatment: how biomarkers can help. Clin Chem 54:1770–1779 Pakkiri P, Lakhani SR, Smart CE (2009) Current and future approach to the pathologist’s assessment for targeted therapy in breast cancer. Pathology 41:89–99 Payne SJ, Bowen RL, Jones JL, Wells CA (2008) Predictive markers in breast cancer–the present. Histopathology 52:82–90 Yip GW, Smollich M, Gotte M (2006) Therapeutic value of glycosaminoglycans in cancer. Mol Cancer Ther 5:2139–2148

and See Also (2012) Adjuvant. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 75. doi:10.1007/978-3-642-16483-5_107 (2012) Antibody. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 208. doi:10.1007/978-3-642-16483-5_312 (2012) Checkpoint. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 754– 755. doi:10.1007/978-3-642-16483-5_1049 (2012) Cytokeratins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1051. doi:10.1007/978-3-642-16483-5_1472 (2012) Differentiation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1113. doi:10.1007/978-3-642-16483-5_1616 (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1291–1292. doi:10.1007/978-3-64216483-5_1958

Breast Cancer Stem Cells (2012) Extracellular matrix. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Familial breast cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1373. doi:10.1007/978-3-642-16483-5_2107 (2012) Fibroblast growth factor 2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1397. doi:10.1007/978-3-64216483-5_2174 (2012) Flusorescence in situ hybridisation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 1436. doi:10.1007/978-3-642-164835_6740 (2012) Glypican 1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1576. doi:10.1007/978-3-642-16483-5_2464 (2012) Glycoprotein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2451 (2012) Glycosaminoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2453 (2012) Heparan sulfate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1647. doi:10.1007/978-3-642-16483-5_2637 (2012) Hyaluronan. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1767. doi:10.1007/978-3-642-16483-5_2876 (2012) Integrin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Ki-67. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1943. doi:10.1007/978-3-642-16483-5_3213 (2012) Metallothionein enzymes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p . doi:225910.1007/978-3-64216483-5_3667 (2012) Mitochondria apoptosis pathway. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2331. doi:10.1007/978-3-642-164835_3764 (2012) Monoclonal antibody therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2367–2368. doi:10.1007/978-3-64216483-5_3823 (2012) Neoadjuvant. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2472. doi:10.1007/978-3-642-16483-5_4003 (2012) Progesterone receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2990. doi:10.1007/978-3-642-16483-5_4754 (2012) Proteoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3100. doi:10.1007/978-3-642-16483-5_4816 (2012) Syndecans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3593. doi:10.1007/978-3-642-16483-5_5623

691 (2012) Targeted therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3610. doi:10.1007/978-3-642-16483-5_5677 (2012) Transcription. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3752. doi:10.1007/978-3-642-16483-5_5899 (2012) Tyrosine kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3822. doi:10.1007/978-3-642-16483-5_6079

Breast Cancer Stem Cells Pranela Rameshwar1 and Shyam Patel2 1 Medicine-Hematology/Oncology, Rutgers, New Jersey Medical School, Newark, NJ, USA 2 Standford University, Palo Alto, CA, USA

Synonyms Breast cancer-initiating cell (breast CIC); Breast tumor-initiating cell (breast TIC)

Definition ▶ Breast cancer stem cells are self-renewing cells that form new tumor cells. They represent a small fraction of cells that are resistant to ▶ chemotherapy and ▶ radiation therapy. Chemotherapeutic agents with some effect on breast cancer stem cells include 5-▶ fluorouracil, platinum-based agents (▶ platinum complexes), ▶ taxol, and doxorubicin. The frequency of tumor stem cells varies, depending on tumor type. However, 70% of the risk of familial breast cancer remains unsettled.

BRCA1 and BRCA2

The BRCA1 gene with a size of around 80 kbp is located on the long (q) arm of chromosome 17 at band 21.31. The 1,863-amino-acid-long Brca1 protein contains a zinc finger (zinc-finger proteins) within the N-terminal RING finger domain, the C3HC4-type domain, and the Brca1 C-terminus domain (Brct domain). This domain is named after Brca1 C-terminal repeat. It consists of a 90–100 amino acid unit that occurs as a single element or as multiple repeats in several proteins involved in the DNA damage response. Heterodimerization between Brct repeats promotes protein-protein interactions. A subset of tandem Brct repeats adopt a conserved head-to-tail structure. Such tandem repeats can function as a phospho-peptide binding module that binds proteins with specific phosphorylation motifs. Brca1 forms the central scaffold for the assembly of a multicomponent complex involved in DNA double-strand break repair and ▶ DNA damage signaling. The BRCA2 gene with a size of around 84 kbp is located on the long arm of chromosome 13 at band 12.3. The Brca2 protein is 3,418 amino acid long and contains eight repeats of the 40 residue Brc motifs. Several out of these motifs in human Brca2 can directly bind to Rad51, a protein that mediates strand exchange, i.e., the central step in ▶ homologous recombination repair. Thus, both Brca1 and Brca2 play important roles in the maintenance of genomic stability by facilitating repair of DNA double-strand breaks, particularly via homologous recombination. More than 2,600 mutations have been found on chromosome 17 in BRCA1 and on chromosome 13 in BRCA2 causing a greater than tenfold relative risk of breast cancer (▶ BRCA1/BRCA2 germline mutations and breast cancer risk). Moreover, they are also associated with an elevated risk of ▶ ovarian cancer. Biallelic mutations in BRCA2 result in ▶ Fanconi anemia, complementation group D1 (FA-D1), with an incidence of 5–10 per million. Fanconi anemia is a ▶ chromosomal instability disease associated with severe familial

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Breast Cancer Susceptibility Genes

Breast cancer: genes with 20–30% BRCA1, BRCA2 sporadio 10–25% 2.5%

8.5 kb, is comprised of ten exons (exon 1 is untranslated), and includes two CpG islands. BRMS1 cDNA is 1485 base pairs and encodes a 246 amino acid protein (MW ~28.5 kDa, although it runs more slowly (Mr ~35 kDa) by SDS-PAGE). The mouse orthologue (Brms1) shares 95% homology at the amino acid level and also suppresses metastasis in murine models of breast cancer. BRMS1 protein contains two putative nuclear localization sequences, a nuclear export sequence, a potential endoplasmic retention signal, two coiled-coil motifs, a CXD motif with E3 ligase activity, imperfect leucine zippers, and a glutamic acid–rich N-terminus. BRMS1 is ubiquitously expressed in human tissues (highest in kidney, placenta, peripheral blood lymphocytes, and testis; lowest in brain and lung). BRMS1 is located predominantly (>90%) in the nucleus, is stabilized by interactions with chaperone proteins, and is regulated by proteasomal degradation.

Modified version of Silveira AC, Welch DR (2012) BRMS1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin Heidelberg, pp 570–571. doi: 10.1007/978-3-642-16483-5_735

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Cellular and Functional Characteristics Depending upon the cell line being studied, BRMS1 restores gap junctional cell-cell communication, decreases growth in soft agar, increases anoikis/apoptosis, reduces cell motility, alters adhesion to specific extracellular matrices, decreases phosphoinositide signaling which, in turn, results in decreased mobilization of intracellular calcium, and downregulates fascin, osteopontin, and urokinase-type plasminogen activator. BRMS1 was shown to coordinately regulate metastasis regulator microRNA (known as metastamiR). The mechanism by which BRMS1 regulates these phenotypes is not fully defined but is thought to be predominantly through regulation of histone deacetylase complexes. BRMS1 directly interacts with SUDS3 and ARID4A which, in turn, interface with large (megadalton) protein complexes including, most notably, class I and class II histone deacetylases (HDAC) and the transcription factor NFkB. BRMS1 is specifically a core member of Sin3-HDAC chromatin remodeling/transcriptional repression complexes, but its involvement has been implicated in other HDAC complexes. Clinical Relevance BRMS1 is regulated at both RNA and protein levels. Several studies evaluated the expression level of BRMS1 in clinical samples. The first study of BRMS1 protein in patient samples found loss of BRMS1 in nearly 25% of breast cancer cases. Loss of BRMS1 correlated with disease-free survival when stratified by loss of estrogen receptor, progesterone receptor, or Her2 overexpression. Loss of BRMS1 expression via promoter methylation is associated with poor survival rates in patients with breast, lung, and nasopharyngeal carcinomas. These studies show that downregulation of BRMS1 by epigenetic silencing has an important role in tumorigenesis. Protein and promoter analyses of BRMS1 in circulating tumor cells (CTCs) show that both parameters predict patient outcomes in breast and non-smallcell lung carcinomas. Importantly, since BRMS1 is regulated at the protein level, looking exclusively at mRNA may be misleading. Nonetheless,

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the majority of studies show high levels of BRMS1 correlates with increased disease-free survival and diminished progression.

Cross-References ▶ Cancer Epigenetics ▶ Circulating Tumor Cells ▶ Class II Tumor Suppressor Genes ▶ Dormancy ▶ Epithelial-to-Mesenchymal Transition ▶ Metastasis ▶ Metastasis Suppressor Gene ▶ Metastatic Colonization ▶ Migration ▶ Motility

References Balgkouranidou I, Chimonidou M, Milaki G et al (2014) Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer. Br J Cancer 110:2054–2062 Edmonds MD, Hurst DR, Vaidya KS, Stafford LJ, Chen D, Welch DR (2009) Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression. Int J Cancer 125(8):1778–1785 Hicks DG, Yoder BJ, Short S et al (2006) Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients. Clin Cancer Res 12:6702–6708 Hurst DR, Welch DR (2011) Unraveling the enigmatic complexities of BRMS1-mediated metastasis suppression. FEBS Lett 585:3185–3190 Liu Y, Smith PW, Jones DR (2006) Breast cancer metastasis suppressor 1 functions as a corepressor by enhancing histone deacetylase 1-mediated deacetylation of RelA/p65 and promoting apoptosis. Mol Cell Biol 26:8683–8696 Liu Y, Mayo MW, Nagji AS et al (2013) BRMS1 suppresses lung cancer metastases through an E3 ligase function on histone acetyltransferase p300. Cancer Res 73:1308–1317 Meehan WJ, Samant RS, Hopper JE et al (2004) Interaction of the BRMS1 metastasis suppressor with RBP1 and the mSin3 histone deacetylase complex. J Biol Chem 279:1562–1569 Samant RS, Clark DW, Fillmore RA et al (2007) Breast cancer metastasis suppressor 1 (BRMS1) inhibits osteopontin transcription by abrogating NF-kappaB activation. Mol Cancer 6:6

BRN-5547136

See Also (2012) Experimental Metastasis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1361. doi:10.1007/978-3-642-16483-5_2062 (2012) Lung Colony Formation Assay. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2115. doi:10.1007/978-3-642-164835_3436 (2012) Macrometastasis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2130. doi:10.1007/978-3-642-16483-5_3483

BRN-5547136 ▶ Temozolomide

Bronchogenic Carcinoma ▶ Lung Cancer ▶ Lung Cancer Clinical Oncology

Brother of the Regulator of Imprinted Sites ▶ BORIS

Bryostatin-1 Bassel El-Rayes Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA Winship Cancer Institute of Emory University, Atlanta, GA, USA

Definition Bryostatins are a class of macrocyclic lactones. Bryostatins are potent modulators of Protein Kinase C (PKC). Bryostatin-1 was isolated from

Bryostatin-1

the marine invertebrate Bugula neritina. Bryostatin-1 is currently not available for commercial use.

Characteristics Rationale for Targeting the PKC PKC is a family of homologous serine/threonine protein Kinases that transduce signals linked to diverse cellular processes including proliferation, differentiation, angiogenesis, and ▶ apoptosis. The PKC family includes 12 isoforms subdivided into three major classes based on their cofactor requirements for activation. Aberrant regulation of the PKC enzymes activity has been demonstrated in a number of malignancies including: breast, colorectal, pancreatic, and non-small cell lung cancer. Preclinical Activity of Bryostatin-1 Treatment of cancer cell lines with bryostatin-1 results in the activation of PKC. However, prolonged exposure to bryostatin-1 induces PKC inhibition most probably through ubiquitinmediated degradation. Inhibition of PKC activity results in cell cycle arrest, apoptosis, cell differentiation, and modulation of chemoresistance. Bryostatin-1 has been shown to potentiate the effects of several classes of cytotoxic agents including: vincristine in diffuse large cell lymphoma, melphalan in Waldenstrom’s macroglobulinemia, gemcitabine in pancreatic and breast cancer, paclitaxel and mitomycin C in gastric cancer cell lines. The synergism between bryostatin and cytotoxic agents is sequence dependent. Single Agent Activity of Bryostatin-1 Phase I trials of bryostatin-1 used two different schedules. The maximal tolerated doses were 25 mg/m2 when infused over 24 h and 120 mg/m2 when infused over 72 h. The most common side effects included myalgia. Other observed toxicities included headache, phlebitis, and transient thrombocytopenia. Single agent bryostatin-1 has been studied in phase II trials for lymphoma, renal, colorectal, head and neck, sarcoma, and melanoma.

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Bryostatin-1 did not demonstrate single agent activity in any of these diseases. Bryostatin-Based Combinations Since PKC activation contributes to chemoresistance, the combinations of bryostatin-1 and cytotoxic agents were tested. In chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin lymphoma, bryostatin-1 was evaluated in combination with fludarabine. Patients received fludarabine daily for 5 days and bryostatin-1 over 24 h infusion either before or after fludarabine. The combination was well tolerated. Partial and complete responses were observed in 6 and 2 patients (total number 27), respectively. Bryostatin-1 and vincristine were evaluated in patients with refractory B-cell lymphoma. Twenty four patients were enrolled on the study. The bryostatin-1 was well tolerated at a dose of 50 mg/m2 over 24 h infusion. The regimen had activity with five patients having objective response and five having stable disease. Bryostatin-1 was also evaluated in combination with cisplatin in two phase I trials. In the first trial, bryostatin-1 (30 mg/m2 over 24 h infusion) had no significant activity. In the second trial, bryostatin-1 was administered at a dose of 15–55 mg/m2 over 72 h. In this study, three responses were reported. A phase I trial of gemcitabine and bryostatin-1 (25–35 mg/m2 over 24 h) revealed that the regimen was well tolerated and resulted in stable disease in 8 out of 36 patients. Bryostatin-1 (15–50 mg/m2 infused over 24 h) was evaluated in combination with paclitaxel. Partial responses were observed in patients with pancreatic and gastroesophageal cancer. The common finding in these trials is that bryostatin-1 can be combined safely with cytotoxic chemotherapy agents. Phase II trials evaluating the activity of bryostatin-1 with cisplatin in cervical cancer had disappointing results. Fourteen patients were enrolled on the trial and there were no treatment responses. Ajani et al. reported on 37 patients with gastroesophageal and gastric cancer treated with bryostatin-1 (40 mg/m2 infused over 24 h) and weekly paclitaxel (80 mg/m2). The response rate was 29% which is higher than the previously

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reported response rates with paclitaxel. In a phase II trial, bryostatin-1 (50 mg/m2 infused over 24 h) and weekly paclitaxel (90 mg/m2) were evaluated in patients with non-small cell lung cancer. Of 11 response evaluable patients, stable disease was seen in 5 patients. Therefore, the bryostatin1 and paclitaxel combination did not demonstrate significant activity in lung cancer. Future Directions Mixed results have been observed in the trials evaluating bryostatin-1 and cytotoxic chemotherapy agents. The future challenges in the development of bryostatin-1 include the identification of biomarkers that can predict activity and the development of combination therapy with other targeted agents. Another approach for the development of bryostatins is through the modification of the chemical structure in order to identify analogues with better safety or efficacy profiles than bryostatin-1.

BSF-2

BSF-2 ▶ Interleukin-6

BTAK ▶ Aurora Kinases

Burkitt Lymphoma Definition Burkitt lymphoma is caused by ▶ Epstein-Barr virus (EBV) and occurs mainly in sub-Saharan Africa.

Cross-References Cross-References

▶ Epstein-Barr Virus

▶ Apoptosis

References Choi SH, Hyman T, Blumberg PM (2006) Differential effect of bryostatin 1 and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by down-regulation of protein kinase Cdelta. Cancer Res 66:7261–7269 Deacon EM, Pongracz J, Griffiths G et al (1997) Isoenzymes of protein kinase C: differential involvement in apoptosis and pathogenesis. Mol Pathol 50:124–131 Kortmansky J, Schwartz GK (2003) Bryostatin-1: a novel PKC inhibitor in clinical development. Cancer Invest 21:924–936

Bystander Effect Olga A. Martin Division of Radiation Oncology and Cancer Imaging, Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia The Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia

Synonyms See Also (2012) Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1943. doi:10.1007/978-3-642-16483-5_3217 (2012) Thrombocytopenia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3678. doi:10.1007/978-3-642-16483-5_5792

Abscopal effect; Distant bystander effect; In vitro bystander effect; In vivo bystander effect; Indirect effect; Nontargeted effect; Out-of-field effect; Radiation-induced bystander effect (RIBE); Stress-induced bystander effect

Bystander Effect

Definition The term “bystander effect” refers to changes in naïve (“bystander”) cells sharing the same milieu with cells that have been damaged. The radiationinduced bystander effect (RIBE) is now a wellestablished consequence of ionizing radiation and is manifested as increased genomic abnormalities and loss of viability of unirradiated cells associated with the targeted cells.

Characteristics The term “bystander effect” was first used to explain the results obtained in cell cultures irradiated with a-particles (energetic helium nuclei with the short range of absorption which can be produced by cyclotrons or synchrotrons). Although only a few cells were traversed by a-particles, many more exhibited sister chromatid exchanges, indicating that nontargeted cells also sustained damage (Nagasawa and Little 1992). Subsequently it was discovered that targeted cells communicate with their bystander neighbors via gap junctions or release of damage-inducing substances into the medium. Medium from irradiated cell cultures was shown to induce various types of ▶ DNA damage in unirradiated cultures (reviewed in Prise and O’Sullivan 2009). This phenomenon was named radiation-induced bystander effect (RIBE). The discovery of RIBE has challenged the central dogma of radiation biology that cells subjected to irradiation are killed through direct DNA damage and the effect is proportional to the dose. Three general protocols have been employed for RIBE studies in cultured cells: • Conditioned medium transfer: medium from irradiated cells is transferred to a culture of unirradiated cells. • Microbeam irradiation (a narrow beam of radiation, of micrometer or sub-micrometer dimensions) of a selected subpopulation of individual cells. • Mixing, with subsequent culturing, of irradiated and unirradiated cells, each of the two

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populations being distinguished by staining with cytoplasmic or nuclear dyes. The in vitro RIBE exhibits a binary (“yes or no”) dose-response, rather than the magnitude of the effect increasing progressively with radiation dose. For example, a few microbeam-irradiated cells can stimulate the RIBE in a large population of their neighbors. Possible mediators of RIBE include various inflammation-related ▶ cytokines that have been found at elevated levels in medium conditioned by irradiated cells. ▶ Reactive oxygen species (ROS) and ▶ nitric oxide (NO) are also implicated in the transmission of RIBE (see Prise and O’Sullivan 2009 for details). Affected bystander cells exhibit increased levels of micronuclei, ▶ apoptosis, mutations, altered DNA damage and repair, as well as senescence and malignant transformation. Results obtained in cell culture indicate that cells under various stress conditions, either exogenous (UV radiation, physical damage, drugs, environmental chemical disbalance, etc.) or endogenous (such as tumor, aging, and apoptotic cells) also influence their bystander neighbors. Thus, RIBE may be a specific instance of a general cell stress response regulated by similar mechanisms. Antitumor Strategies The detrimental consequences of the bystander effect have been employed in antitumor therapy strategies. When tumors contain cells pre-labeled with lethal doses of DNA-incorporated 125I (an Auger electron emitter, the decay of which is characterized by induction of highly focused radiochemical damage, over subcellular dimensions), a distinct inhibitory effect on the tumor growth is observed. This inhibition is a consequence of a bystander effect that is generated by factors released from the 125I -damaged cells (Xue et al. 2002). Another strategy is antitumor suicide gene therapy. It consists of introduction into cancer cells of a “suicide” gene (in particular, thymidine kinase (TK) gene from herpes simplex virus (HSV)) which codes an enzyme that converts a nontoxic prodrug (ganciclovir (GCV)) into a toxic drug (Mesnil

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and Yamasaki 2000). This is an attractive approach since gene transfer remains limited to a part of a total cell population. In this case, HSV-TK-expressing cells pass to surrounding tumor cells not only bystander signals but also toxic metabolites of GCV. The strategy also helps avoid systemic drug toxicity. Both phenomena described are mediated through gap junction cell communication. Epigenetic Changes For decades, radiation oncologists have reported reactions in normal unirradiated tissues after radiotherapy (RT) of a particular part of the body. These “out-of-field” or abscopal effects have been described as “an action at a distance from the irradiated volume but within the same organism.” Abscopal effects have been also shown to be a general phenomenon; they result from a number of other localized stimuli aside from ionizing radiation, e.g., hyperthermia and laser immunotherapy. The discovery of RIBE has prompted the description of abscopal effects as distant in vivo bystander effects. Several studies reported in vivo RIBEs in animal models. Using strategies that involve partial body X-ray irradiation, profound genetic and epigenetic changes, including tumor formation, were identified in shielded organs. The results of the in vivo RIBE can be transmitted to future generations and manifested as epigenetic dysregulation in the unexposed progeny conceived after paternal exposure (reviewed in Xue et al. 2002). RIBE potentially contributes to the well-documented clinical phenomenon of secondary cancers, a major concern in cancer radiotherapy, affecting more than 1% of patients. Cell-Cell Communication The cell-cell communication involved in in vivo RIBE is mediated by the immune system and can be more complex. Many cytokines, such as ▶ IL-6, IL-8, IL-1a, IL-1b, TNF-a, TGF-a, ▶ TGF-b, MCP-1/CCL2, and others, mediate this systemic response. The presence of a tumor also induces inflammation and DNA damage responses in its immediate and distant microenvironment, due to the production of ROS and

Bystander Effect

cytokines, similar to RIBE signaling (Mesnil and Yamasaki 2000). Chronic activation of the immune system promotes both changes in immune homeostasis and normal aging progression and is responsible for abscopal effects related to cancer and other human diseases. Thus, the bystander effect can be considered as a manifestation of a more generalized systemic communication/response phenomenon.

Cross-References ▶ Abscopal Effects ▶ Apoptosis ▶ Cancer ▶ Cancer Epigenetics ▶ Chemokines ▶ Cytokine ▶ DNA Damage ▶ DNA Damage Response ▶ DNA Oxidation Damage ▶ Genomic Instability ▶ Inflammation ▶ Inflammatory Response and Immunity ▶ Interleukin-4 ▶ Interleukin-6 ▶ Ionizing Radiation Therapy ▶ Macrophages ▶ Nitric Oxide ▶ Oxidative Stress ▶ Radioimmunotherapy ▶ Reactive Oxygen Species ▶ Repair of DNA ▶ Stress Response ▶ Suicide Gene Therapy ▶ Transforming Growth Factor-Beta ▶ gH2AX

References Ilnytskyy Y, Kovalchuk O (2011) Non-targeted radiation effects-an epigenetic connection. Mutat Res 714:113–125 Mesnil M, Yamasaki H (2000) Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. Cancer Res 60:3989–3999

Bystander Effect Nagasawa H, Little JB (1992) Induction of sister chromatid exchanges by extremely low doses of alpha-particles. Cancer Res 52:6394–6396 Prise KM, O’Sullivan JM (2009) Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer 9:351–360 Redon CE, Dickey JS, Nakamura AJ, Kareva IG, Naf D, Nowsheen S, Kryston TB, Bonner WM, Georgakilas AG, Sedelnikova OA (2010) Tumors induce complex DNA damage in distant proliferative tissues in vivo. Proc Natl Acad Sci USA 107:17992–17997 Xue LY, Butler NJ, Makrigiorgos GM, Adelstein SJ, Kassis AI (2002) Bystander effect produced by radiolabeled tumor cells in vivo. Proc Natl Acad Sci USA 99:13765–13770

See Also (2012) Alpha-particles. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 147. doi:10.1007/978-3-642-16483-5_208 (2012) DNA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1129. doi:10.1007/978-3-642-16483-5_1663 (2012) Epigenetic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1283. doi:10.1007/978-3-642-16483-5_1940 (2012) Interleukin-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1892. doi:10.1007/978-3-642-16483-5_3095 (2012) Interleukin-8. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1896. doi:10.1007/978-3-642-16483-5_3100

709 (2012) Ionizing radiation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1907. doi:10.1007/978-3-642-16483-5_3139 (2012) MCP-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2192. doi:10.1007/978-3-642-16483-5_3576 (2012) Micronucleus. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2300. doi:10.1007/978-3-642-16483-5_3726 (2012) Mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Neoplastic cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2474. doi:10.1007/978-3-642-164835_4013 (2012) Senescence. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3370. doi:10.1007/978-3-642-16483-5_5236 (2012) Sister chromatid exchange. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3418. doi:10.1007/978-3-642-164835_5328 (2012) TGF-b. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3661. doi:10.1007/978-3-642-16483-5_5753 (2012) TNF-a. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3713. doi:10.1007/978-3-642-16483-5_5841 (2012) Tumor necrosis factor-a. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6041

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C.elegans Cell Death 4 Homolog

Ca2+ Homeostasis

▶ APAF-1 Signaling

Olivier Dellis Signalisation Calcique et Interactions Cellulaires dans le Foie, INSERM UMR-S 1174, Université Paris-Sud 11, Orsay, France

C.I. 75300 ▶ Curcumin

Definition

C/EBP-Epsilon-Regulated MyeloidSpecific Secreted Cysteine-Rich Protein (XCP1) ▶ Resistin

c-erb-B2 ▶ HER-2/neu

C21H2006 ▶ Curcumin

C33 ▶ Metastasis Suppressor KAI1/CD82 # Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3

Ca2+ homeostasis is the dynamic equilibrium of Ca2+ ion concentration in the body or in the cell (“cellular Ca2+ homeostasis”).

Characteristics Even if 99% of the Ca2+ ions of the body are trapped in the bones and could be considered as stable, Ca2+ homeostasis refers to the control of the free Ca2+ ion concentrations at the extracellular (“[Ca2+]ex”) and the intracellular (“[Ca2+]i”) levels. Control of [Ca2+]ex is important because Ca2+ ions stabilize numerous ionic channels like the voltage-gated ones: thus a decrease of [Ca2+]ex could induce spontaneous contraction of muscles, implying that [Ca2+]ex must be tightly controlled in the blood. This is mainly done by parathyroid hormone that controls the uptake of Ca2+ ions by the gusts, the release by the bones, and the reabsorption by the kidneys.

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Cellular Ca2+ Homeostasis At the cellular level, [Ca2+]i is an equilibrium between the Ca2+ ion concentration of the different compartments, mainly the cytosol ([Ca2+]cyt), endoplasmic reticulum ([Ca2+]ER), and mitochondria ([Ca2+]m). Even if [Ca2+]ER is important for the synthesis and the right folding of proteins and [Ca2+]m for the activity of the mitochondria, the control of [Ca2+]cyt must be tightly done by the cell as it directly regulates almost all the cellular processes, from the egg fertilization to activation, neurotransmission, proliferation, and apoptosis. As depicted by Berridge et al. (2003), [Ca2+]cyt is the result of an equilibrium between “on” reactions allowing entry of Ca2+ ions in the cytosol and “off” reactions extruding Ca2+ ions from the cytosol. Indeed, the [Ca2+]ex is commonly of 1–2 mM, and [Ca2+]cyt must remain at 100 nM to avoid any uncontrolled activation of cellular process: due to this huge gradient of concentration, reinforced by the fact that cells are negatively charged, the Ca2+ electrochemical gradient is in favor of a massive Ca2+ ion entry. Thus, even in resting conditions, cells face a Ca2+ ion entry. To avoid the fast rise of [Ca2+]cyt, Ca2+ ions are rapidly exited from the cytosol by two ways: (i) Plasma membrane Ca2+ ATPases (“PMCA”) pump the Ca2+ ions out of the cells, with the help of Na+/Ca2+ exchangers (“NCX”), (ii) And/or Ca2+ ions are mainly pumped in the lumen of the ER by the sarcoplasmicendoplasmic reticulum Ca2+ ATPases (“SERCA”). However, according to the cell types, Ca2+ ions can be also uptake in the apparatus of Golgi by the secretory pathway Ca2+ ATPases (“SPCA”). Furthermore, the cytosol contains different type of proteins able to buffer the Ca2+ concentration. Thus, each type of cells expresses different isoforms of these Ca2+ transporters and buffers allowing cell type control of resting [Ca2+]cyt. Cellular Ca2+ Homeostasis During Cell Stimulation During the cell stimulation, the Ca2+ homeostasis will reach a new equilibrium due to the entry of

Ca2+ Homeostasis

Ca2+ ions from the extracellular medium and/or the Ca2+ ions release by internal stores. Ca2+ influx is allowed by a large variety of plasma membrane channels with different types of opening: membrane depolarization for voltageoperated channels (L, N, P/Q, R, and T types), fixation of a ligand for receptor-operated channels (NMDA receptors or ATP receptor P2X), fixation of a second messenger for cyclic nucleotide-gated (CNG) channels, interaction with an ER protein for Ca2+ release-activated channels (CRAC, allowing the Ca2+ influx known as store-operated calcium entry (SOCE), formally controlled by the ER), etc. Ca2+ release by internal stores, mainly by the ER (or its derivative the sarcoplasm in muscle cells), is controlled by Ca2+ itself or by second messengers like inositol 1,4,5-triphosphate (“IP3”) allowing the opening of intracellular Ca2+ channels. The expression spectra of this different kind of plasma membrane and internal channels in the different type of cells create different types of Ca2+ homeostasis change during cell stimulation, inducing different response from the cells. For example, in cells like T lymphocytes, the stimulation of the T-cell receptor induces the synthesis of IP3, allowing the release of Ca2+ ions by the ER through IP3 receptors. This release induces the opening of the plasma membrane ORAI1, allowing the massive entry of extracellular Ca2+ ions. This increase activates different proteins like calmodulin and calcineurin, which one induces the translocation of the nuclear factor NFAT to the nucleus. Nuclear NFAT can then activates the transcription of various genes, like the one of interleukin-2. In the absence of this [Ca2+]cyt rise due to a default in the signal transduction from the plasma membrane to the ER, or the non-expression of ORAI1 channels, T lymphocytes cannot be activated, and the immune system is shut down. Inhibition of calcineurin by cyclosporin impairs the signal transduction between the Ca2+ rise and the activation of NFAT. Thus in every kind of cells, the defect of only one Ca2+ transporters could have huge impacts on the cell activity and properties.

Cachexia

Cellular Ca2+ Homeostasis in Cancer Even if the role of Ca2+ ions is well established in a large number of cellular processes like proliferation and apoptosis, two processes implied in the appearance of cancer, its role in cancerogenesis and metastasis formation is surprisingly poorly documented. However, since few years, some works have clearly implied Ca2+ ions in the proliferation of cancerous cells and metastasis. Thus, since the discovery of the ORAI1 channels in 2006 and its important role for the proliferation of non-excitable cells, it seems that ORAI1 channel activity may control the proliferation of cancerous cells. For example, inhibition of ORAI1 channel impairs the proliferation and migration of melanoma cells (Umemura et al. 2014) or the formation of bone metastasis of breast cancer cells (Chantôme et al. 2013). Some other compounds acting on ORAI1 seem to induce apoptosis. To conclude, Ca2+ homeostasis appears modified in numerous cancer cells. In the near future, new molecules able to control Ca2+ channels to avoid the cancer cell proliferation and formation of metastases will probably appear.

Cross-References ▶ Apoptosis Induction for Cancer Therapy ▶ Ion Channels ▶ Membrane Transporters ▶ Signal Transduction

References Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529 Chantôme A, Potier-Cartereau M, Clarysse L, Fromont G, Marionneau-Lambot S, Guéguinou M, Pagès JC, Collin C, Oullier T, Girault A, Arbion F, Haelters JP, Jaffrès PA, Pinault M, Besson P, Joulin V, Bougnoux P, Vandier C (2013) Pivotal role of the lipid raft SK3-Orai1 complex in human cancer cell migration and bone metastases. Cancer Res 73(15):4852–4861 Umemura M, Baljinnyam E, Feske S, De Lorenzo MS, Xie LH, Feng X, Oda K, Makino A, Fujita T, Yokoyama U, Iwatsubo M, Chen S, Goydos JS, Ishikawa Y, Iwatsubo K (2014) Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration. PLoS One 9(2):e89292

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See Also (2012) Ca 2+ aTPase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin, p 577. doi:10.1007/ 978-3-642-16483-5_763 (2012) Ca 2+ -release channels. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 577. doi:10.1007/978-3-642-16483-5_764 (2012) Calcineurin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 584. doi:10.1007/978-3-642-16483-5_775 (2012) Cyclosporin A. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1036. doi:10.1007/978-3-642-16483-5_1440 (2012) Inositol 1,4,5-trisphosphate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 1870. doi:10.1007/978-3-642-16483-5_3071 (2012) T lymphocyte. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3600. doi:10.1007/978-3-642-16483-5_5654

Ca2+-Activated PhospholipidDependent Protein Kinase ▶ Protein Kinase C Family

CaBP3 ▶ Calreticulin

Cachectin ▶ Tumor Necrosis Factor

Cachexia Chen Bing Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK

Definition Cachexia came from the Greek “kakos” and “hexis” meaning “bad conditions.” Cachexia is a

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complex metabolic syndrome characterized by progressive weight loss with extensive loss of skeletal muscle and adipose tissue, which is secondary to the growing malignancy.

Characteristics Most cancer patients develop cachexia at some point during the course of their disease, and nearly one-half of all cancer patients have weight loss at diagnosis. Cachexia prevents effective treatments for cancer and predicts a poor prognosis because the severity of wasting inversely correlates with survival. The consequences of cachexia are detrimental and cachexia is considered to be the direct cause of about 20% of cancer deaths. The pathogenesis of cancer cachexia remains to be fully understood, but it is evidently multifactorial. Weight Loss Clinically, cachexia should be suspected if involuntary weight loss of more than five percent of premorbid weight occurs within a 6-month period. Weight loss is not simply caused by competition for nutrients between tumor and host as the tumor burden may be only 1–2% of total body weight. The frequency of weight loss varies with the type of malignancy, being more common and severe in patients with cancers of the gastrointestinal tract (▶ gastrointestinal stromal tumor) and lung (▶ lung cancer). Gastric and pancreatic cancer patients may lose large amounts of weight, up to 25% of initial body weight. Over 15% of weight loss in patients is likely to cause significant impairment of respiratory muscle function, which probably contributes to premature death. Weight loss can arise from several metabolic changes that take place during malignancy, for example, reduced food intake, increased energy expenditure, and tissue breakdown. Poor Appetite Loss of the desire to eat or lack of hunger is common in cancer patients. It can be related to the mechanical effect of the tumor such as obstructions (especially of the upper gastrointestinal tract), side-effects of chemotherapy or

Cachexia

radiotherapy (▶ chemoradiotherapy), and emotional distress. Some tumors may secrete products which act on the brain to inhibit appetite. Regulation of food intake involves the integration of the peripheral and neural signals in the hypothalamus and other brain regions. In the hypothalamus, the orexigenic signals such as neuropeptide Y (NPY), the most potent appetite stimulant, increase food intake, and the anorexigenic signals including the pro-opiomelanocortin/cocaine and amphetamine regulated transcript (POMC/CART) inhibit appetite. Dysregulation of NPY in the hypothalamic pathway can lead to decreased energy intake but higher metabolic demand for nutrients. It has been demonstrated that NPY-immunoreactive neurons in the hypothalamus are decreased in experimental model of cancer anorexia. In contrast, reduced food consumption can be restored to normal levels by blocking the POMC/CART pathway in tumorbearing animals. High level of leptin, a hormone primarily secreted by adipocytes, inhibits the release of hypothalamic NPY. In cancer cachexia the leptin feedback loop appears to be deranged, altering the signaling pathway of NPY. Cytokines such as interleukin 1b (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor-a (TNFa) are implicated to be involved in cancer anorexia, possibly by stimulating corticotrophin-releasing factor, a neurotransmitter which suppresses food intake at least in rodents, and/or by inhibiting neurons that produce NPY in the hypothalamus. Increased Metabolism and Energy Expenditure Maintaining normal body weight requires energy intake to equal energy expenditure. In some patients with cancer cachexia, energy balance becomes negative as reduced food intake is not accompanied by a parallel decrease in energy expenditure. For example, patients with lung and pancreatic cancers generally have higher resting energy expenditure (REE) compared with normal control subjects; however, REE is usually normal in patients with colorectal cancer. The mechanisms of increased energy expenditure are not clear although studies suggest that it might be through the upregulation of uncoupling proteins, a family of mitochondrial membrane proteins,

Cachexia

which are proposed to be involved in the control of energy metabolism. Uncoupling protein-1 (UCP-1), which decreases the coupling of respiration to ADP phosphorylation thereby generating heat instead of ATP, is only expressed in brown adipose tissue (BAT). UCP-1 mRNA levels in BAT are increased in mice bearing the MAC16 colon adenocarcinoma. Although BAT is uncommon in adults, the prevalence of BAT has been found to be higher in cancer cachectic patients than the age-matched control subjects. mRNA levels of UCP-2 (expressed ubiquitously) and UCP-3 (expressed in skeletal muscle and BAT) in skeletal muscle are upregulated in rodent models of cancer cachexia. In humans, skeletal muscle UCP-3 mRNA levels are over fivefold higher in cachectic cancer patients compared with patients without weight loss and health controls. Elevated expression of UCP-2 and UCP-3 has been suggested to contribute to lipid utilization rather than whole-body energy expenditure. Cytokines such as TNFa and/or other tumor products may be responsible for the changes in UCP expression at least in rodents. Additional energy consumption could arise from the metabolism of tumor-derived lactate via “futile cycles” between the tumor and the host. The main energy source for many solid tumors is glucose, which is converted to lactate and transferred to the liver to convert back into glucose. This “futile cycle” requires large amount of ATP, resulting in an extra loss of energy in cancer patients. Loss of Adipose Tissue Fat constitutes 90% of normal adult fuel reserves, and depletion of adipose tissue together with hyperlipidemia becomes a hallmark of cancer cachexia. Computed tomography (CT) scanning has revealed that cachectic cancer patients with gastrointestinal carcinoma had significantly smaller visceral adipose tissue area than control subjects. Increased lipolysis is implicated in cancer-associated adipose atrophy. The activity of hormone-sensitive lipase, a rate-limiting enzyme of the lipolytic pathway, is increased in cancer cachectic patients, which causes elevated plasma levels of free fatty acids and triglycerides. Meanwhile, there is a fall in lipoprotein lipase (LPL) activity in white

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adipose tissue, thus inhibiting cleavage of triglycerides from plasma lipoproteins into glycerol and free fatty acids for storage, causing a net flux of lipid into the circulation. Finally, glucose transport and de novo lipogenesis in the tissue are reduced in tumor-bearing state, leading to a decrease in lipid deposition. There is also evidence that loss of adipose tissue in cancer cachexia could be the result of impairment in the formation and development of adipose tissue. The expressions of several key adipogenic transcription factors including CCAAT/enhancer-binding protein alpha, CCAAT/ enhancer-binding protein beta, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein-1c are markedly reduced in adipose tissue of cancer cachectic mice. Various factors produced by tumors or the host’s immune cells responding to the tumor can disturb lipid metabolism. TNFa has been shown to affect adipose tissue formation by inhibiting the differentiation of new adipocytes, causing dedifferentiation of mature fat cells and suppressing the expression of genes encoding key lipogenic enzymes. TNFa has also been associated with increased lipolysis probably through suppression of LPL activity in adipocytes. In addition, both TNFa and IL-1b are able to inhibit glucose transport in adipocytes and consequently decrease the availability of substrates for lipogenesis. Certain prostate, gut, and pancreatic tumors secrete a lipid-mobilizing factor (LMF), also produced by a mouse adenocarcinoma model. LMF has been shown to be identical to the plasma protein zinc-a 2-glycoprotein (ZAG). It is found to be secreted by human adipocytes and upregulated in adipose tissue of mice with cancer cachexia. ZAG causes rapid lipolysis in vitro and in vivo, possibly through activation of intracellular cyclic AMP. ZAG also stimulates expression of UCPs in brown fat of mice, which may contribute to increased energy expenditure as well as lipid catabolism during cachexia. Moreover, ZAG expression and secretion by adipose tissue is enhanced weight loss patients with gastrointestinal cancer. Given its lipid-mobilising effect, ZAG could contribute to adipose tissue loss associated with cancer cachexia in humans.

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Loss of Muscle Protein Weakness, commonly seen in cancer cachectic patients, is directly related to wasting of muscle that accounts for almost half the body’s total protein and bears the brunt of enhanced protein destruction. Reduced protein synthesis together with enhanced proteolysis has been observed in experimental animal models and in muscle biopsies from cancer patients with cachexia, and whole-body protein turnover can be markedly increased in cachectic cancer patients. Some mediators and pathways of excessive protein breakdown have been incriminated in cancer cachexia. TNFa appears to be involved, as treatment with recombinant TNFa enhances proteolysis in rat skeletal muscle and activates the ubiquitin–proteasome system. Ubiquitin, an 8.6 kD peptide, is crucially involved in targeting of proteins undergoing cytosolic ATP-dependent proteolysis. There is an increase in ubiquitin gene expression in rat skeletal muscle after incubation with TNFa in vitro. Tumors also produce cachectic factors such as proteolysis-inducing factor (PIF), a 24 kD glycoprotein initially isolated from a cachexia-inducing tumor (MAC16) and the urine of cachectic cancer patients. PIF induces muscle protein breakdown by stimulation of the ubiquitin–proteasome proteolytic pathway. There is increasing evidence that both cytokines and PIF cause protein degradation by activation of ▶ nuclear factor kappa B (NFkB), a transcription factor that regulates the expression of a number of proinflammatory cytokines. TNFa and PIF can upregulate components of the ubiquitin–proteasome pathway in an NFkBdependent manner. Activation of NFkB by TNFa in murine muscle cells suppresses mRNA of the transcription factor MyoD, inhibiting skeletal muscle cell differentiation as well as preventing the repair of damaged skeletal muscle fibers.

Cachexia

cancer patients. Appetite stimulants such as megestrol acetate and medroxyprogesterone acetate are commonly used at present in the treatment of anorexia and cachexia. These agents are believed to stimulate orexigenic peptide NPY in the hypothalamus and inhibit the synthesis and release of proinflammatory cytokines. Their effects on appetite and well being are short-termed and they do not influence lean body mass and survival. ▶ Cannabinoids have also been studied as potential appetite stimulants. However, dronabinol has failed to prevent progressive weight loss in patients with advanced cancer. Therapeutic interventions include anticytokines such as thalidomide with multiple immunomodulatory properties. It suppresses the production of TNFa, IL-1b, IL-12, and cyclooxygenase-2, which is probably through inhibiting NFkB activity. Thalidomide has been shown to attenuate total weight loss and loss of lean body mass in cachectic patients with advanced pancreatic cancer. Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid from fish oil, has attracted attention as a potential anticachectic agent. EPA has been shown to attenuate the increased expression of the components of the ubiquitin–proteasome proteolytic pathway in skeletal muscle of mice with cancer cachexia, and EPA can block PIF-induced protein degradation in vitro. In randomized clinical trials, cachectic patients with unresectable pancreatic cancer receiving EPA have shown a stabilization in the rate of weight loss, fat and muscle mass, as well as the REE. Data from animal studies suggest that EPA combined with the leucine metabolite beta-hydroxybeta-methylbutyrate seems to be more effective in the reverse of muscle protein wasting.

Cross-References Treatment Current treatment designed to ameliorate cancer cachexia has limited benefit. Nutritional supplementation (oral or parenteral) alone has little effect and, critically, does not restore muscle mass and improve quality of life or prognosis in

▶ Cannabinoids ▶ Chemoradiotherapy ▶ Gastrointestinal Stromal Tumor ▶ Lung Cancer ▶ Nuclear Factor-κB

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References

Cadherin-1 Argiles JM, Busquets S, Lopez-Soriano FJ (2006) Cytokines as mediators and targets for cancer cachexia. Cancer Treat Res 130:199–217 Bing C, Brown M, King P et al (2000) Increased gene expression of brown fat uncoupling protein (UCP)1 and skeletal muscle UCP2 and UCP3 in MAC16-induced cancer cachexia. Cancer Res 60:2405–2410 Bing C, Russell S, Becket E et al (2006) Adipose atrophy in cancer cachexia: morphologic and molecular analysis of adipose tissue in tumour-bearing mice. Br J Cancer 95:1028–1037 Fearon KC, Moses AG (2002) Cancer cachexia. Int J Cardiol 85:73–81 Mracek T, Stephens NA, Gao D et al (2011) Enhanced ZAG production by subcutaneous adipose tissue is linked to weight loss in gastrointestinal cancer patients. Br J Cancer 104:441–7. doi: 10.1038/sj.bjc.6606083 Tisdale MJ (2002) Cachexia in cancer patients. Nat Rev Cancer 2:862–871

See Also (2012) Brown adipose tissue. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 572. doi:10.1007/978-3-642-16483-5_742 (2012) Eicosapentaenoic acid. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1212. doi:10.1007/978-3-642-164835_1836 (2012) Hyperlipidemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1784. doi:10.1007/978-3-642-16483-5_2909 (2012) Lipogenesis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2055. doi:10.1007/978-3-642-16483-5_3378 (2012) MyoD. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2440. doi:10.1007/978-3-642-16483-5_3942 (2012) Neuropeptide Y. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2504. doi:10.1007/978-3-642-16483-5_4043 (2012) Resting energy expenditure. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3264. doi:10.1007/978-3-642-164835_5060 (2012) Uncoupling protein-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3846. doi:10.1007/978-3-642-16483-5_6106

▶ E-Cadherin

C Cafe-Au-Lait Macule ▶ Cafe-Au-Lait Spots

Cafe-Au-Lait Spots Synonyms Cafe-au-lait macule

Definition Coffee-with-milk-colored spots on the skin that are seen characteristically in the neurofibromatosis type 1 (NF1) syndrome.

Cajal Bodies Vincenzo de Laurenzi Department of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, Rome, Italy

Synonyms Coiled bodies

Definition

Cachexia-Inducing Agent ▶ Leukemia Inhibitory Factor

Small nuclear organelles (0.1–2.0 mM in diameter), present in all eukaryotic cells, involved in a number of different nuclear functions.

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Characteristics The nucleus of eukaryotic cells contains a number of different highly specialized organelles. Unlike cytoplasmic organelles these nuclear structures are not delimited by a membrane but are by all means compartments that contain a number of specific proteins. Most of the organelles can be clearly identified through immunostaining using antibodies directed against specific marker proteins; however, it should be kept in mind that these organelles are highly dynamic structures that often exchange components and therefore many proteins can be found in more than one organelle. Among these organelles are Cajal bodies (CBs), described over a century ago by Ramon y Cajal. CBs were originally described in neuronal cells but have since been described in a variety of cell types, both in animals and in plants, suggesting that they are involved in some fundamental cellular process. Due to their characteristic ultrastructural appearance as a tangle of coiled fibrillar strands, they have also been called coiled bodies. They usually vary in size from 0.2 to 2 mM, but can be occasionally larger. The number of CBs is usually between 0 and 4 in normal diploid cells; however, many more can be found in some cancer cells. The number of CBs per cell is regulated during the cell cycle. Indeed, CBs disappear in prophase nuclei, to reappear in G1 at the same time of the nucleolus. Their number is then doubled, usually reaching the number of four, in the S phase. It has been suggested that in these cells the number of CBs depends on the ploidy of the cells or more specifically on the number of chromosomes 1 and 6. CBs can be found associated with specific gene loci such as snoRNA, snRNA, and histone gene clusters. In addition, CBs can also be found in association with other nuclear bodies such as cleavage bodies and PML bodies, suggesting that there is an exchange of components between the different nuclear organelles. CBs have a heterogeneous composition, containing different small nuclear ribonucleoproteins (snRNPs), small nucleolar ribonucleoproteins (snoRNPs), cell cycle regulating proteins,

Cajal Bodies

and transcription factors, as well as other proteins, whose function still needs to be determined. The generally recognized marker of CBs is p80 coilin. The function of this protein is still unknown; its deletion in mice results in reduced coilin / animal litters, suggesting a developmental defect; however surviving animals appear normal. Deletion of coilin results in residual bodies that still contain some components such as fibrillarin, Nopp140, and FLASH, but not others like splicing snRNPs. While their function is still in part elusive, recent work suggests that they are involved in several nuclear functions. CBs are supposed to be the site of assembly of the three eukaryotic RNA polymerases (pol I, pol II, and pol III) with their respective transcription and processing factors that are then transported as multiprotein complexes to the sites of transcription. They are also involved in the modification of small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs), which are important for spliceosome formation. Indeed CBs contain newly assembled snRNPs and snoRNPs that later accumulate in speckles and nucleoli, and it has been suggested that CBs are sites of modification for snRNPs and particularly sites where 20 -O-methylation and pseudouridine formation occur. This process requires a novel class of small CB-specific RNAs (scaRNAs) that pair with the snRNAs and function as guides for 20 -O-methylation. The reaction is probably mediated by the fibrillarin, a CB, and nucleolarassociated protein with methyl transferase activity. CBs have also been implicated in replication-dependent histone gene transcription, and a subset of CBs is physically associated with histone gene clusters on chromosomes 1 and 6. Phosphorylation of a CB component p220/ NPAT by cyclin E/Cdk2 is required for activation of histone transcription, exit from G1, and progression through S phase of the cell cycle. Moreover it has been shown that another CB component FLASH is essential for this function. Downregulation of FLASH results in structural alteration of CBs, reduction of replicationdependent histone gene transcription, and block of cells in the S phase (▶ S-phase damage-sensing

Calcitonin

checkpoints) of the cell cycle. In addition, CBs are involved in U7 snRNA-dependent cleavage of the 30 end of histone pre-mRNA before the mature mRNA can be exported to the cytoplasm. Finally, a role for CBs in regulating ▶ telomerase function has been proposed. Based on the presence of the RNA component of telomerase (hRT) in CBs of cancer cells, it has been suggested that CBs play a role in the maturation of hRT or in the assembly of the telomerase complex. However, CBs might represent only a site of accumulation of hRT; alternatively, this could be an altered localization only present in cancer cells; therefore further studies are required to clarify this potential CB function. Alteration of CB structure, as well as other nuclear structure alterations, has been observed in various diseases; however, in most cases it is not clear if these defects are a consequence of altered nuclear functions or play a role in the disease pathogenesis. CBs have been found associated with the aggregates formed in CAG triplet expansion diseases and ataxin-1; mutated in spinocerebellar ataxia type 1 (SCA1), it has been shown to interact with coilin. The role of these findings in the disease pathogenesis is yet to be established. Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by motor neuron degeneration associated with muscular atrophy and paralysis; it is usually caused by mutations of the surviving motor neuron 1 (SMN1) gene. SMN is a 294 amino acid protein, ubiquitously expressed; it bears no homology to other known proteins and its function is still unknown. It is localized both in the cytoplasm and in the nucleus where it is found in two different nuclear organelles: Cajal bodies (CBs) and Gems (for Gemini of CBs). Pathogenesis of SMA is not clearly understood, but reduction of SMN levels results in an alteration of CB structure. Alteration of CBs in cancer has not been thoroughly studied yet, and a role for these organelles in cancer has not been clearly established. However cancer cell lines often show an increased number of CBs, and some alteration of CBs can be found in specific cancers. In MLL-ELL

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leukemia (▶ acute myeloid leukemia) the presence of the MLL-ELL fusion protein results in alteration of CB structure and altered localization of coilin. The TLS/CHOP fusion protein generated by the t(12;16) translocation (▶ chromosomal translocations), found in liposarcomas, shows high transforming capacity and is in part localized in CBs. In conclusion, while studies have started to shed light on the function of CBs and on the interrelationship between these organelles and other nuclear structures, more work is required to clearly understand the molecular mechanisms involved in their formation and clarify their different roles in nuclear function. This in turn will provide information on their potential role in the pathogenesis of a range of human diseases.

References Cioce M, Lamond AI (2005) Cajal bodies: a long history of discovery. Annu Rev Cell Dev Biol 21:105–131 Gall JG (2000) Cajal bodies: the first 100 years. Annu Rev Cell Dev Biol 16:273–300 Ogg SC, Lamond AI (2002) Cajal bodies and coilin – moving towards function. J Cell Biol 159(1):17–21

CAK1 Antigen ▶ Mesothelin

Calcitonin Girish V. Shah Department of Pharmacology, University of Louisiana College of Pharmacy, Monroe, LA, USA

Definition Calcitonin (CT) is a 32-amino acid peptide synthesized in mammals by the C cells of the thyroid

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gland. Several extrathyroidal sites including the ▶ prostate gland, gastrointestinal tract, thymus, ▶ bladder, ▶ lung, pituitary gland, and central nervous system (CNS) also produce this peptide molecule (Ball 2007).

Characteristics Almost all cells of human body synthesize and secrete procalcitonin (proCT), a precursor of the CT peptide, in response to infection/▶ inflammation. Only cells of the thyroid and neuroendocrine organs can process proCT to produce mature CT molecule (Davies 2015). CT sequence among various species shows remarkable divergence (Steinwald et al. 1999). However, all sequences contain 32 amino acids, a carboxy-terminal proline amide, and a disulfide bridge between cysteine residues at positions 1 and 7. In addition to CT, other biologically and chemically diverse molecules such as CT gene-related peptide (CGRP), ▶ adrenomedullin (AFP-modern), and amylin (AMY) are considered as CT family of peptides because of their ability to interact with CT receptor (CTR) and induce biological response. Each of these peptides displays selective tissue distribution and distinct physiological effects (Findlay and Saxton 2003). For example, CGRP is predominantly present in central and peripheral nervous system and is important for neurotransmission and neuromodulation. ADM is relatively abundant in vascular space, and plays an important role in the regulation of cardiovascular and respiratory functions, and CT is essential for calcium balance. However, CT does not regulate calcium in extrathyroidal tissues but is implicated to play an important role in cell growth, cell differentiation, and other regulatory functions. Biosynthesis Four CT genes, CALC-I, CALC-II, CALC-III, and CALC-IV with significant nucleotide homologies have been identified. However, CT is encoded by only CALC-I gene. CALC-I and CALC-II encode two different forms of CGRP, CGRP-I and CGRP-2. CALC-III is thought to be a

Calcitonin

pseudo gene, and CALC-IV produces AMY. Human (h) CT (CALC-I) gene is located in the p14qter region of chromosome 11. CT gene encodes two distinct peptides CT and CGRP, which arise by tissue-specific alternative splicing of the same primary mRNA transcript. The primary mRNA transcript is spliced almost exclusively to CT mRNA in thyroid and to CGRP in the nervous system. CT is synthesized as part of a larger precursor protein of 136 amino acids. The DNA sequence of the hCT gene predicts that the hormone is flanked in the precursor by N- and C-terminal peptides. Both N-terminal and C-terminal flanking peptides are detected in the plasma and thyroidal tissues of both normal and medullary thyroid carcinoma (MTC) patients. However, no biological function for either of these two peptides has been conclusively determined. Cyclic adenosine monophosphate (cAMP), pentagastrin, and progesterone are potent stimulators of CT gene expression. In contrast, testosterone and estrogens have inhibitory effect. There is evidence for polymorphisms in CALC-I gene that leads to increased risk for ovarian ▶ cancer in carrier women (T-C 624 bp upstream of translation initiation codon); and 16 bp microdeletion polymorphism has been reported in a family with multiple cases of unipolar and bipolar depressive disorders. Biological Actions Actions on Bone

CT is the only hormone that inhibits bone resorption by direct action on osteoclasts in the bone. It is characterized by the rapid loss of osteoclast ruffled borders, reduced cytoplasmic spreading, decreased release of lysosomal enzymes, and inhibition of collagen breakdown. This role is physiologically more relevant at times of stress on skeletal calcium conservation such as pregnancy, lactation, and growth, when bone remodeling by osteoclasts and the consequent release of calcium stores in the bone need to be tightly regulated to prevent unnecessary bone loss. In normal adult humans, even large dose of CT has little effect on serum calcium. However, in pathologies created

Calcitonin

by increased bone turnover such as thyrotoxicosis, metastatic bone disease, or Paget’s disease, CT treatment effectively inhibits bone resorption and lowers serum calcium. Renal Actions

CT increases urinary excretion rate of sodium, potassium, phosphorus, and magnesium. CT also enhances 1-hydroxylation of 25-hydroxy vitamin D in the proximal straight tubule by stimulating the expression of 25-hydroxy vitamin D 1-hydroxylase. Central Actions

Central administration of CT produces analgesia, affects sleep cycles producing insomnia, major reduction in slow wave sleep and long period of alteration of rapid eye movement (REM) sleep and wakening. The centrally mediated actions of CT correlate well with the location of CT binding sites. CT also demonstrates multiple hypothalamic actions such as modulation of hormone release, decreased appetite, gastric acid secretion, and intestinal motility. Administration of CT in clinical situations of bone pain is very effective in ameliorating the pain symptoms. Other Actions

CT and its receptors have been identified in a large number of other cell types and tissue sites suggesting multiple roles for CT–CTR axis. CTR-binding sites have been identified in the kidneys, brain, pituitary, testis, prostate, spermatozoa, lung, and lymphocytes. There is evidence to suggest the involvement of CT in cell growth and differentiation, tissue development and tissue remodeling. CT appears to be important for blastocyst implantation and development of the early blastocyst. CT in Cancer

Overexpression of CT has been reported in cancer-derived cells from thyroid, ▶ lung, ▶ Brms1, ▶ prostate, ▶ pancreas, pituitary, bone (osteoclastoma, osteogenic sarcoma), and embryonal carcinoma, suggesting the deregulation of CT expression is an important event in several malignancies. The results from our laboratory

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have shown that CT and CTR are present in undifferentiated basal cells but absent in differentiated secretory cells of normal human prostate gland. However, CT and CTR become detectable in malignant secretory epithelium suggesting malignancy-associated deregulation of CT/CTR expression. CT and CTR transcripts in malignant human prostate become detectable as early as high-grade PIN and progressively increase with increase in tumor grade. In human pancreas, CTR is present in benign as well as malignant regions but CT is exclusively detected in malignant sections of multiple pancreatic carcinomas, including ductal adenocarcinomas. Mechanism of CT Action Receptors

CT acts by binding to receptors on the plasma membrane of responding cells. CTR cDNA has been cloned in multiple mammalian species. Analysis of the protein translated from CTR cDNA sequence reveals the size of approximately 500 amino acids, and the receptor belongs to the class B family of G protein-coupled receptors (GPCRs), which also includes numerous potentially important drug targets. The human CTR gene is located on chromosome 7 at 7q21.3. The CTR gene exceeds 70 Kb in length, comprises of at least 14 exons, separated by introns ranging in size from 70 nucleotides to >20,000 nucleotides. Multiple polymorphic sites in CTR gene have been identified, and several of them lead to lower bone mineral density in postmenopausal women. Receptor Isoforms

Human CTR (hCTR) is known to exist in mutiple isoforms that arise from alternative splicing of the same primary transcript. The two most common hCTR variants arise by alternative splicing of intracellular domain 1 (ondel 2000). The most common variant (type 1 hCTR) leads to the addition of a 16 amino acid insert in the first intracellular loop. Alternative splicing of this small exon leads to the expression of type 2 hCTR, which differs from type 1 hCTR (abundant in the brain and the kidneys) by the absence of a 16-amino acid insert in the first intracellular loop. Type

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2 CTR is predominantly expressed in malignant prostate and pancreatic cells. It has been shown that the lack of 16-amino acid insert in first intracellular loop enables type 2 hCTR to coactivate both adenylyl cyclase and phospholipase C. In addition, another receptor referred as calcitonin receptor-like receptor (CRLR) has been reported (Barwell et al. 2012). Modulation of CTR Specificity

CTR displays high affinity for CT but low affinities for other CT family peptides. However, the ligand specificity of CTR is significantly altered when it binds to a RAMP protein. Several RAMPs have been identified but three (RAMP1, RAMP2, RAMP3) are investigated. hCTR displays low affinity for AMY, but association with RAMPs enables hCTR to bind AMY with high affinity. Similarly, ligand specificity of CRLR depends on the complexed RAMP. For example, CRLRRAMP1 serves as CGRP receptor whereas CRLR-RAMP2 acts as ADM receptor. CRLRRAMP3 displays high affinity for ADM as well as CT. This phenomenon opens up a possibility that ligand specificity of CTRs can be regulated by modulation of RAMPs expression. CTR Signaling G Protein-Mediated Signaling

The intracellular mechanisms by which CTR produces biological effects are still being elucidated. However, the signaling pathways appear to vary with cell type as well as animal species. As with most other GPCRs, the CTRs show coupling with multiple G proteins, which also depends on the isoform of CTR. For example, type 1 CTR preferentially couples to Gas, leading to the activation of adenylyl cyclase and elevation in the intracellular levels of cAMP. The inhibitory action of CT on osteoclasts is accompanied by increase in cAMP levels. Forskolin, a direct activator of adenylyl cyclase, as well as dibutyryl cAMP, which elevates intracellular cAMP levels independent of adenylyl cyclase, mimic CT actions on bone resorption. Similarly, CTR is known to activate adenylyl cyclase in kidney as well as in cancers of lung, breast, and bone.

Calcitonin

Unlike type 1 CTR, type 2 CTR simultaneously couples with Gas and Gaq, leading to the coactivation of adenylyl cyclase and phospholipase C. This results in the elevation of intracellular levels of cAMP, as well as inositol triphosphates, and thence increased cytosolic calcium levels. This, together with coliberated diacyl glycerols, activates protein kinase C. In brain tissue, CT couples to G proteins other than Gas as indicated by limited activation of adenylyl cyclase in neural tissues. In hepatocytes, CT increases cytosolic calcium levels without activating adenylyl cyclase. In LLC-PK1 kidney cells, CT increases either intracellular cAMP levels or cytosolic calcium levels in a cell cycle-dependent manner; and in pituitary lactotrophs, CT inhibits TRH-induced increases in cytosolic levels and activation of protein kinase C. In ▶ prostate cancer cells, CT coactivates protein kinases A and C and pathways activated by these enzymes play an important role in CT-stimulated growth, invasiveness, and tumorigenicity of prostate cancer cells. G Protein-Independent Signaling

Evidence suggests that GPCRs also activate G protein-independent signaling by interacting with proteins referred as GPCR-interacting proteins (GIPs). GPCRs activate this signaling by binding to GIPs through one or more of structural interacting domains such as Src homology 2 (SH2) and SH3, plackstrin homology, PDZ and Eva/WASP homology (EVH) domains. Examination of CTR sequence reveals that the last four amino acids at the extreme C-terminus of the C-tail form E-S-S-A tetramer (amino acids 447–50), which conforms to the canonical type I PDZ ligand. A single serine-to-alanine substitution in the PDZ ligand of prostate CTR almost abolished CT-elicited increase in invasiveness and tumorigenicity of PC-3 prostate cancer cell line, raising a strong possibility that metastasizing ability of CTR is dependent upon its ability to interact with intracellular proteins containing PDZ domain(s). CTR seems to induce ▶ metastasis by disassembly of tight junctions on prostate cancer cells, which leads to the loss of cell polarity, activation of proteases such as urokinase type

Calcitonin

plasminogen activator, matrix metalloproteinases 2 and 9. These results raise a possibility that the prevention of interaction between CTR and its intracellular partner through the PDZ ligand can be an effective strategy to prevent CT-mediated metastasis. With current advances in medicinal chemistry and peptide mimetics, it should be possible to design a small peptide of 4–6 amino acids or a small molecule to prevent this interaction. CT also activates phosphoinositol-3-kinase (PI3K)–Akt–Survivin pathway and induces chemoresistance and ▶ apoptosis in multiple prostate cancer cell lines through as yet uncharacterized mechanism. CT activated protein kinase A plays a key role in multiple actions of CT on prostate cancer cell lines, suggesting that both, G protein-dependent and G protein-independent, actions of CTR may act in concert to increase oncogenicity of prostate cancer cell lines. Significance of CT–CTR Axis in Cancer: Clinical Aspects CT Is “Oncogene” for Prostate Cancer but “Tumor Suppressor” for Breast Cancer

Although growing body of evidence suggests elevated expression of CT and CTR in multiple cancers, extensive studies on CT actions have been conducted only in ▶ prostate cancer and ▶ breast cancer (▶ tumor suppressor genes) cell lines. Interestingly, CT displays sexual dimorphism in these two cancers, raising a possibility of the modulatory role of sex hormones on CT actions in these two organs. For example, CT is a potent ▶ oncogene for prostate cancer as indicated by the progressive increase in CT and CTR expression in primary prostate cancers with tumor progression, and potent stimulatory actions of CT on tumorigenicity of prostate cancer cell lines. In contrast, CT and CTR is constantly expressed in normal mammary ductal epithelium, the loss of CTR expression is associated with the progression of breast cancer to ▶ metastatic phenotype, and CT inhibits growth of some breast cancer cell lines. Although opposing actions of CT on prostate and breast cancer cell lines remain to be thoroughly investigated, initial studies in the author’s laboratory suggest that CT protects junctional

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complexes in breast cancer cell lines, and estradiol attenuates the actions of CT in estradiol receptorpositive breast cancer cell lines (Han et al. 2006; Seck et al. 2005). These results emphasize the importance of CTR actions on junctional complexes in cancer and its significance in cancer cell growth and metastasis. CT Is an Angiogenic Factor

▶ Angiogenesis, the process of new vessel formation or neovascularization, has aroused increasing interest over the last 25 years. Expansion of the tumor cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Our results have demonstrated the presence of CTR in HMEC-1 cell line, and that CT stimulates in vivo angiogenesis in nude mice, and directly stimulates all major phases of in vitro angiogenesis including endothelial cell migration, invasion, proliferation, and tube morphogenesis. The stimulatory actions of CT on in vitro angiogenesis are comparable to the actions of ▶ vascular endothelial growth factor (VEGF). Importantly, silencing of CTR in HMEC-1 cells completely abolishes CT-induced tube morphogenesis. Furthermore, prostate and thyroid cancer cell lines expressing high levels of CT form large, highly vascular tumors. In contrast, the silencing of CT expression in these cell lines markedly reduces tumor growth and vascularity. These results may also explain the findings that malignancies displaying high levels of CT expression (such as MTCs and multiple endocrine neoplasias) also produce highly vascular tumors. Considering that therapeutic use of CT for pain relief is fairly widespread in cancers as well as other diseases, it will be important to consider oncogenic and angiogenic effects while determining CT therapy in these patients. In summary, CT and CTR expression has been well investigated in breast and prostate carcinomas. CT is a potent stimulator of tumor growth, angiogenesis, and metastasis in prostate cancer cell lines. In contrast, CTR expression is lost with breast cancer progression, and CTR attenuates growth of breast cancer cell lines. Significant expression of CT and CTR has also been reported in MTCs, multiple endocrine neoplasias, and

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carcinomas of lung, ▶ pancreas, gastrointestinal tract, thymus, and ▶ bladder. However, the significance of CT–CTR axis in these carcinomas remains to be investigated.

Calcium-Binding Proteins

Calcium-Binding Proteins Meenakshi Dwivedi and Joohong Ahnn Department of Life Science, Hanyang University, Seoul, South Korea

Cross-References Definition ▶ Bladder Cancer ▶ Lung Cancer ▶ Medullary Thyroid Cancer Targeted Therapy ▶ Metastasis ▶ Multiple Endocrine Neoplasia Type 1 ▶ Pancreatic Cancer ▶ SH2/SH3 Domains ▶ Src ▶ Thyroid Carcinogenesis

References Ball DW (2007) Medullary thyroid cancer: therapeutic targets and molecular markers. Curr Opin Oncol 19(1):18–23 Barwell J, Gingell JJ, Watkins HA, Archbold JK, Poyner DR, Hay DL (2012) Calcitonin and calcitonin receptorlike receptors: common themes with family B GPCRs? Brit J Pharmacol 166(1):51-65. doi:10.1111/j.14765381.2011.01525.x. Davies J (2015) Procalcitonin. J Clin Pathol 68: 675–79 Findlay DM, Saxton PM (2003) Calcitonin. In: Henry HL, Normon AW (eds) Encyclopedia of hormones and related cell regulators. Academic, New York, pp 220–230 Han B, Nakamura M, Zhou G et al (2006) Calcitonin inhibits invasion of breast cancer cells: involvement of urokinase-type plasminogen actor and uPA receptor. Int J Oncol 28(4):807–814 ondel M (2000) Calcitonin and calcitonin receptors: bone and beyond. Int J Exp Pathol 81(6):405–422 Seck T, Pellegrini M, Florea AM et al (2005) The delta e13 isoform of the calcitonin receptor forms six transmembrane domain receptor with dominant negative effects on receptor surface expression and signaling. Mol Endocrinol 19(8):2132–2133 Steinwald PM, Whang KT, Becker KL, Snider RH, Nylen ES, White JC (1999) Elevated calcitonin precursor levels are related to mortality in an animal model of sepsis. Critical Care 3(1):11–16 Thomas S, Chigurupati S, Anbalagan M, Shah G (2006) Calcitonin increases tumorigenicity of prostate cancer cells: evidence for the role of protein kinase A and urokinase-type plasminogen receptor. Mol Endocrinol 20(8):1894–1911

Calcium-binding proteins are proteins that participate in calcium signaling pathways by binding to Ca2+. The most ubiquitous Ca2+-binding protein, found in all eukaryotic organisms including yeasts, is calmodulin. With their role in signal transduction, Ca2+-binding proteins contribute to all aspects of the cell’s functioning, from homeostasis to ▶ cancer.

Characteristics Normal cell cycle division is a highly coordinated progression of molecular events that is subject to control mechanisms from both outside and inside the cell. Commitment to cell cycle initiation is made from outside and occurs as a response to extracellular signals such as growth factors. Inside the cell, control mechanisms exist to determine the timing of intracellular events such as nuclear and cytoplasmic cleavage. Under normal conditions, growth-regulating mechanisms endeavor to maintain homeostasis. Homeostasis within a cell is regulated by the balance between proliferation, growth arrest, and ▶ apoptosis. Intracellular Ca2+ is an important modulator of a variety of biochemical processes associated with cell cycle progression. With few exceptions, the controls exerted by intracellular Ca2+ are transduced through sitespecific interactions with specialized Ca2+binding proteins. There exist at least three main families of Ca2+-binding proteins. The first of these is represented by proteins that possess one or more EF-hand helix–loop–helix structural motifs predicting Ca2+-binding domains as typically found within calmodulin. The second class of Ca2+-binding proteins is known generically as annexins. A possible third family of Ca2+-binding proteins is the “calreticulin-like” group of proteins

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that include ▶ calreticulin, Grp78, endoplasmin, and protein disulfide isomerase. Ca2+ has also been implicated in cell growth under pathological states. An altered cellular response to extracellular calcium ion concentration is one of the earliest changes induced in mouse epidermal cells by chemical carcinogens. However, whereas some human breast cancer cell lines and leukemia cell lines exhibit Ca2+-induced cell proliferation, other carcinoma cell lines exhibit retarded growth in the presence of Ca2+ or no sensitivity to Ca2+ at all. In a human breast cancer cell line, which is sensitive to Ca 2+, the administration of calcium channel antagonists lowered intracellular Ca2+ and inhibited cell proliferation. A sustained physiological elevation of intracellular calcium ion concentration (Ca2+i) may be responsible for a loss of proliferative potential in neoplasmic keratinocytes. It appears from preliminary evidence that Ca2+ is not only important to cell cycling and growth in normal cells, but the abnormal regulation of Ca2+ may also contribute to changes in these processes in disease conditions like cancer.

S100A7L2–S100A7L4 are present at other genomic locations (21q22, xp22, 4p16, 5q14, and 1q22, respectively). Their gene structure is highly conserved, in general comprising three exons and two introns, of which the first exon is noncoding. The S100 family is a remarkable group of proteins that acquired highly specialized functions during their evolution, even though they are small proteins (9–13 kDa acidic proteins) with a single functional domain. S100 proteins, which exhibit dramatic changes in the expression, are involved in tumor progression and include S100A1, S100A4, S100A6, S100A7, and S100B (11–14), whereas S100A2 has been postulated to be a tumor suppressor (Table 1). Such changes might be caused by rearrangements and deletions in chromosomal region, which are frequently observed in tumor cells. Although in most cases, the function of S100 proteins in cancer cells is still unknown, the specific expression patterns of these proteins can be used as a valuable prognostic tool. The other members of EF-hand motif Ca2+binding family involved in cancer are calcineurin, recoverin, calretinin, oncomodulin, etc. (Table 2).

EF-Hand Motif Calcium-Binding Proteins The EF-hand protein structural motif was first discovered in the crystal structure of parvalbumin. It consists of two alpha helices positioned roughly perpendicular to one another and linked by a short loop region (usually about 12 amino acids) that often binds calcium ions. A consensus amino acid sequence for this motif has aided the identification of new members of this family that now has over 200 members. A few of these proteins are present in all cells, whereas the vast majorities are expressed in a tissue-specific fashion. Some members, like S100 family, calcineurin, calmodulin, etc., have proved to be useful therapeutic markers for a variety of cancers. S100 family: The S100 (“Soluble in 100% saturated solution with ammonium sulfate”) family, the largest family within the EF-hand protein, comprises at least 26 members, 19 of which (S100A1–16, profillagrin, trychohyalin, and repetin) are located in the epidermal differentiation complex situated at 1q21, while S100B, S100G, S100P, S100Z, and

Annexins and Other Non-EF-Hand Motif Proteins Annexin (AnxA1) is a Ca2+-binding and acidic phospholipid binding protein with antiinflammatory properties. AnxA1 has been found in leukocytes, tissue macrophages, T-lymphocytes, and epithelial cells of the respiratory and urinary systems. Cellular functions of AnxA1 include regulation of membrane trafficking, cellular adhesion, cell signaling, and membrane fusion in exocytosis and endocytosis. The AnxA1 protein is involved in maintaining normal breast biology. The AnxA1 gene expression may provide data about the future therapeutic plan of breast carcinomas. The decreased expression of AnxA1 gene in normal histological sections of breast may warn the clinician that a malignant version of the cancer is about to form from the benign gland. This observation carries an important prognostic clinical value on microscopic reading of the surgical specimen, especially if these normal glands are adjacent to surgical margins. Similar result was reported as a prognostic

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Calcium-Binding Proteins

Calcium-Binding Proteins, Table 1 S100 proteins involved in cancer with characteristic features S100 protein S100A2

Previous name CAN19, S100L

S100A3

S100E

S100A4

CAPL, Calvasculin, MTS1, Metastasin, p9Ka, FSP1

S100A5

S100D

S100A6

CABP, CACY, Calcyclin, MLN 4, PRA, Prolactin receptorassociated protein

S100A7

PSOR1, Psoriasin

Cancer type Esophageal SCC Thyroid Oral SCC Laryngeal SCC Melanoma Skin tumors (other) NSCLC Lung SCC Gastric Lymphoma Prostate Ovarian Breast Astrocytomas Thyroid carcinoma NSCLC Colorectal Gastric Prostate Breast Gallbladder Bladder Pancreas Esophageal SCC Melanoma Oral SCC Meningioma Thyroid Pancreas Breast Lung Melanoma Colorectal Breast Esophageal SCC Bladder SCC Melanoma Skin SCC Gastric

Characteristic features Function in carcinogenesis is dependent upon the context of tissue and associated tumor type as well as stage of malignancy

Marker of patient prognosis

Level of S100A3 protein expression identified pilocytic astrocytomas Stimulate angiogenesis Overexpression is associated with many different cancer types Poor patient prognosis in breast, colorectal, NSCLC, and bladder cancers

Expression can be associated with prognostic value in recurrence of meningiomas Upregulation of S100A6 appears to be an early event in progression towards pancreatic cancer

Potentially act as a predictor of clinical outcome Expression is restricted to keratinocytes and breast epithelial cells

Overexpression has a role in early breast tumor progression

(continued)

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Calcium-Binding Proteins, Table 1 (continued) S100 protein S100A8

Previous name Calgranulin A, MRP-8, MIF, NIF, P8, CFAG, CGLA, CP-10, Calprotectin

S100A9

Calgranulin B, MRP-14, P14, Calprotectin

S100A10

CAL1L, CLP11, GP11, p10, ANX2LG

S100A11

Calgizzarin, MLN70, S100C

S100A12 S100A14

Calgranulin C, MRP6, p6, CAAF1, ENRAGE BCMP84, S100A15, S114

S100A16

S100F, DT1P1A7

S100B

S100, S100 protein (beta chain), NEF S100E, MIG9

S100P

Cancer type Prostate Breast Esophageal SCC HNSCC Gastric Pancreas Bladder TCC Endometrial Ovarian Colorectal Prostate Breast HNSCC Esophageal SCC Liver (hepatocellular) Gastric Lung Ovarian NSCLC Gastric Renal cell carcinoma Lymphoma Breast Bladder Prostate Thyroid Lymphoma Gastric Colon Esophageal SCC Esophageal SCC Circulating tumor cells Circulating tumor cells Melanoma NSCLC Pancreas Prostate Breast Colon

Characteristic features Upregulated in PIN and in prostatic adenocarcinomas

C Early involvement of the proteins in prostate cancer

Upregulated in PIN and in prostatic adenocarcinomas

Annexin 2 protein ligand Overexpressed in human renal cell carcinoma

Downregulated at transcriptional level in malignant bladder cells Expression may be involved in tumor suppression and better prognosis Cytoplasmic staining pattern in papillary carcinomas Downregulated Used for CTC monitoring in peripheral blood

Used for CTC monitoring in peripheral blood Putative cancer biomarker Isolated from placenta Upregulation is early event in pancreatic cancer valuable marker for the prediction of clinically relevant early pancreatic lesions

SCC squamous cell carcinoma, HNSCC head and neck SCC, NSCLC nonsmall cell lung carcinoma, TCC transitional cell carcinoma, CTCC circulating tumor cells

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Calcium-Binding Proteins

Calcium-Binding Proteins, Table 2 Other EF-hand motif family members EF-hand motif protein Recoverin

Calretinin

Cancer type Cancerassociated retinopathy Colon adenocarcinoma and mesothelioma of epithelial type

Sorcin

Ovarian carcinoma

Calcineurin B

Squamous cell carcinoma of cervix, pancreatic cancer

Oncomodulin/ parvalbumin

Carcinoma cell lines characterized by translocative activity

Calmodulin

Osteoclast apoptosis

Characteristic features Autoimmune response Belongs to the calbindin subgroup, autoantigen in a paraneoplastic disease Overexpression leads to paclitaxel resistance Calcineurin B subunit appears to be a significant biological response modifier due to its anticancer effects Related to motile behavior of carcinoma cells, can be a possible candidate for tumor marker Calcium ion receptor in neoplastic cells

factor with downregulation of AnxA1 and other Anxs in the development of the lethal prostatic carcinoma phenotype. The other major protein that belongs to this category is clusterin (CLU). CLU is a disulfidelinked heterodimeric protein associated with the clearance of cellular debris and apoptosis. In prostate, breast, and colorectal cancers, the CLU was found to have anti- or proapoptotic activity regulated by calcium homeostasis. Reports so far suggest “two faces” of CLU activity: the calcium-dependent cytoplasmic localization of CLU positively correlates with cell survival, whereas nuclear translocation of this protein promotes cell death in calcium-deprived cells. The cytoplasmic retention and high level of the 50-kDa CLU protect tumor cells from apoptotic stimuli induced by chemotherapeutic drugs or natural ligands, such as FasL, whereas its nuclear

localization (nCLU) enhances cell apoptosis. The 50-kDa CLU isoform is mainly overexpressed in cancer cells and retained in the cytoplasm, promoting cancer progression and aggressiveness. Cytoplasmic CLU could easily translocate into the nucleus in the presence of various inducers, such as IR, chemotherapy, hormones, or cytokines, or depletion of cellular calcium. These findings support CLU as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer. Calreticulin-Like Proteins Calreticulin is a 46-kDa Ca2+-binding chaperone protein found across a diverse range of species. The human gene for calreticulin is located on chromosome 19 at locus p13.3–p13.2 and the homologous gene in the mouse maps to chromosome 8. Calreticulin at the cell surface may play a role in cell adhesion, cell–cell communication, and apoptosis. Calreticulin has also been implicated in the pathology of some cancers. The protein also plays an important role in autoimmunity and cancer. For example, it appears that calreticulin might be an excellent molecular marker for prostate cancer. The expression of calreticulin is downregulated in metastatic melanoma and ▶ squamous cell carcinoma, whereas significantly upregulated in colon cancer. Further, the N-domain of the protein has been reported to have inhibitory effects on tumors and to inhibit ▶ angiogenesis on endothelial cells. This observation is of great interest because the development of angiogenesis inhibitors is currently a highly promising approach in anticancer therapy.

Cross-References ▶ Angiogenesis ▶ Apoptosis ▶ Calreticulin ▶ Cancer ▶ Squamous Cell Carcinoma

Calpain

References Donato R (2003) Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 60:540–551 Kretsinger RH, Tolbert D, Nakayama S et al (1991) The EF-hand, homologs and analogs. In: Heizmann CW (ed) Novel calcium-binding proteins: fundamentals and clinical implications. Springer, New York, pp 17–37 Pfyffer GE, Haemmerlit G, Heizmann CW (1984) Calcium-binding proteins in human carcinoma cell lines. Proc Natl Acad Sci U S A 81:6632–6636 Pfyffer GE, Humbel B, Strauli P et al (1987) Calciumbinding proteins in carcinoma, neuroblastoma and glioma cell lines. Virchows Arch 412:135–144

See Also (2012) EF-Hand Proteins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1211. doi: 10.1007/978-3-642-16483-5_1819 (2012) Proteins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3099. doi: 10.1007/978-3-642-16483-5_4812

Calcium-Binding Reticuloplasmin of Molecular Weight 55 kDa ▶ Calreticulin

CALI ▶ Chromophore-Assisted Laser Inactivation

CALM

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proteases was named “calpains” to reflect their dependency upon calcium ions for proteolytic activity, and homology to the papain family of cysteine proteases. In mammalian species, the calpain protein family is comprised of 15 members, of which nine are ubiquitously expressed in all tissues and the remainder are expressed in a tissue-specific manner. The ubiquitously expressed calpain 1 and calpain 2 are the most well characterized isoforms. Calpain 1 and calpain 2 function as heterodimeric enzymes composed of a large catalytic subunit (calpain 1 and calpain 2) bound to a small regulatory subunit (calpain 4). Calpain activity in vivo is tightly regulated by the ubiquitously expressed endogenous inhibitor calpastatin. Calpains result in the proteolysis of a broad spectrum of cellular proteins. No unique consensus amino acid sequence has been identified as a calpain-binding or -cleavage site, rather, it appears that calpains target substrates for cleavage by recognition of unidentified tertiary structure motifs. Another distinguishing feature of calpain proteases is their ability to confer limited cleavage of protein substrates into stable fragments, rather than complete proteolytic digestion. Thus, the calpain-calpastatin proteolytic system represents a major pathway of posttranslational modification of proteins that influences various aspects of cellular physiology. The application of pharmacological and molecular intervention strategies against calpain activity demonstrates a broad role for this class of proteases in the control of proliferation, ▶ migration, and ▶ apoptosis in most cell types.

▶ PICALM

Characteristics

Calpain Neil O. Carragher Drug Discovery Group, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh, UK

Definition The calpains represent a unique class of intracellular protein degrading enzymes. This class of

Cell proliferation, migration, and apoptosis are key processes that have to be tightly regulated in order to maintain optimal tissue homeostasis, required for development and viability of multicellular organisms. Deregulation of any of these cellular processes will ultimately result in pathological outcomes, such as cancer. A number of studies have identified a correlative link between modulation of calpain gene expression and/or activity with cancer development and progression in vivo. For example, in human renal cell

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carcinomas, significantly higher levels of calpain 1 expression are found in tumors that metastasized to peripheral lymph nodes relative to tumors that had not metastasized. In addition, elevated calpain activity was detected in breast cancer tissues relative to normal breast tissues and was determined to be greater in estrogen receptor (ER)-positive tumors than ER negative tumors. Calpainmediated proteolysis of the tumor suppressor protein neurofibromatosis type 2 (NF2 or ▶ Merlin) is associated with the development of schwannomas and menigiomas. Experimental studies performed in vitro demonstrate that total cellular calpain activity is elevated upon transformation induced by the v-src, v-jun, v-myc, k-ras, and v-fos oncogenes. Furthermore, calpain activity is necessary for full cellular transformation induced by such oncogenes. A number of intervention studies utilizing small molecule inhibitors or oligonucleotides that impair calpain activity have demonstrated a role for calpain during tumor cell progression in vitro and in vivo. Cell proliferation, migration, and apoptosis are controlled by a plethora of regulatory proteins that participate in complex biochemical signaling cascades, of which calpain is a pivotal regulator. Thus, targeting calpain activity may represent an effective strategy for cancer prevention and/or treatment. Calpain and Cell Proliferation Studies using pharmacological inhibitors of calpain activity, overexpression of calpastatin, and cells expressing depleted levels of calpain activity have all implicated calpain in the promotion of cell proliferation. Sequential progression through G1, S, G2, and M phases of the cell cycle is required for mitosis and cell proliferation. Several studies indicate that calpain can cleave a number of cell cycle control proteins such as ▶ cyclin D, cyclin E, and p27kip1 all of which regulate progression through G1 and S phase. Calpain has also been demonstrated to cleave upstream regulators of cell cycle control proteins such as p53 and p107. Consequently, elevated calpain levels and activity in tumors may contribute to cancer cell proliferation through cleavage of cell cycle control proteins and deregulation of normal cell cycle control.

Calpain

More detailed mechanistic studies also demonstrate that calpain 2 is an important downstream component of many growth factor receptor and non-receptor tyrosine kinase signaling pathways that include signaling kinases such as, ▶ epidermal growth factor receptor (EGFr), ▶ platelet derived growth factor receptor (PDGFr), Src, and ▶ focal adhesion kinase (FAK). Such signaling molecules play an important role in transmitting extracellular signals to intracellular mediators that control cell proliferation and are often constitutively activated in cancer cells. Activation of receptor and non-receptor kinases subsequently leads to activation of the Ras/MAPK pathway, which results in ERK-mediated phosphorylation of calpain 2 on a serine residue (Ser 50). Phosphorylation of calpain 2 on Ser 50 initiates a conformational switch culminating in enhanced proteolytic activity. This evidence, together with pharmacological and molecular intervention studies targeting calpain activity, suggests that activation of calpain, in part, mediates growth factor receptor and non-receptor induced cell proliferation and migration of cancer cells. Calpain and Cell Migration The interaction between cell surface adhesion receptors known as integrins and their extracellular matrix substrates controls the migration of all cells. Integrin-linked focal adhesions are large complexes of structural and signaling proteins that provide both a structural and biochemical link between the extracellular environment and intracellular proteins. Dynamic spatial and temporal regulation of focal adhesion assembly and disassembly is required for optimal cell motility. Several studies indicate that calpains localize to integrin-associated adhesions. Furthermore, many of the protein components of focal adhesions are known substrates of calpain. Calpain-mediated cleavage of the focal adhesion components, FAK, paxillin, talin, and possibly others promotes the disassembly of these complexes, contributing to reduced cell adhesion and increased migration. In fact, calpain-mediated cleavage of talin has been reported to represent the rate-limiting step in adhesion turnover. In addition to mediating focal adhesion turnover, emerging evidence

Calpain

suggests a role for calpain in regulating components of the actin cytoskeleton involved in cell spreading and membrane protrusion, mechanisms that are also essential for persistent and directed cell migration. It is likely that calpain cleavage of actin-binding and actin-regulatory proteins such as, ezrin, rhoA, and cortactin influences the dynamic formation and retraction of membrane structures known as filipodia and lamellipodia thereby influencing cancer cell migration and ▶ invasion. Pharmacological and molecular inhibition of calpain activity has been shown to impair cancer cell migration across experimental two-dimensional substrates and invasion into three-dimensional extracellular matrix substrates in vitro. Furthermore, an intervention study demonstrates that antisense-mediated suppression of calpain 2 gene expression reduced the invasion of prostate cancer cells both in vitro and in a mouse model in vivo. Thus, evidence strongly indicates that calpain activity contributes to the invasion and ▶ metastasis of cancer cells. Calpain and Apoptosis Apoptosis is defined as the process of programmed cell death. Apoptosis often follows activation of the caspase family of cysteine proteases, which degrade numerous proteins that are essential for cell viability. Regulated apoptosis is critical for the development of multicellular organisms and also restricts the growth and spread of malignant cancer cells. Conflicting roles for calpain activity in the promotion and suppression of cell apoptosis have been proposed. Calpain activity has previously been shown to play a pro-apoptotic role through the activation of caspase 3 and caspase 12 and cleavage of Bax and ▶ Bid proteins to their pro-apoptotic forms. Enhanced calpain activity has also been implicated as the major proteolytic pathway resulting in breakdown of essential proteins during caspase-independent mechanisms of apoptosis. Conversely, calpain-mediated cleavage of caspase 7 and caspase 9 has been found to suppress their activity and subsequent apoptosis. In addition, calpain-mediated cleavage of IkBa can lead to activation of the NFkB transcription factor resulting in subsequent expression of anti-

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apoptotic survival proteins. Many chemotherapeutic agents such as cisplatin induce their tumoricidal effect via inducing apoptosis of cancer cells. Tumor cell resistance to cisplatininduced apoptosis is a common feature frequently encountered during chemotherapy of cancer patients. Inhibition of calpain activity has been shown to sensitize resistant tumor cells to cisplatin-induced death, whereas other studies suggest that calpain potentiates cisplatin-induced cell death. Thus, the role of calpain during cell apoptosis is context dependent and determined by cell type, the apoptotic stimuli, and status of intrinsic regulators of cell apoptosis. In contrast to the aforementioned studies suggesting a pro-tumorigenic role for calpain activity, an anti-tumorigenic role is also supported by studies indicating that calpain degrades a number of oncogene-generated protein products such as PDGFr, EGFr, c-Jun, c-Fos, c-Src, and c-Mos. Also, calpain-mediated cleavage of protein kinase C (PKC), a downstream effecter for tumor promoting phorbal esters, inhibits malignant transformation. Furthermore, specifically calpain 9 (nCL-4) activity contributes to the suppression of cell transformation in vitro and gastric tumors in vivo. Although the calpain 9 substrates that mediate this antitumor effect remain to be determined. A substantial body of evidence has accumulated demonstrating that activity of the calpain family of proteases plays a broad and important role in the physiology of both normal and cancer cells. Further investigation into the complex and multifaceted role of calpain in cancer may lead to the discovery of novel therapeutic approaches targeting calpain activity that may impact on the development, progression, and prevention of cancer.

Cross-References ▶ Apoptosis ▶ Cyclin D ▶ Focal Adhesion Kinase ▶ Epidermal Growth Factor Receptor

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▶ Invasion ▶ Merlin ▶ Metastasis ▶ Migration ▶ Platelet-Derived Growth Factor

References Carragher NO, Frame MC (2002) Calpain: a role in cell transformation and migration. Int J Biochem Cell Biol 34:1539–1543 Franco SJ, Huttenlocher A (2005) Regulating cell migration: calpains make the cut. J Cell Sci 118(17):3829–3838 Goll DE, Thompson VF, Li H et al (2003) The calpain system. Physiol Rev 83:731–801

Calreticulin

Calreticulin Yoshito Ihara Department of Biochemistry, School of Medicine, Wakayama Medical University, Wakayama, Japan

Synonyms CaBP3; Calcium-binding reticuloplasmin of molecular weight 55 kDa; Calsequestrin-like protein; CRP55; CRT; ERp60; HACBP; High affinity Ca2+-binding protein; Reticulin

See Also (2012) Calpastatin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 598. doi:10.1007/978-3-642-16483-5_783 (2012) EGFR. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1828 (2012) ERK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1307-1308. doi:10.1007/978-3-642-16483-5_1987 (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1362. doi:10.1007/978-3-642-164835_2067 (2012) Filipodia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1407. doi:10.1007/978-3-642-16483-5_2189 (2012) Focal Adhesion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1436-1437. doi:10.1007/978-3-642-16483-5_2227 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Lamellipodia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1971. doi:10.1007/978-3-642-16483-5_3267 (2012) MAPK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) Papain. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2781. doi:10.1007/978-3-642-16483-5_4366 (2012) Platelet-Derived Growth Factor Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2910. doi:10.1007/9783-642-16483-5_4612 (2012) Proteases. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3080. doi:10.1007/978-3-642-16483-5_4786

Definition Calreticulin (CRT) is a Ca2+-binding multifunctional ▶ molecular chaperone in the endoplasmic reticulum (ER). CRT is a 46-kDa soluble protein with a cleavable N-terminal amino acid signal sequence and the C-terminal sequence Lys-Asp-Glu-Leu (KDEL), a retrieval signal in the ER. Calnexin (CNX), a membranebinding paralog of CRT, shares the ▶ chaperone function in the ER. CRT is expressed in a variety of tissues and organs, but its levels are particularly high in the pancreas, liver, and testis. It is also a highly conserved protein with over 90% amino acid identity in mammals including humans, rabbits, rats, and mice. The CRT gene has been mapped to human chromosome 19 at p13.2, and its expression is up-regulated by ▶ ER stress such as unfolded protein responses and deprivation of Ca2+ in the ER.

Characteristics Structure of CRT Based on structural and functional studies, CRT can be divided into three distinct domains: N-terminal [N], proline-rich [P], and C-terminal [C]. The proline-rich P-domain shows a characteristic structure with an extended and curved arm

Calreticulin

733 N

P

C KDEL

[P] [N]

Amino terminal domain (N-domain) *Chaperone functions *Lectin site

[C] KDEL

Proline-rich domain (P-domain or Arm domain) *Chaperone functions *ERp57 binding *High affinity Ca2+− binding site

Carboxyl terminal domain (C-domain) *Chaperone functions *Low affinity Ca2+− binding site

Calreticulin, Fig. 1 Schematic structure of calreticulin

connected to a globular N-domain. The N-terminal region encompassing the N- and P-domains of CRT interacts with misfolded proteins and glycoproteins, binds ATP, Zn2+, and Ca2 + with high affinity and low capacity, and is likely to be involved in the chaperone function of the protein. The C-domain binds Ca2+ with high capacity and plays a role in the storage of Ca2+ in the ER in vivo, though no structural information is available at present (Fig. 1). Functions of CRT in the Cell CRT is involved in a number of biological processes including the regulation of glycoprotein folding, Ca2+ homeostasis and intracellular signaling, cell adhesion, gene expression, and nuclear transport (Fig. 2).

and the glycoprotein is released from the CNX/CRT chaperone cycle. However, if the glycoprotein is not properly folded, the terminal glucose is once again attached by the action of UDP-Glc: glycoprotein glucosyltransferase, which discriminates between folded and unfolded substrates. Together, CRT and CNX form a specific chaperone cycle for the biosynthesis of glycoproteins in the ER. Because of the preference of CNX/CRT for oligosaccharides as substrates, CNX and CRT are called “lectin-like chaperones.” CRT and CNX function with the help of other chaperones such as ERp57 and BiP/GRp78. The binding site for ERp57 has been identified in the P-domain of CRT or CNX. As a chaperone, CRT plays an important role in the formation of major histocompatibility complex (MHC) class I to aid in antigen presentation.

CRT, a Lectin-Like Molecular Chaperone in the ER

A molecular chaperone function of CRT has been reported for several protein substrates. In the biosynthesis of glycoproteins bearing N-linked glycans in the ER, the oligosaccharide Glc3Man9GlcNAc2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) is attached to the Asn residue contained in the consensus sequence Asn-X-Ser/Thr, of newly synthesized polypeptides. CRT or CNX binds the Glc1Man5–9GlcNAc in glycoproteins after the processing of sugar chains. The N-domain of CRT and CNX is speculated to be the oligosaccharide-binding site (lectin site). If the glycoprotein is completely folded in the ER, the terminal glucose is removed by glucosidase-II

CRT, a Regulator of Ca2+ Homeostasis in the ER

The ER is the main reservoir of intracellular Ca2+ and plays an important role in ▶ Ca2+ homeostasis. CRT has two Ca2+-binding sites and this characteristic contributes to the function of the ER as a Ca2+ reservoir. Ca2+ is released from the ER by receptors for inositol-1,4,5-trisphosphate (IP3) and ryanodine, and taken up into the ER by sarcoplasmic and endoplasmic reticulum Ca2+-ATPase (SERCA). With respect to the regulation of the Ca2+ level, the involvement of CRT and SERCA2b or the IP3-receptor has been reported. Furthermore, the store-operated release of Ca2+ from the ER was shown to be suppressed by overexpression of CRT protein. These findings indicate that CRT is not

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Calreticulin

Vasostatin (CRT aa1-180) Ca2+

Antiangiogenesis and tumor suppressor

Phagocytosis Ca2+ homeostasis Tumor immunity

Ca2+

∗Integrin

MHC SERCA

IP3-R Cell signaling

ER quality control

∗Akt/

PKB ∗Calcineurin / NF-AT

E-cadherin Cell adhesion and motility

N-cadherin Vinculin

Gene expression Calreticulin

Cell growth, death and differentiation

Nuclear transport

EMT

Calreticulin, Fig. 2 Functions of calreticulin in the cell. CRT is involved in a variety of cellular processes, including the quality control of glycoprotein synthesis in the ER, Ca2+ homeostasis, intracellular signaling, gene expression, and nuclear transport. In cancer cells, the altered

expression of CRT may lead to alterations in cellular characteristics, such as growth, adhesion, motility, immune responses, and susceptibility to apoptosis. Furthermore, extracellular CRT fragments (i.e., vasostatin) elicit antiangiogenic or tumor-suppressing activities

only a reservoir of Ca2+ but also a regulator of Ca2 + -homeostasis in the ER.

in 40% of patients with systemic lupus erythematosus, patients with secondary Sjogrens syndrome, rheumatoid arthritis, celiac disease, complete congenital heart block, and halothane hepatitis. CRT is known to bind to complement, C1q, and compete with antibodies for binding to C1q and inhibition of C1q-dependent hemolysis. In autoimmune diseases, impairment of the classical pathway of compliment causes a failure to clear immune complex, resulting in progression of the disease. Therefore, extracellular CRT may contribute to the progression of autoimmune diseases by preventing the clearance of immune complex. Furthermore, it has been reported that cell surface CRT is involved in the mechanism for clearance of viable or apoptotic cells through the transactivation of LDL-receptor-related protein (LRP) on phagocytes. However, it is still controversial whether CRT is exported from necrotic cells or apoptotic cells under pathologic conditions.

Other Miscellaneous Functions of CRT In and Out of the ER

CRT is involved in cell ▶ adhesion by affecting integrin-related cell signaling. In CRT-deficient embryonic stem cells, integrin-mediated Ca2+ influx was impaired leading to a decrease in cell adhesion to fibronectin and laminin. It is still not clear whether CRT affects integrin directly or indirectly to regulate cell adhesion signaling. Cell surface expression of CRT has also been reported in various cell types and may be related with cell adhesion and ▶ migration. The cell surface CRT may modulate cell adhesion by binding with extracellular matrix proteins, such as ▶ fibrinogen, ▶ laminin, and ▶ thrombospondin. Furthermore, extracellular CRT is implicated in the pathological processes of autoimmune diseases. ▶ Autoantibodies against CRT were found

Calreticulin

Cytosolic CRT functions as an export factor for multiple nuclear hormone receptors, such as steroid hormone, nonsteroid hormone, and orphan receptors. This function is consistent with previous findings that CRT suppresses the transactivation of nuclear hormone receptors including ▶ androgen receptor and vitamin D. However, the mechanisms by which CRT molecules are transported into, and retained in, the cytosol/nucleus are not fully defined. CRT and Development CRT is essential for cardiac and neural development in mice. CRT-deficient embryonic cells showed an impaired nuclear import of nuclear factor of activated T cell (NF-AT3), a transcription factor, indicating that CRT functions in cardiac development as a component of the Ca2+/ calcineurin/NF-AT/GATA-4 transcription pathway. Actually, cardiac-specific expression of calcineurin reversed the embryonic lethality of CRT-deficient mouse. CRT transgenic mice suffer a complete heart block and sudden death, and CRT-dependent cardiac block involves an impairment of both the L-type Ca2+ channel and gap junction ▶ connexins (Cx40 and Cx43). Phosphorylated Cx43 was also decreased in CRT transgenic heart, suggesting that the functions of protein kinases are altered via the regulation of Ca2+ homeostasis. Collectively, CRT plays a vital role in cardiac differentiation and function, though how has not been fully clarified. CRT and Cancer Expression of CRT in Cancer

In terms of the relationship between CRT and cancer, proteomic analysis has revealed a new functional role of CRT in the early diagnosis of cancers. CRT is proposed to be a new tumor marker of bladder cancer. In addition, it was reported that the expression of CRT is up-regulated in a variety of malignant cells or tissues including progressive fibrosarcoma cells, colorectal cancer cells, and pituitary adenomas. Furthermore, autoantibodies to CRT isoforms have utility for the early diagnosis of pancreatic

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cancer. These reactions are not indicative of malignant properties of CRT, but rather are markers of immunogenicity and anticancer responses. On the other hand, another report demonstrated that CRT is overexpressed in the nuclear matrix in ▶ hepatocellular carcinoma, compared with normal liver tissue, suggesting a relationship between overexpressed CRT and malignant transformation. In contrast, it was also reported that CRT expression correlates with the differentiation of ▶ neuroblastomas to predict favorable patient survival. Pathophysiological Relevance of CRT in Malignant Disease

Susceptibility to ▶ apoptosis is important in terms of cancer treatments including the use of antibiotics and irradiation. In embryonic fibroblasts from CRT knockout mice, susceptibility to apoptosis was significantly suppressed, indicating that CRT functions in the regulation of apoptosis. Furthermore, it was found that overexpression of CRT modulates the ▶ radiation sensitivity of human glioma U251MG cells by suppressing Akt/protein kinase B signaling for cell survival via alterations of cellular Ca2+ homeostasis. These findings suggest that the expression level of CRT is well correlated with the susceptibility to apoptosis. In contrast, overexpression of CRT provides resistance to oxidant-induced cells death in renal epithelial LLC-PK1 cells. The function of CRT in the regulation of apoptosis may differ in specific cell types and is still controversial. As for cell adhesion, it was reported that CRT expression modulates cell adhesion by coordinating up-regulation of N-cadherin and vinculin. It has been reported that overexpression of CRT induces ▶ epithelial-mesenchymal transition (EMT)-like morphological changes and enhances cellular invasiveness in renal epithelial MDCK cells. The enhanced invasiveness mediated through ▶ E-cadherin gene repression was regulated by the gene repressor, Slug, via altered Ca2+ homeostasis caused by overexpression of CRT in MDCK cells. This study suggests that expression of CRT may play some causative role in the gain of invasiveness during the process of malignant transformation. In addition, it has been reported

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that cellular migration and binding to collagen type V are apparently suppressed in embryonic fibroblasts from CRT knockout mice, indicating that the cellular level of CRT is important for the regulation of cell motility. Furthermore, CRT protein binds to GCN repeats in mRNA of the myeloid transcription factor CCAAT/enhancer-binding protein a (CEBPA), and thereby impedes translation of the CEBPA mRNA, suggesting that CRT plays a functional role in the differentiation block in ▶ acute myeloid leukemia through suppression of CEBPA by the leukemic ▶ fusion gene AML1-MDS1-EVl1. Together, these findings suggest that CRT is involved in the regulation of cancer characteristics, although the overall mechanisms are still not clear.

Calsequestrin-Like Protein

References Eggleton P, Michalak M (2003) Introduction to calreticulin. In: Eggleton P, Michalak M (eds) Calreticulin, 2nd edn. Landes Biosciences/Eurekah. com/Kluwer/Plenum Publishers, Georgetown/New York, pp 1–8 Gelebart P, Opas M, Michalak M (2005) Calreticulin, a Ca -binding chaperone of the endoplasmic reticulum. Int J Biochem Cell Biol 37:260–266 Johnson S, Michalak M, Opas M et al (2001) The ins and outs of calreticulin: from the ER lumen to the extracellular space. Trends Cell Biol 11:122–129 Michalak M, Corbett EF, Mesaeli N et al (1999) Calreticulin: one protein, one gene, many functions. Biochem J 344:281–292 Williams DB (2006) Beyond lectins: the calnexin/ calreticulin chaperone system of the endoplasmic reticulum. J Cell Sci 119:615–623

CRT as a Tool for Cancer Therapy

CRT can form complexes with peptides in vitro to elicit peptide-specific CD8+ ▶ T cell responses. In addition, peptide-bound CRT purified from tumor extracts elicits an antitumor effect specific to the source tumor. Antigen-specific cancer immunotherapy is an attractive approach to the eradication of systemic tumors at multiple sites in the body. It has been reported that vaccination with DNA encoding chimera for CRT and a tumor antigen, human papilloma virus type-16 (HPV-16) E7 [CRT/E7], resulted in a significant reduction in the number of lung tumor nodules in immunocompromised mice. All together, the use of CRT represents a feasible approach for enhancing tumor-specific T cell-mediated immune responses. Therapeutic agents that target the tumor vasculature may prevent or delay tumor growth and even promote tumor regression or dormancy. As another approach to cancer therapy, CRT or a fragment thereof (amino acids 1–180) (i.e., vasostatin) inhibits ▶ angiogenesis and suppresses tumor growth. The combination of vasostatin and IL-12 as well as vasostatin and interferon-inducible protein-10 had a suppressing effect on the cell growth of Burkitt lymphoma and colon carcinoma in mouse metastasis models. Although this suggests some potential for use in cancer therapy, the molecular mechanism of CRT actions at the cell surface is not fully understood.

Calsequestrin-Like Protein ▶ Calreticulin

CAM ▶ Chorioallantoic Membrane

Campto

®

▶ Irinotecan

Camptosar

®

▶ Irinotecan

CAMs ▶ Cell Adhesion Molecules

CAMTA1

CAMTA1 Kai-Oliver Henrich and Frank Westermann DKFZ, German Cancer Research Center, Heidelberg, Germany

Definition CAMTA1 is a candidate ▶ tumor suppressor gene encoding a member of a protein family designated as calmodulin-binding transcription activators (CAMTAs). It resides within a distal portion of chromosomal arm 1p that is frequently deleted in a wide range of human malignancies.

Characteristics CAMTA1 maps to 1p36.31-p36.23 and its 23 exons are spread over 982.5 kb. The 6,582 bp cDNA encodes a protein of 1,673 amino acids. The primary structure of protein contains a nuclear localization signal, two DNA-binding domains (CG-1 and TIG), a transcription activation domain, calmodulin binding motifs (IQ motifs), and ankyrin domains. Although the expression of CAMTA1 is seen in various organs, the highest levels are found in neuronal tissues. Information on the physiologic roles of CAMTAs is scarce and most data derive from plant and drosophila studies. CAMTAs are transcription factors that typically bind to CGCG boxes via their CG-1 domain. An alternative mechanism of transcriptional activation has been described for CAMTA2, the second human CAMTA homolog. It acts as a coactivator of another transcription factor, Nkx2-5, to stimulate gene expression. This function is inhibited by binding of class II histone deacetylases to the ankyrin-repeat region of CAMTA2. Upstream signaling components can activate CAMTA2 by promoting the export of class II histone deacetylases to the cytoplasm, relieving their repressive influence on CAMTA2. The sole fly homolog of CAMTA1 induces the expression of an F-box gene, the product of which

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inhibits a Ca2+-stimulating G-protein-coupled receptor (GPCR). The controlled deactivation of Ca2+-stimulating GPCRs is needed to tune Ca2+mediated signaling and prevents abnormal cell proliferation. As CAMTA activity is increased by the Ca2+-sensor calmodulin, the Ca2+/ calmodulin/CAMTA/F-box protein pathway may mediate a negative feedback loop controlling the activity of Ca2+-stimulating GPCRs. This regulatory loop is of special interest taking into account the fundamental links between GPCR-mediated pathways and cancer biology. Clinical Relevance Deletions within 1p occur in various types of human malignancies, ranging from virtually all types of solid cancers to leukemias and myeloproliferative disorders. Functional evidence for a role of 1p in tumor suppression derives from experiments in which the introduction of 1p chromosomal material into ▶ neuroblastoma cells resulted in reduced tumorigenicity. In neuroblastoma and other cancers, deletion of 1p36 is a predictor of poor patient outcome. Therefore, it is widely assumed that distal 1p harbors a gene (or genes) with tumor suppressive properties. To define the DNA, deleted from 1p, more precisely in pursuit of identifying the gene(s) of interest, substantial mapping efforts have been undertaken with the most detailed picture being worked out for neuroblastoma. In this tumor entity, the combination of loss of heterozygosity (LOH) fine mapping studies allowed to considerably narrow down a smallest region of consistent deletion spanning only 261 kb at 1p36.3 and pinpointing the CAMTA1 locus. Sequence analysis revealed no evidence for somatic mutations in the remaining CAMTA1 copy of neuroblastomas with 1p deletion. However, a rare sequence variant leading to amino acid substitution within the ankyrin domain was seen in a subgroup of neuroblastomas. More importantly, low CAMTA1 expression is significantly associated with markers of unfavorable tumor biology and is itself a marker of poor neuroblastoma patient outcome. Moreover, CAMTA1 expression is a neuroblastoma predictor variable that is independent of the established molecular markers including 1p

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Cancer

deletion. Thus, the measurement of this variable should allow an additional biological stratification of neuroblastomas and help to assign patients to the appropriate therapy. Additional evidence for a role of CAMTA1 in tumor development comes from glioma and colon cancer in which 1p is frequently deleted. In glioma, a 1p minimal deleted region spans 150 kb and resides entirely within CAMTA1. In colorectal cancer, a genome-wide analysis of genomic alterations revealed that loss of a 2 Mb recurrently deleted genomic region encompassing CAMTA1 has the strongest impact on survival when compared with other genomic changes. Furthermore, as in neuroblastoma, the low expression of CAMTA1 is an independent marker of poor patient outcome. The high prevalence of CAMTA1 deletion in neuroblastoma, glioma, and colorectal cancer together with the independent predictive power of low CAMTA1 expression for neuroblastoma and colorectal cancer outcome is consistent with the idea that low CAMTA1 levels mediate a selective advantage for developing tumor cells.

Kim MY, Yim SH, Kwon MS et al (2006) Recurrent genomic alterations with impact on survival in colorectal cancer identified by genome-wide array comparative genomic hybridization. Gastroenterology 131(6):1913–1924

Cross-References

Definition

▶ Neuroblastoma ▶ Tumor Suppressor Genes ▶ Ubiquitin Ligase SCF-Skp2

Cancer is a deregulated multiplication of cells with the consequence of an abnormal increase of the cell number in particular organs. Initial stages of the developing cancer are usually confined to the organ of origin whereas advanced cancers grow beyond the tissue of origin. Advanced cancers invade the surrounding tissues that are initially connected to the primary cancer. At a later stage, they are distributed via the hematopoetic and lymphatic systems throughout the body where they can colonize in distant tissues and form ▶ metastasis. The development of cancers is thought to result from the damage of the cellular genome, either due to random endogenous mechanisms or due to environmental influences. The origin of cancers can be traced back to alterations of cellular genes. Genetic damage can be of different sorts:

References Barbashina V, Salazar P, Holland EC et al (2005) Allelic losses at 1p36 and 19q13 in gliomas: correlation with histologic classification, definition of a 150-kb minimal deleted region on 1p36, and evaluation of CAMTA1 as a candidate tumor suppressor gene. Clin Cancer Res 11(3):1119–1128 Bouche N, Scharlat A, Snedden W et al (2002) A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277(24):21851–21861 Henrich KO, Fischer M, Mertens D et al (2006) Reduced expression of CAMTA1 correlates with adverse outcome in neuroblastoma patients. Clin Cancer Res 12(1):131–138 Henrich KO, Claas A, Praml C et al (2007) Allelic variants of CAMTA1 and FLJ10737 within a commonly deleted region at 1p36 in neuroblastoma. Eur J Cancer 43(3):607–616

See Also (2012) Glioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1557. doi:10.1007/978-3-642-16483-5_2423 (2012) G-protein Couple Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1587. doi:10.1007/978-3-642-16483-5_2294 (2012) Loss of Heterozygosity. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2075-2076. doi:10.1007/978-3-642-164835_3415

Cancer Manfred Schwab German Cancer Research Center (DKFZ), Heidelberg, Germany

• Recessive mutations in ▶ tumor suppressor genes

Cancer

• Dominant mutations of ▶ oncogenes • Loss-of-function mutations in genes, involved in maintaining genomic stability and ▶ repair of DNA (resulting in ▶ genomic instability)

History Human cancer is probably as old as the human race. It is obvious that cancer did not suddenly start appearing after modernization or industrial revolution. The world’s oldest documented case of cancer comes from ancient Egypt, in 1500 BC. The details were recorded on a papyrus, documenting eight cases of tumors occurring on the breast. It was treated by cauterization, a method to destroy tissue with a hot instrument called “the fire drill.” It was also recorded that there was no treatment for the disease, only palliative treatment. The word cancer came from the father of medicine, Hippocrates, a Greek physician (460–370 BC). Hippocrates used the Greek words, carcinos and carcinoma to describe tumors, thus calling cancer “karkinos.” The Greek terms actually were words to describe a crab, which Hippocrates thought a tumor resembled. Hippocrates believed that the body was composed of four fluids: blood, phlegm, yellow bile, and black bile. He believed that an excess of black bile in any given site in the body caused cancer. This was the general thought of the cause of cancer for the next 1,400 years. Autopsies done by Harvey in 1628 paved the way to learning more about human anatomy and physiology. By about the same time period, Gaspare Aselli discovered the lymphatic system, and this led to the end of the old theory of black bile as the cause of cancer. The new theory suggested that abnormalities in the lymph and lymphatic system as the primary cause of cancer. The lymph theory replaced Hippocrates’ black bile theory on the cause of cancer. The discovery of the lymph system gave new insight to what may cause cancer, it was believed that abnormalities in the lymphatic system were the cause. Other theories surfaced, such as cancer being caused by trauma, or by parasites, and it was thought that cancer may spread “like a liquid” (Bentekoe, 1687, Heinrich Vierling, “personal

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communication”). The belief that cancer was composed of fermenting and degenerating lymph fluid was predominant. The discovery of the microscope by Leeuwenhoek in the late seventeenth century added momentum to the quest for the cause of cancer. By late nineteenth century, with the development of better microscopes to study cancer tissues, scientists gained more knowledge about the cancer process. It wasn’t until the late nineteenth century that Rudolph Virchow, the founder of cellular pathology, recognized that cells, even cancerous cells, derived from other cells. The early twentieth century saw great progress in our understanding of microscopic structure and functioning of the living cells. Researchers pursued different theories to the origin of cancer, subjecting their hypotheses to systematic research and experimentation. John Hill first recognized an environmental cause from the dangers of tobacco use in 1761 and published a book “Cautions Against the Immoderate Use of Snuff.” Percivall Pott of London in 1775 described an occupational cancer of the scrotum in chimney sweeps caused by soot collecting under their scrotum. This led to identification of a number of occupational carcinogenic exposures and public health measures to reduce cancer risk. This was the beginning of understanding that there may be an environmental cause to certain cancers. A virus causing cancer in chickens was identified in 1911 (Rous sarcoma virus). Existence of many chemical and physical carcinogens was conclusively identified during the later part of the twentieth century. The later part of the twentieth century showed tremendous improvement in our understanding of the cellular mechanisms related to cell growth and division. The identification of ▶ transduction of oncogenes with the discovery of the ▶ SRC gene, the transforming gene of Rous sarcoma virus, led to formulating the oncogene concept of tumorigenesis and can be viewed as the birth of modern molecular understanding of cancer development. Subsequently, tumor suppressor genes were identified. Many genes that suppress or activate the cell growth and division are known to date, their number is ever growing. It is conceivable that in the end the

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confusing situation may arise to recognize that all genes of the human genome, in one way or another, take part in signaling normal or cancerous cellular growth.

Characteristics A large proportion of genetic changes appears to arise by mechanisms endogenous to the cell, such as by errors occurring during the replication of the ~3  109 base pairs present in the human genome. Environmental factors have a major role as well, predominantly as: • Chemical carcinogens (e.g. aflatoxin B1 in liver cancer) (▶ hepatocellular carcinoma molecular biology), tobacco smoke in ▶ lung cancer; (▶ tobacco carcinogenesis) • Radiation (▶ radiation carcinogenesis) • Viruses (such as ▶ hepatitis B virus in liver cancer, or human papillomavirus in ▶ cervical cancers) Types of Genetic Damage Damage to oncogenes and tumor suppressor genes can be of different sorts: • Point mutations resulting in the activation of a latent oncogenic potential of a cellular gene (e.g. RAS) or in the functional inactivation of a tumor suppressor gene by generating an intragenic stop codon that leads to premature translation termination with the consequence of an incomplete truncated protein (e.g. p53) or the failure for maintaining genomic stability (mismatch repair genes in HNPCC). • ▶ Amplification leading to an increase of the gene copy number beyond the two alleles normally present in the cell (copy number can reach 500 and more; example: MYCN in human ▶ neuroblastoma). • Translocation, which is defined as an illegitimate recombination between nonhomologous chromosomes, the result being either a fusion protein (where recombination occurs between two different genes such as BCR-ABL in chronic myclogenous leukemia) or in the

Cancer

disruption of normal gene regulation (where the regulatory region of a cellular gene is perturbed by the introduction of the distant genetic material such as MYC in Burkitt lymphoma (▶ Epstein-Barr virus)). • Viral insertion by the integration of viral DNA into the regulatory region of a cellular gene. This integration can occur after a virus has infected a cell. Viral insertion is well documented in animal tumors (HBV integration in the vicinity of MYCN in liver cancer in experimental animals; liver cancer, molecular biology). Cellular Aspects Cancer in solid tissues (solid cancer) usually develops over long periods (often 20–30 years latency period) of time. An exception is solid cancers (such as neuroblastoma) in children, which often are diagnosed shortly after birth. Malignant cancers are characterized by their ability to develop metastasis (i.e. secondary cancers at distance from the primary tumor), often they also show multidrug resistance, which means that they hardly react to conventional chemotherapy. It is thought that the development of a normal cell to a metastatic cell is a continuous process driven by genetic damage and genomic instability, with the progressive selection of cells that have acquired a selective advantage within the particular tissue environment (▶ multistep development). Studies of colorectal cancers have identified 6–7 genetic events required for the conversion of a normal cell to a cell with metastatic ability. This is in contrast to leukemias, which usually require one genetic event, most often a translocation, for disease development. Sporadic Versus Familial Cancer The vast majority of cancers are “sporadic,” which simply means that they develop in an individual. Descendants of this individual do not have an increased risk because the cellular changes that have resulted in cancer development are confined to this individual. In contrast, ~10% to 15% of cancer cases have a clearly recognizable hereditary background, they show familial clustering (prominent examples include retinoblastoma

Cancer and Cadmium

(▶ retinoblastoma, cancer genetics), breast cancer, FAP (▶ APC gene in familial adenomatous polyposis) and HNPCC as familial forms of colorectal cancer (colon cancer), ▶ melanoma). This is not to say that “sporadic” cancers never are related to heredity. In fact, it is well possible that an undetermined fraction, if not all, “sporadic” cancers may be related to an individual inherited susceptibility that does not appear as a strong single gene determinant, but rather as a genetic constitution consisting of complex balance of polymorpic genes. Familial cancers have been identified to result from germline mutation of genes. These germ line mutations do not always directly dictate cancer development, although they are considered “strong” hereditary determinants. They represent susceptibility genes that confer a high risk for cancer development to the gene carrier. The relative risk of the individual carrying the mutant gene can vary considerably. For instance, the risk of carriers of one of the breast cancer susceptibility genes BRCA1 or BRCA2 for breast cancer development can vary between approximately 60% and 90%. In reality, this means that the risk for cancer development is difficult to predict, and individuals may not develop cancer at all in spite of the presence of a mutated gene in their germ line. The molecular basis for the differences in risk is unknown. Formally the activity of modifying factors, either environmental or genetic, has been suggested. Such modifying factors appear to be less important for some other familial cancers, such as retinoblastoma, where the risk is constant between 90% and 95% for gene carriers. Polygenic Determinants of Risk The relative risk of the individual for cancer development can also be determined by the so-called weak genetic factors. Normal cells contain a number of genes involved in ▶ detoxification reactions. Different allelic variants of these genes exist in the human population that encodes proteins with slightly different enzymatic activities. Although the exact contribution of individual allelic variants to cancer development is difficult to assess, it is reasonable to assume that individuals that have inherited “weak” enzymatic

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activities in different detoxification systems are likely to have a higher risk. It is likely, therefore, that the risk for such cancers is “polygenic.”

Cross-References ▶ Cohesins ▶ Toxicological Carcinogenesis

Cancer (or Tumor) Stroma ▶ Tumor Microenvironment

Cancer and Cadmium Tim S. Nawrot and Jan A. Staessen Division of Lung Toxicology, Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.), Division of Hypertension and Cardiovascular Rehabilitation, Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium

Definition Cadmium is a metal that has the symbol Cd and atomic number 48 in the periodic table. Cadmium has high toxic effects, an elimination half-life of 10–30 years, and accumulates in the human body, particularly the kidney. Roughly 15,000 t of cadmium is produced worldwide each year for nickel–cadmium batteries, pigments, chemical stabilizers, metal coatings, and alloys.

Characteristics Urinary excretion of cadmium over 24 h is a biomarker of lifetime exposure. Exposure to cadmium occurs through intake of contaminated food

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or water, or by inhalation of tobacco smoke or polluted air. Occupational exposures can be found in industries such as electroplating, welding, smelting, pigment production, and battery manufacturing. Other exposures to cadmium can occur through inhalation of cigarette smoker. Gastrointestinal absorption of cadmium is estimated to be around 5–8%. Inhalation absorption is generally higher, ranging from 15% to 30%. Absorption after inhalation of cadmium fume, such as cigarette smoke, can be as high as 50%. Once absorbed, cadmium is highly bound to the metalbinding protein, metallothionein. Cadmium is stored mainly in the kidneys and also the liver and testes, with a half-life in the body of 10–30 years. In general, nonsmokers have urinary cadmium concentrations of 0.02–0.7 mg/g creatinine, which increase with age in parallel with the accumulation of cadmium in the kidney. Cadmium is a global environmental contaminant. Populations worldwide have a low-level intake through their food, causing an age-related cumulative increase in the body burden of this toxic metal. Environmental exposure levels to cadmium, that are substantially above the background, occur in areas with current or historical industrial contamination, for instance, in regions of Belgium, Sweden, the UK, Japan, and China. As an environmental carcinogen, cadmium could have substantial health implications. Three lines of evidence explain why the International Agency for the Research on Cancer classified cadmium as a human carcinogen. First, as reviewed by Verougstraete and colleagues, several, albeit not all studies in workers, showed a positive association between the risk of lung cancer and occupational exposure to cadmium; discrepancies between these studies should not be ascribed to the better design of the studies. Verougstraete and colleagues suggested that such inconsistencies might be attributed to the high relative risk of cancer in the presence of coexposure to ▶ arsenic, nickel, or toxic fumes and that the increasingly stringent regulations with regard to levels of exposure permissible at work might be a confounding factor (▶ lead

Cancer and Cadmium

exposure, ▶ nickel carcinogenesis). Second, data from rats showed that the pulmonary system is a target site for carcinogenesis after cadmium inhalation. However, exposure to toxic metals in animal studies has usually been much higher than those reported in environmentally exposed humans to toxic metals. Third, several studies done in vitro have shown plausible pathways, such as increased oxidative stress, modified activity of transcription factors, and inhibition of DNA repair. Most errors that arise during DNA replication can be corrected by DNA polymerase proof reading or by postreplication mismatch repair. In fact, inactivation of the DNA repair machinery is an important primary effect, because repair systems are required to deal with the constant DNA damage associated with normal cell functions. The latter mechanism might indeed be relevant for environmental exposure because Jin et al. found that chronic exposure of yeast to environmentally relevant concentrations of cadmium can result in extreme hypermutability. In this study the DNA-mismatch repair system is already inhibited by 28% at cadmium concentrations as low as 5 mM. For example, the prostate of healthy unexposed humans accumulates cadmium to concentrations of 12–28 mM, and human lungs of nonsmokers accumulate cadmium to concentrations of 0.96 mM. Further, in vitro studies provide evidence that cadmium may act like an estrogen, forming high-affinity complexes with estrogen receptors, suggesting a positive role in breast cancer carcinogenesis. Along with this experimental evidence, two epidemiological studies in 2006 gave important positive input into the discussion on the role of exposure to environmental cadmium in the development of cancer in human beings. First, the results of a population-based case–control study noticed a significant twofold increased risk of breast cancer in women in the highest quartile of cadmium exposure compared with those in the lowest quartile. Second, we conducted a population-based prospective cohort study with a median follow-up of 17.2 years in an area close to three zinc smelters. Cadmium concentration in

Cancer Causes and Control

soil ranged from 0.8 to 17.0 mg/kg. At baseline, geometric mean urinary cadmium excretion was 12.3 nmol/day for people in the high-exposure area, compared with 7.7 nmol/day for those in the reference (i.e., low exposure) area. The risk of lung cancer was 3.58 higher than in a reference population from an area with low exposure. 24-h urinary excretion is a biomarker of lifetime exposure to cadmium. The risk for lung cancer was increased by 70% for a doubling of 24-h urinary cadmium excretion. Confounding by coexposure by arsenic could not explain the observed association. Epidemiological studies did not convincingly imply cadmium as a cause of prostate cancer. Of 11 cohort studies, only three (33%) found a positive association. In conclusion, experimental and epidemiological studies strongly suggest environmental exposure to cadmium as a causal factor in the development of cancer of the lung and breast.

References Jarup L, Berglund M, Elinder CG et al (1998) Health effects of cadmium exposure – a review of the literature and a risk estimate. Scand J Work Environ Health 24(Suppl 1):1–51 Jin YH, Clark AB, Slebos RJ et al (2003) Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat Genet 34(3):326–329 McElroy JA, Shafer MM, Trentham-Dietz A et al (2006) Cadmium exposure and breast cancer risk. J Natl Cancer Inst 98(12):869–873 Nawrot T, Plusquin M, Hogervorst J et al (2006) Environmental exposure to cadmium and risk of cancer: a prospective population-based study. Lancet Oncol 7(2):119–126 Verougstraete V, Lison D, Hotz P (2003) Cadmium, lung and prostate cancer: a systematic review of recent epidemiological data. J Toxicol Environ Health B Crit Rev 6(3):227–255

Cancer Antigen 3 ▶ NY-ESO-1

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Cancer Causes and Control Graham A. Colditz Washington University in St. Louis, St. Louis, MO, USA

Synonyms Cancer etiology; Cancer prevention; Prevention

Definition The process of identifying causes of cancer and developing strategies to change cancer risk through health-care providers, regulations that reduce risk, or individual and community level changes.

Characteristics Over six million people around the world die from cancer each year. There is overwhelming evidence that lifestyle factors impact cancer risk and that positive population-wide changes can significantly reduce the cancer burden. Current epidemiologic evidence links behavioral factors to a variety of diseases, including the most common cancers diagnosed in the developed world – ▶ lung cancer, colorectal cancer, ▶ prostate cancer, and ▶ breast cancer. These four cancers account for over 50% of all cancers diagnosed on western countries. As summarized in Fig. 1, tobacco causes some 30% of cancer, lack of physical activity 5%, obesity 15%, diet 10%, alcohol 5%, viral infections 5%, and UV light by excess sun exposure 3%. Because of the tremendous impact of modifiable factors on cancer risk, especially for the most common cancers, it has been estimated that at least 50% of cancer is preventable. Currently in the US not all risk factors are equally distributed across race and social class. Trends in risk factors should also be

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Cancer Causes and Control Cancer causes Tobacco Diet Obesity Inactivity Occupation Viruses Sun Alcohol Family history

Cancer Causes and Control, Fig. 1 Causes of cancer

considered when assessing potential for prevention. To bring about dramatic reductions in cancer incidence, widespread lifestyle changes will be necessary. Rose advocates the need for population approaches for prevention of chronic disease. He emphasizes that when the relation between a lifestyle factor and biological predictor of risk is continuous, the majority of cases attributable to the exposure will likely arise in those who are not classified as being at high risk. He illustrates this with examples of blood pressure and rates of coronary heart disease. Specifically, even small changes in blood pressure at the population level can translate into large reduction in the rates of coronary disease and stroke. To reduce the risk of disease in the population, substantial benefits can be achieved by a small reduction for all members of the society rather than just focusing on the high-risk groups. Because population wide trends in cardiovascular risk factors show continuing improvement, the rate of coronary heart disease incidence and mortality continues to decrease. When we consider population approaches to cancer prevention, we must address the etiologic process, which covers a different time course and sequence from coronary heart disease. Although cardiovascular disease is the end point of the chronic process of atherosclerosis, treatment focuses on the reversal and subsequent prevention of the acute thrombotic process of myocardial infarction. Cancer, on the other hand, is the result of a long process of accumulating DNA damage (▶ multistep development), leading ultimately to

clinically detectable lesions such as in situ and invasive cancer. For example, studies of the progression in ▶ colon cancer from first mutation to invading malignancy suggest that DNA changes accumulate over a period of as long as 40 years. The goal of cancer prevention is to arrest this progression; different interventions interrupt carcinogenesis at different points in the process. Further, most cancers do not have a late “acute” event, analogous to thrombosis, which can be prevented with medical interventions. The benefits of cancer prevention and control programs take time to be observed. The fact that different interventions will impact at different points along the pathway to cancer, that can stretch over nearly half a century, has implications for when we can expect to see pay-off in terms of lower cancer rates. Research has demonstrated that those who initiate smoking during early adolescence greatly increase risk of lung cancer even when one takes into account both the dose and duration of smoking. If we could delay the age at which most adolescents first start to smoke, we would probably substantially reduce lung cancer rates, but this benefit will not be observable for 20–40 years after the intervention. Adult cessation, on the other hand, reduces risk more rapidly, but fails to address the continuing recruitment of the next generation of smokers. Declines in the incidence of lung cancer among younger men and women in the United States reflect reductions in the rate of smoking among younger adults. Other lifestyle interventions may act as preventive early in the DNA pathway to cancer. For example, ▶ aspirin and folate appear to act early in the pathway inhibiting colon cancer. Population-wide prevention strategies for cancer do work. For example, reductions in lung cancer rates in the United States mirror changes in cigarette smoking patters, with marked decreases seen first in young men, then older men, and finally in women. Introduction of the Papaniculou test for cervical cancer in the 1950s was followed by a dramatic decline in cervical cancer in those countries that made widespread screening available. The decline in Australian ▶ melanoma mortality for those born after 1950 is an additional example of effective intervention

Cancer Causes and Control

at the population level. Behavior change is possible and offers great potential for cancer prevention. The recommendations for cancer risk reduction include reducing tobacco use, increasing physical activity, maintaining a healthy weight, improving diet, limiting alcohol, avoiding excess sun exposure, utilizing safer sex practices, and obtaining routine cancer screening tests. Age is the dominant factor that drives cancer risk; for all major malignancies, risk rises markedly with age. The importance of age is exemplified by the fact that the aging U.S. population together with projected population growth will result in a doubling of the total number of cancer cases diagnosed each year by the year 2050, assuming that incidence rates remain constant. With this estimated growth in cancer from 1.3 million to 2.6 million cases per year, it is expected that that both the number and proportion of older persons with cancer will also rise dramatically. Tobacco Tobacco is the major cause of premature death around the world accounting for some 5 M deaths each year. In the United States, adult smokers lose an average of 13 years of life because of smoking, and approximately half of all smokers die of tobacco-related disease. Smoking is well known to cause over 90% of ▶ lung cancers in addition to a range of other malignancies (▶ tobacco carcinogenesis; ▶ tobacco-related cancers). It causes about 30% of all the cancer in the developed world, including lung cancer, mouth cancer, larynx cancer, esophagus cancer, ▶ pancreas cancer, cervix cancer, kidney cancer, and ▶ bladder cancer. Smoking also confers increased risk of colorectal cancer, ▶ gastric cancer, ▶ liver cancer, and prostate cancer, as well as to leukemia. In addition, smoking leads to many other health problems, including heart disease, stroke, lung infections, emphysema, and pregnancy complications. Tobacco may act on multiple stages of carcinogenesis; it delivers a variety of carcinogens, causes irritation and ▶ inflammation, and interferes with the body natural protective barriers. The health risks of tobacco use are not limited to cigarette smoking. Cigar and pipe use increase the

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risk of disease, as does exposure to second-hand smoke and smokeless tobacco use (▶ lung cancer and smoking behavior). Avoiding initiation of tobacco use clearly offers the greatest potential for disease prevention. However, for those who use tobacco products, there are substantial health benefits that come with quitting. There are numerous effective cessation methods, and in the past 25 years, 50% of all living Americans who have ever smoked, have successfully quit. Quitting smoking has immediate and significant health benefits for men and women of all ages. For example, former smokers live longer than individuals who continue smoking. Those who quit before age 50 have approximately half the risk of dying in the next 15 years. This decline in mortality risk is measurable shortly after cessation and continues for at least 10–15 years. Strategies to assist smoking cessation and decreasing youth initiation from both a population and clinical perspective are essential steps to reducing the burden of cancer. Trends Current smoking among US adults has remained steady over the past decade. Once quite pronounced, gender disparity in smoking rates is now relatively small and has been stable since 1990. In 2002, 25.7% of men were current smokers compared to 20.8% of women. Given the profound impact of smoking on cancer, disparities in smoking rates and in access to effective cessation methods will continue to translate directly into differences in the burden of smoking-related cancers. Physical Activity Lack of physical activity causes over 2 M deaths each year around the world. People in the US and in other developed nations are extremely inactive – over 60% of the US adult population does not participate in regular physical activity, which includes 25% of adults who are almost entirely sedentary. Fortunately, the negative effects of a sedentary lifestyle are reversible: increasing one’s level of physical activity, even after years of inactivity, can reduce mortality risk.

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Lack of physical activity increases the risk of colon and breast cancer and likely endometrial cancer, as well as diabetes, osteoporosis, stroke, and coronary heart disease. Overall, sedentary lifestyles have been linked to 5% of deaths from cancer. Among both men and women, high levels of physical activity may decrease the risk of colon cancer by as much as half. Using a variety of measures of activity, studies have consistently shown higher physical activity lowers risk of colon cancer. Physical activity also appears to lower the risk of large adenomatous polyps, precursor lesions for colon cancer, suggesting that it may influence the early stages of the adenomacarcinoma sequence. In addition, the relationship between physical activity and breast and colon cancer are seen across levels of obesity, indicating that physical activity and obesity have separate or independent effects on cancer incidence. Growing evidence suggests that physical activity may also be protective against lung and prostate cancer. Several mechanisms have been proposed to explain these associations. Physical activity reduces circulating levels of insulin, a growth factor for colonic epithelial cells. Additionally, it is postulated that cancer risk is reduced through alterations in prostaglandin levels, improvement in immune function, and modification of bile acid metabolism. Potential mechanisms for the reduction of breast cancer risk include physical activity’s lowering of the cumulative lifetime exposure to circulating estrogens and improving immune pathways. The benefits of physical activity include the prevention of cancer and a large number of other chronic diseases. Increasing levels of physical activity, even after years of inactivity, reduces mortality risk. As little as 30 min of moderate physical activity (such as brisk walking) per day significantly reduces disease risk. Trends One major determinant of activity level that has changed over time is the amount of activity required for work and daily living. With advances in technology and the development of labor-saving devices, there is now a greatly reduced need for

Cancer Causes and Control

physical activity for transportation, household tasks, and occupational requirements. Overall, the prevalence of physical inactivity in the United States is remarkably high; in 1996, about 28% of Americans reported absolutely no participation in leisure-time physical activity. In addition, physical activity in schools has declined, and almost half of young Americans between the ages of 12–21 are not vigorously active on a routine basis. Given the trends in our society, it is unlikely that this decreasing energy expenditure will reverse rapidly. Accordingly, the burden of cancer due to lack of physical activity will increase in the years ahead unless new strategies to promote activity are rapidly implemented. Weight Control and Obesity Prevention Overweight and obesity (▶ Obesity and Cancer Risk) is increasing at epidemic rates in the United States, around the world, and is estimated to account for 2.6 M deaths each year. Currently almost 65% of American adults are overweight (body mass index (BMI) 25 kg/m2), and over 30% are considered obese (BMI  25 kg/m2). Overweight and obesity cause a variety of cancers; colorectal cancer, postmenopausal ▶ breast cancer, ▶ endometrial cancer, ▶ renal cancer, and ▶ esophageal cancer. The proportion of cancer caused by obesity ranges from 9% for postmenopausal breast cancer to 39% for endometrial cancer. One large US study suggested that obesity influences an even broader range of cancers, increasing the risk of death from cancers of the colon and rectum, prostate, breast, esophagus, liver, gallbladder, pancreas, kidney, stomach, uterus, and cervix in addition to non-Hodgkin lymphoma and multiple myeloma. Overall, obesity causes 14% of cancer deaths among men and 20% of cancer deaths among women. Excess body fat may act by altering levels of hormones and tumor growth factors. It is clear that excess weight has severe health consequences. In addition to raising the risk of cancer, overweight and obesity also increase the risk of a multitude of other diseases and chronic conditions, such as stroke, cardiovascular disease, type 2 diabetes, osteoarthritis, and pregnancy complications.

Cancer Causes and Control

The International Agency for Research on Cancer has proposed a comprehensive set of recommendations to address the issue of weight control at multiple levels, including steps by health care providers, regulatory approaches to create adequate access to safe places for exercise (including school, worksite, and community), and family and community level actions. Trends In the USA, the prevalence of overweight and obesity has increased so dramatically and so rapidly, it is frequently referred to as an obesity epidemic. The trend is also being seen among children and adolescents. This epidemic has affected people of all ages, races, ethnicities, socioeconomic levels, and geographic regions. Given limited long-term success in weight reduction programs, the cancer burden due to obesity will likely continue to follow the rising prevalence of this risk factor in the coming years. Dietary Improvements Fruit and vegetable intake has been most consistently evaluated as a cancer prevention strategy. The global burden of inadequate intake is estimated to account for over 2 M deaths each year. While evidence for cardiovascular benefits and reduced risk of diabetes are clear, evidence for cancer risk reduction has become less convincing with the results of numerous prospective cohort studies showing weaker associations with cancer risk. Low intake of fruits and vegetables are probably related to increased risk of pancreas, bladder, lung, colon, mouth, pharynx, larynx, esophagus, and stomach cancer. Although the effect of fruit and vegetable consumption on the risk of prostate cancer has been examined in nearly twenty studies, data remain inconsistent. The majority of studies suggest that overall fruit and vegetable intake has a little effect if any on the risk of prostate cancer. However, individual fruits and vegetables may offer the potential for greater risk reduction, with tomatoes being the most promising, with a 40–50% reduction in risk among men who consumed large amounts of tomatoes and tomato products. The

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carotenoid lycopene is hypothesized to be responsible for the protective effect. A number of mechanisms have been suggested to explain the protective effect of fruits and vegetables, but it is not known if specific agents, such as ▶ carotenoids, folic acid, and vitamin C, or a special combination of factors create anticarcinogenic effects. It is also possible that diet in childhood and adolescence is more important than later in life in driving risk of cancer. A number of studies have found that as folate intake increases, the risk of colorectal cancer (as well as polyps) decreases. The Nurses’ Health Study found that a high intake of folate from fruits and vegetables was sufficient to lower risk but that supplementation with a multivitamin that contained folate offered even greater reductions. The underlying biologic role of folate and its interaction with the MTHFR gene add support to the causal relation between low folate and colon cancer. In addition to the reduction in risk of colon cancer, growing evidence points to folate also reducing the adverse effect of alcohol on breast cancer. Based on this evidence and the benefits for prevention of neural tube defect and cardiovascular disease, use of a daily vitamin supplement containing folate is recommended. Dietary Fat Variations in international cancer rates have often been attributed to differences in total fat intake, yet evaluation has shown no clear link between dietary fat and breast, colon or prostate cancer. Although dietary fat overall does not appear to impact cancer risk, there is some evidence to suggest that certain types of fat, such as animal fat, may increase risk. Fiber has been shown to reduce the risk of heart disease and diabetes, but it does not appear to offer protection against cancer. Long believed to help prevent colon cancer, the data do not support this hypothesis. Red Meat High intake of red meat, including beef, pork, veal, and lamb is associated with an elevated risk of colorectal cancer. The mechanism of this

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increased risk is not well understood, but it may be related to the high concentrations of animal fat or to carcinogens such as heterocyclic amines produced when the meat is cooked at high temperatures. Calcium Higher calcium intake has been linked to a reduced risk of colorectal adenomas and colorectal cancer. However, increased dietary calcium is also associated with an increased risk of prostate cancer. Research indicates that there may be a moderate intake of calcium that provides protection against colorectal cancer risk without causing a large increase in prostate cancer risk. Excess Caloric Intake One consistent dietary finding is that excess calories from any source result in weight gain and increased cancer risk. As the obesity epidemic continues to spread, the importance of balancing caloric intake with caloric expenditure becomes even more evident for the prevention of cancer and other chronic diseases. Whole Grains Although grain products in general have not been shown to affect cancer risk, whole-grain foods may provide some protection against stomach cancer. Grains such as wheat, rice, and corn form the basis for most diets worldwide. Some grain products, such as whole-wheat bread and brown rice, are consumed in the “whole-grain” form, while others, like white bread and white rice, are more refined. During the process of refining grain, most of the fiber, vitamins, and minerals are removed, thus whole-grain foods tend to be more nutrient-rich than refined foods and may offer more in terms of disease prevention. The benefits of whole-grain foods in reducing cardiovascular disease and ischemic stroke are well established. Vitamin A and Carotenoids Isolated vitamin A and carotenoids are not likely to play a large role in cancer prevention. Some observational data support a probable inverse relation with lung cancer risk, but randomized trials of

Cancer Causes and Control

beta-carotene intake found either no effect or an increased risk of lung cancer. It has also been suggested that beta-carotene impacts breast cancer risk, however, it seems that at best, there is only a small decrease in breast cancer risk associated with a high intake of carotenoids. Selenium Ecological studies have suggested that increased ▶ selenium intake is associated with decreased risk of colon and breast cancer. A randomized control trial of selenium for skin cancer prevention showed no effect of selenium on skin cancer incidence, however it did show a reduction in incidence of lung, colon, and prostate cancer. Despite these promising results, the impact of selenium remains unclear. Fortification of the soil in Finland in the mid-1980s led to higher blood selenium levels, but no decline in incidence or mortality has been noted for prostate or colon cancer. Vitamin D Growing evidence relates lower levels of ▶ vitamin D to increased risk of cancer and to poor survival after diagnosis. Trends The proportion of adults consuming the recommended five servings of fruits and vegetables a day varies between 8 and 32%. While these estimates are clearly low for the entire population, certain groups, as defined by gender, race/ethnicity, education, and income, are of particular concern. Given constraints due to both financial resources and physical access to markets that provide fresh fruit and vegetables, it remains likely that SES gradients in diet will continue. Interventions are needed to overcome these existing barriers and make healthy foods readily available to all. Limitation of Alcohol Use Globally, alcohol intake in excess is responsible for 1.8 M deaths each year. Clear benefits of moderate alcohol intake have been shown in terms of reducing cardiac and diabetes risk, but alcohol remains a risk factor for cancer mortality.

Cancer Causes and Control

Alcohol is a known carcinogen that may raise cancer risk in several ways. For example, it may act as an irritant, directly causing increased cell turnover, or it may allow for improved transport and penetration of other carcinogens into cells. Alcohol use is a primary cause of esophageal and oral cancer, and it is associated with an increased risk of breast, liver, and colorectal cancer. Multiple other risks are also associated with alcohol use, including the risk of hypertension, addiction, suicide, accident, and pregnancy complication. To balance the cardiovascular benefits with the risks of cancer and other negative consequences, it is recommended that those who drink alcohol should do so only in moderation. Intake should be limited to less than one drink per day for women and less than two drinks per day for men. Safer Sex and Decreased Viral Transmission Unsafe sex is responsible for 2.9 M deaths each year, primarily due to the transmission of HIV. However, unprotected sexual contact also results in the spread of multiple other sexually transmitted infections including oncogenic viruses. Some of these viruses may also be spread through exposure to blood and blood products. Human papillomavirus causes cervical cancer, vulvar, penile, and anal cancer; hepatitis B virus and ▶ hepatitis C virus cause hepatocellular cancer; human lymphotropic virus-type 1 is associated with adult T-cell leukemia (▶ human T-lymphotropic virus); human immunodeficiency virus-type 1 causes ▶ Kaposi sarcoma and non-Hodgkin lymphoma; and human herpes virus causes Kaposi sarcoma and body cavity lymphoma. Prevention strategies to contain the spread of these viruses should include behavioral and educational interventions to modify sexual behavior, and structural and regulatory changes to promote safer sex and make condoms readily available. Biomedical interventions to administer vaccines are also needed. For example, it is estimated that vaccination programs could reduce the global burden of liver cancer by 60%. Additional strategies to prevent viral spread include needle exchange programs for intravenous drug users; regulation of tattooing and acupuncture; and screening of blood

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donors and the development of artificial blood products. Trends and Disparities Current U.S. data on the prevalence of these different viruses is not adequate to predict trends in cancer incidence. In addition, the development of new technologies such as vaccines against HPV suggest a new era in prevention of cervical cancer. However, for success, such vaccines must be available and accessible to the entire population. Assuring access remains a policy priority to maximize the potential benefit of this cancer prevention strategy. Sun Protection The American Cancer Society estimates over 50,000 melanoma diagnoses each year in the USA. The incidence of melanoma is rising more rapidly than that of any cancer in this country. Exposure to the sun (▶ UV radiation) is the major modifiable cause of melanoma and other skin cancers. For most people, the majority of lifetime sun exposure occurs during childhood and adolescence, and migrant studies clearly show that age at migration to high-risk countries has a strong impact on risk of this malignancy. For this reason, early intervention has the greatest potential for prevention. The risk of melanoma and other less aggressive forms of skin cancer exists for all racial and ethnic groups, but skin cancers occur predominantly in the non-Hispanic white population. Constitutional characteristics including hair color, mole count, and family history contribute to risk of melanoma. However, studies show that established risk factors alone do not identify a sufficient proportion of cases to focus prevention efforts on only a subset of the population. Because identifying high-risk individuals will miss the majority of cases, population-based efforts provide greater protection. There is tremendous potential to substantially reduce the burden of this common malignancy through effective prevention efforts. Screening Screening for cancer can provide protection in several ways. In the case of colorectal and cervical

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cancers, screening can detect premalignant changes that can be treated to prevent cancer from developing. This primary prevention has the potential to substantially reduce the burden of cancer. With colorectal screening, the mortality from colon cancer is reduced by a half or more. If cancer is already present, screening can act as a secondary prevention, such as mammography for breast cancer, facilitating early diagnosis and treatment, thereby decreasing morbidity and mortality. This type of prevention is an added benefit of colorectal and cervical screening, and is the main goal of breast cancer and prostate cancer screening. Trends and Disparities Trends in cervical cancer screening have been impacted by the breast cancer cervical cancer screening act which provided resources to states, via the Centers for Disease Control and Prevention, to bring screening services to low income women. Despite these efforts, national data suggest that low income and Hispanic women are less likely to be current with screening recommendations. Lack of access to care, defined as not having a usual source of health care, was associated with significantly lower compliance with cervical screening. Evidence from the 1998 Health Interview Survey indicates that all US born women have comparable and high compliance with screening for cervical cancer. Foreign born women, however, appear to be under-screened, accounting for the disparity among Hispanic women and suggesting a priority area for prevention as the USA continues to have a large immigrant population at risk of cancer. Surveys of colorectal screening suggest that the rates of screening have been rising and that Caucasians are more likely to be up to date with screening than other racial or ethnic groups. Conclusion Lifestyle changes offer tremendous potential for prevention of cancer and multiple other chronic conditions. This potential is often underestimated. To achieve the maximal benefit through behavioral change, interventions are necessary at multiple levels. Societal changes are needed to support

Cancer Causes and Control

and encourage the behavior modification of individuals. Approaches are needed to target individuals, communities, and systems, and create an environment less inductive to high-risk lifestyles. Social systems and regulatory efforts must complement individual behavior changes if these changes are to be sustained and the benefits of reduced disease burden realized. Overall, the major lifestyle factors considered here account for the majority of cancer and could be modified to prevent at least half of all cancers. However, the burden of cancer is not limited to just the major lifestyle factors considered here. For example, occupational and environmental exposures also account for a relatively small number of cancer cases compared to the lifestyle factors considered above. Yet the burden of exposure to these harmful agents may be disproportionately high among low-income populations, accentuating their cancer risk. In large part, these exposures can be prevented through adequate enforcement of regulatory changes, and this should remain a high priority. Small individual changes can result in large population benefits, but efforts to create prevention programs for only certain members of our society limits the potential for prevention. We must largely reframe our approach to the issue. Identifying risk factors and setting goals for reduction is only the beginning. Research and policy must now focus on bringing about population-wide lifestyle change, addressing the issues of disparities, and leaving no group or community behind.

Cross-References ▶ Alcohol Consumption ▶ Aspirin ▶ Bladder Cancer ▶ Breast Cancer ▶ Carotenoids ▶ Cervical Cancers ▶ Colorectal Cancer ▶ Endometrial Cancer ▶ Esophageal Cancer

Cancer Epidemiology

▶ Gastric Cancer ▶ Hepatitis C Virus ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Hepatocellular Carcinoma ▶ Human T-Lymphotropic Virus ▶ Inflammation ▶ Kaposi Sarcoma ▶ Lung Cancer ▶ Lung Cancer and Smoking Behavior ▶ Multistep Development ▶ Obesity and Cancer Risk ▶ Pancreatic Cancer ▶ Prostate Cancer ▶ Prostate Cancer Clinical Oncology ▶ Renal Cancer Clinical Oncology ▶ Renal Cancer Genetic Syndromes ▶ Selenium ▶ Tobacco Carcinogenesis ▶ UV Radiation ▶ Vitamin D

751 (2012) Lycopene. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2116. doi:10.1007/978-3-642-16483-5_3441 (2012) MTFR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2383–2384. doi:10.1007/978-3-642-16483-5_6767 (2012) Renal cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3225– 3226. doi:10.1007/978-3-642-16483-5_6575 (2012) Tobacco. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3716– 3717. doi:10.1007/978-3-642-16483-5_5844 (2012) Ultraviolet light. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3841. doi:10.1007/978-3-642-16483-5_6101 (2012) Viruses. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3924. doi:10.1007/978-3-642-16483-5_6203 (2012) Vitamin A. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3925. doi:10.1007/978-3-642-16483-5_6205 Möslein G (2009) Colon cancer. In: Schwab M (ed) Encyclopedia of cancer, 2nd edn. Springer, Berlin/Heidelberg, pp 722–727. doi:10.1007/978-3-540-476481_126

References Colditz GA, DeJong D, Emmons K et al (1997) Harvard report on cancer prevention. volume 2. Prevention of human cancer. Cancer Causes Control 8:1–50 Curry S, Byers T, Hewitt M (2003) Fulfilling the potential of cancer prevention and early detection. National Academy Press, Washington, DC International Agency for Research on Cancer (2002) Weight control and physical activity, vol 6. International Agency for Research on Cancer, Lyon Rose G (1981) Strategy of prevention: lessons from cardiovascular disease. Br Med J (Clin Res) 282:1847–1851 U.S. Department of Health and Human Services (1996) Physical activity and health: a report of the Surgeon General. US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Atlanta

See Also (2012) Folate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1440. doi:10.1007/978-3-642-16483-5_2231 (2012) Hepatitis B Virus. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1663. doi:10.1007/978-3-642-16483-5_2659 (2012) HIV. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1706. doi:10.1007/978-3-642-16483-5_2764

Cancer Cell Cytotoxicity ▶ Artemisinin

Cancer Cell-Platelet Microemboli ▶ Tumor Cell-Induced Platelet Aggregation

Cancer Epidemiology Paolo Boffetta Icahn School of Medicine at Mount Sinai, New York, NY, USA

Synonyms Population-based cancer research

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Definition Knowledge about causes and preventive strategies for malignant neoplasms has greatly advanced during the last decades. This is largely attributable to the development of cancer epidemiology. In parallel to the identification of the causes of cancer, primary and secondary preventive strategies have been developed. A careful consideration of the achievements of cancer research, however, suggests that the advancements in knowledge about causes and mechanisms have not been followed by an equally important reduction in the burden of cancer. Part of this paradox is explained by the long latency occurring between exposure to carcinogens and development of the clinical disease. In addition, the most important risk factors of cancer are linked to lifestyle, and their modification entails cultural, societal, and economic consequences. The failure to identify valid biomarkers of cancer risk is another reason of the limited success in cancer control. Cancer epidemiology investigates the distribution and determinants of cancer in human populations. Although the main tool in cancer epidemiology is the observational study, the intervention study, of experimental nature, is conducted to evaluate the efficacy of prevention strategies, such as screening programs and chemoprevention trials (clinical trials are usually considered outside the scope of epidemiology). Intervention studies follow the randomized trial design. Observational epidemiology can be broadly divided in descriptive epidemiology and analytical studies. Analytical studies can be based on data collected at the individual or population level. The former consist of ▶ cohort study, ▶ case–control study, and crosssectional study (and a few variations on these themes), the latter of the so-called ecological study. Family-based studies are used in genetic epidemiology to identify hereditary factors. An additional useful distinction among etiological studies concerns the nature of the information on exposure: while some studies use information routinely collected for other purposes, such as censuses and medical records, in other circumstances exposure data are collected ad hoc following a

Cancer Epidemiology

variety of approaches including questionnaires, pedigrees, environmental measurements, and measurement of biological markers. A method-oriented (rather than subject- or design-oriented) approach has led to the identification of specific subdisciplines such as molecular epidemiology.

Characteristics A distinctive feature of cancer epidemiology is the availability in many countries of a populationbased cancer registry, which allow the calculation of valid and reliable estimate of the occurrence of cancer (incidence, mortality, prevalence, survival). Typically, registries collect routinely demographic data of patients, which are used to generate statistics according to period of diagnosis, age, sex, and other characteristics. These studies of descriptive epidemiology have been critical in developing etiological hypotheses. One particular type of descriptive studies concerns migrants from low- to high-risk areas or vice versa: the repeated demonstration of rapid changes in the risk of many cancers among migrants (from that prevalent in the area of origin toward that of the host area) provided very strong evidence of a predominant role of modifiable factors in the etiology of human cancer. While cancer registries were initially established in high-resource countries, a growing number of populations in middleand low-resource countries are now covered by good-quality registries, thus providing a solid infrastructure for most ambitious research projects. Other routinely collected data are used in epidemiology. Mortality statistics are available in many countries of the world and provide a good approximation of the incidence of the most fatal cancers. In a growing number of populations, automatic linkage is possible between incidence or mortality data and other population-based registries (e.g., hospital discharges, use of medications). Record-linkage study may represent an efficient alternative to investigations based on ad hoc collection of data. The number of new cases of cancer which occurred worldwide in 2012 has been estimated

Cancer Epidemiology

at about 14,100,000. Of them, 7,400,000 occurred in men and 6,700,000 in women. About 6,100,000 cases occurred in more developed countries (North America, Japan, Europe including Russia, Australia, and New Zealand) and 8,000,000 in less developed countries. Among men, lung, prostate, colorectal, stomach, and liver cancers are the most common malignant neoplasms, while breast, colorectal, lung, cervical, and stomach cancers are the most common neoplasms among women. The number of deaths from cancer in 2012 was estimated at about 8,200,000 and that of 5-year prevalent cases at about 32,500,000. Epidemiology has been instrumental to identify the causes of human cancer. In several cases, the epidemiological results preceded the elucidation of the underlying mechanisms. In other areas, however, epidemiological techniques are not sufficiently sensitive and specific to lead to conclusive evidence on the presence or absence of an increased risk. As for other branches of the discipline, the observational nature of epidemiology represents an opportunity for bias, including that generated by confounding, to generate spurious results. Techniques have been developed to prevent, control, and assess the presence and extent of bias in epidemiological studies. Cancer epidemiology has led to the identification of tobacco smoking and use of smokeless tobacco products, chronic infections, overweight, alcohol drinking, and reproductive factors as major causes of human cancer. Other important causes include medical conditions, some drugs, perinatal factors, physical activity, occupational exposures, and ultraviolet and ionizing radiation. A role of diet in cancer risk has been suggested, but for very few dietary factors, there is a conclusive evidence of an effect on cancer risk. With a few exception of little relevance in most populations, the role of pollutants on cancer is not established. Tobacco smoking is the main single cause of human cancer worldwide. It is a cause of cancers of the oral cavity, pharynx, esophagus, stomach, liver, pancreas, nasal cavity, larynx, lung, cervix, kidney, and bladder and of myeloid leukemia. The proportion of cancers in a population attributable to tobacco smoking

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depends on the distribution of the habit a few decades earlier. Therefore, in populations in which the tobacco epidemic has not fully matured (e.g., men in many low-income countries and women in most European countries), the full effect of tobacco smoking on cancer burden is not yet observed. The notion that genetic susceptibility plays an important role in human cancer is old, and genetic epidemiology studies that have characterized familial conditions entailing a very high risk of cancer have been identified, such as the Li–Fraumeni syndrome and the familial polyposis of the colon, and have identified high-risk cancer genes responsible for these syndromes. However, such high-risk conditions explain only a small fraction of the role of inherited susceptibility to cancer. The remaining fraction of genetic predisposition is likely explained by the combination of common variants in genes involved in one or more steps in the carcinogenic process, such as preservation of genomic integrity, repair of DNA damage. The identification of such low-penetrance susceptibility genes and of their interactions with exogenous factors (so-called gene–environment interactions) represents a challenge to genetic epidemiology. Advances in molecular biology and genetics offer new tools for epidemiological investigations and have led to the development of new methodological approaches, broadly defined as molecular epidemiology. The application of biomarkers to epidemiology has led to advances in the identification of human carcinogens (e.g., the role of aflatoxin in liver cancer) and in the elucidation of mechanisms of carcinogenesis (e.g., TP53 mutations in tobacco-related carcinogenesis). Exposure to most known carcinogens – at least in theory – should be avoided or reduced. This is true in particular for tobacco smoking and chronic infections, the two major known causes of cancer. Tobacco control measures have been implemented in most countries, and effective vaccination is available today against two of the main carcinogenic viruses, hepatitis B and human papilloma. Control of workplace exposure to known and suspected carcinogens in high-resource countries is another example of successful primary

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prevention of cancer. In many instances, however, primary prevention of cancer would require major changes in lifestyle, which are difficult to achieve. Detection of preclinical neoplastic lesions before they have developed the full malignant phenotype and notably the ability to metastasize is a highly appealing approach to control cancer. The effectiveness of screening has been demonstrated via epidemiological studies for cervical cancer (cytological smear), breast cancer (mammography), and colorectal cancer (colonoscopy). The development of effective strategies for the early detection of other neoplasms is an active area of research. Cancer epidemiology exemplifies the strengths and the weaknesses of the discipline at large. Cancer epidemiology has the privilege of using complete and good-quality disease registries available in many populations and covering a broad spectrum of rates and exposures. In many occasions, cancer epidemiology has been the key tool to demonstrate the causal role of important cancer risk factors. The best example is the association between tobacco smoking and lung cancer, which led in the early 1960s to the establishment of criteria for causality in observational research. These findings have brought important regulatory and public health initiatives as well as lifestyle changes in many countries of the world. These epidemiological “discoveries” share two important characteristics: they involve potent carcinogens, and methods are available to reduce misclassification of exposure to the risk factor of interest and to major possible confounders. It has been therefore possible to demonstrate consistently an association in different human populations. Note that it is not necessary for the prevalence of exposure to be high (although this obviously has an impact on the population attributable risk): examples are the many occupational exposures and medical treatments for which conclusive evidence of carcinogenicity has been established on the basis of epidemiological studies conducted in small populations of individuals with well-characterized exposure. When these conditions are not met, however, the evidence accumulated from epidemiological studies is typically inconsistent and difficult to

Cancer Epidemiology

interpret. The history of cancer epidemiology presents many examples of premature conclusions, which have not been confirmed by subsequent investigations and have damaged the reputation of the discipline. Exposure misclassification, uncontrolled confounding, emphasis of positive findings generated by chance, and inadequate statistical power are the most common limitations encountered in epidemiological studies. Several solutions have been proposed to overcome these problems. First, epidemiological studies should be very large in size. This is achieved either by conducting multicentric studies including thousands of cases of cancer or by performing pooled and meta-analyses of independent investigations. Second, as mentioned above, the use of biological markers of exposure and early effect might contribute to reduce exposure misclassification, increase the prevalence of the relevant outcomes, and shed light on the underlying mechanisms. Finally, guidelines that have been developed to improve and standardize the conduct are report of observational epidemiological studies. Although relatively young, epidemiology has become a key component in cancer research. Most cancer centers have an epidemiological research group, and cancer is a major subject of research in most academic departments of epidemiology. Epidemiologists are more and more often invited to meetings of clinicians and basic researchers not only to provide an introduction to the distribution and the risk factors of a given cancer but to participate in interdisciplinary discussions on clinical, preventive, or mechanistic aspects of the disease. The strongest cancer epidemiology groups in the world are those combining different lines of expertise, from biostatistics to molecular biology and genetics to medical oncology. Despite its limitations, cancer epidemiology remains one of the most powerful tools at the disposal of the research community to combat cancer at all levels.

Cross-References ▶ Case Control Association Study ▶ Epidemiology of Cancer

Cancer Epigenetics

References Doll R, Peto R (2003) Epidemiology of cancer. In: Warrell DA, Cox TM, Firth TD (eds) Oxford textbook of medicine, 4th edn. Oxford University Press, London, pp 193–218 Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F (2013) GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide. International Agency for Research on Cancer, Lyon, France Last JM (1983) A dictionary of epidemiology. Oxford University Press, New York STROBE Statement. STrengthening the Reporting of OBservational studies in Epidemiology. http://www. strobe-statement.org/

Cancer Epigenetics Berna Demircan and Kevin Brown University of Florida, College of Medicine, Gainesville, FL, USA

Definition Epigenetics is defined as chromatin modifications that can alter gene expression, are heritable during cell division, but do not involve a change in DNA coding sequence.

Characteristics In the context of normal biological processes, epigenetic mechanisms establish regions within the genome containing transcriptionally active (termed euchromatin) and silent (termed heterochromatin) DNA. Further, epigenetic mechanisms are responsible for stably inherited patterns of gene expression such as X chromosome inactivation and genomic imprinting (i.e., selective expression of maternal or paternal alleles). Chromatin modifications that alter gene expression are

This entry was first published in the 2nd edition of the Encyclopedia of Cancer in 2009.

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both changes to the methylation state of DNA and posttranslational modifications to histone complexes. It is well recognized that genetic mutations occur in cancer cells and that these events can exert profound and disease-associated changes in gene expression and/or function. However, it is becoming widely accepted that cancer cells also exhibit aberrant epigenetic alterations and that these changes can play a prominent role in disease initiation and progression. Epigenetic changes are potentially as important as genetic mutations in causing cancer since chromatin alterations can exert an influence regional gene expression, thereby changing the transcriptional profile of multiple genes. In this chapter, we summarize the principal epigenetic alterations that occur in cancer cells: regional DNA hypermethylation and ▶ histone modifications and global DNA hypomethylation. DNA Hypermethylation Chromatin structure is influenced by cytosine ▶ methylation, the only known naturally occurring base modification in DNA. Cytosine methylation occurs at 50 -CG-30 dinucleotides (referred to as CpGs) and is catalyzed by a class of enzymes termed DNA methyltransferases (DNMTs). Several DNMTs have been characterized in mammalian cells including DNMT1, DNMT3a, and DNMT3b. These enzymes catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the 5-carbon position of cytosine, forming 5-methycytosine. DNMT3a and DNMT3b appear to be principally involved in methylating previously unmodified cytosines (termed de novo methylation). In contrast, DNMT1 preferentially methylates hemimethylated DNA and is thus viewed as the DNA methyltransferase principally responsible for continuation of DNA methylation patterns in daughter cells (termed maintenance methylation). From a statistical standpoint, the human genome is depleted in CpG dinucleotides; however, 60% of genes in our genome are associated with regions ranging from 200 to 4,000 bases in length containing high density of CpG dinucleotides relative to the bulk genome. These regions

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Cancer Epigenetics

Cancer Epigenetics, Table 1 Genes subject to epigenetic silencing in cancer Gene APC

MGMT

Function Regulation of b-catenin, cell adhesion DNA repair Homotypic epithelial cell–cell adhesion DNA repair

MLH1

DNA repair

CDKN2A (p16)

Cell cycle control

PTEN

Regulation of cell growth and apoptosis Inhibits angiogenesis, regulates transcription DNA damage response

BRCA1 CDH1 (E-cadherin)

VHL

ATM

Tumor types Colorectal, gastrointestinal Breast, ovarian Bladder, breast, colon, liver

Brain, colorectal, lung, head, and neck Colorectal, endometrial, ovarian Lung, brain, breast, colon, bladder, melanoma prostate Prostate, brain, endometrial, melanoma Renal cell carcinoma

Breast, colorectal, head, and neck

are referred to as ▶ CpG islands and are usually located within upstream promoter regions or gene transcriptional start sites. In normal somatic cells, gene-associated CpG islands are usually unmethylated and associated with genes in a transcriptionally active euchromatic state. In cancer cells, hypermethylation of such CpG islands is strongly correlated with the transcriptional silencing of genes. Thus, through this epigenetic mechanism, tumor cells can dramatically downregulate expression of numerous genes, including ▶ tumor suppressor genes (TSGs). At present, numerous TSGs have been characterized as targets for epigenetic silencing through hypermethylation of associated CpG islands. Table 1 is a partial listing of characterized TSG whose promoter regions have been shown to be hypermethylated in various tumor types. Given the wide spectrum of tumor types that display epigenetic silencing of TSGs, mounting evidence clearly supports the assertion that epigenetic silencing is a prominent mechanism driving the process of tumorigenesis.

Cancer cells often overexpress DNMTs. Compared to normal tissues, the expression of DNMT1 is almost always increased in tumors. However, since DNMT1 expression is normally regulated during the cell cycle with increased abundance paralleling entry into S-phase, much of this increased expression may simply reflect increased cell proliferation within the tumor. Although demonstrable in model experiments, it remains unresolved if increased expression of DNMT1 is responsible for aberrant methylation in cancer cells. In contrast, increased expression of DNMT3a and DNMT3b observed in some tumors is likely significant, since these enzymes are normally expressed at low levels in somatic cells. However, it is still unclear to what extent overexpression of these enzymes is responsible for cancer-associated DNA hypermethylation especially when one considers that cancer cells exhibit overall genome hypomethylation (discussed below). Thus, it remains unclear how CpG islands associated with specific TSG are targeted for hypermethylation during the process of tumorigenesis. One mechanism by which DNA methylation can negatively impact gene expression is by simply blocking the binding of essential transcription factors to gene promoter sequences. While several examples of this are documented, it is also apparent that CpG methylation is also capable of directing transcriptional repression through promoting additional layers of chromatin alteration. Specifically, several proteins have been characterized that bind to methylated CpG dinucleotides and are capable of promoting further chromatin condensation and consequential transcriptional repression through recruitment of chromatinmodifying activities. Histone Modification Owing to technical considerations, DNA methylation is the most widely analyzed type of epigenetic alteration in human tumors. However, another extremely important epigenetic modification capable of altering gene expression during carcinogenesis involves various types of histone modifications. The fundamental packing unit of chromatin within the nucleus is termed the nucleosome.

Cancer Epigenetics

A single nucleosome unit contains 146 base pairs of DNA wrapped around eight histone subunits (histone octamer). The histone octamer contains two copies each of histones H2A, H2B, H3, and H4. Structural studies have determined that each histone possesses an amino-terminal tail rich in the amino acid lysine. These lysine residues can undergo a variety of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitination. Such modifications are recognized by various proteins and protein complexes, and combinations of histone modifications constitute a proposed histone code important in establishing a given gene’s transcriptional profile. Perhaps the best-studied histone modification is acetylation of the e-amino group of lysine residues within the amino-terminal tail of H3 and H4 although acetylation of both H2A and H2B occurs as well. This is a reversible modification that is carefully controlled by two large enzyme families: histone acetyltransferases (HATs) and histone deacetylases (HDACs). The net positive charges carried by these lysine residues are proposed to contribute to the high affinity of histones for negatively charged DNA. Acetylation of lysine residues by HATs neutralizes this positive charge, thus decreasing histone/DNA interaction. This raises molecular access to DNA and promotes gene transcription. Conversely, HDACs promote transcriptional repression by supporting chromatin condensation into a heterochromatic conformation. Several proteins that bind specifically to methylated CpG through a conserved methyl-binding domain (MBD) motifs have been discovered. The first such protein to be characterized, termed MeCP2, is capable of recruiting the corepressor molecule mSin3 to the sites of methylated DNA. In turn, mSin3 binds to HDAC1 and HDAC2, thus promoting localized histone deacetylation. The importance of HDAC activity in transcriptional repression is underscored by the observation that repression of several gene promoters can be partially relieved by HDAC inhibitors. A functionally similar complex termed MeCP1 binds to methylated DNA via an associated protein termed MBD2. The MeCP1 complex

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contains multiple subunits besides MBD2, including components of the NuRD complex, a characterized repressor complex containing both chromatin remodeling and HDAC activities. In addition to acetylation, lysine residues within histone tails can be methylated and exist in either mono-, di-, or trimethylated states. Similar to the effects of histone acetylation/ deacetylation, methylated lysine 4 of H3 (H3K4) is associated with transcriptionally active chromatin, while transcriptionally silent chromatin generally contains methylated lysine 9 of H3 (H3K9). H3K9 is methylated by a number of histone methyltransferases including ESET, Eu-HMTase, G9a, and the closely related methyltransferases SUV39-H1 and SUV39-H2. Histone methylation is likely a dynamic process since a histone demethylase, termed LSD1, was characterized. Methylated H3K9 binds to the chromodomain protein heterochromatin protein 1 (HP1) which promotes heterochromatin formation and gene silencing. Moreover, since H3K9 methylation cannot occur when this position is acetylated, it is clear that H3K9 acetylation and methylation represent opposing forces in determining chromatin conformation. In a broad view, it is reasonable to propose that CpG methylation and histone acetylation/ deacetylation act synergistically in the progressive silencing of genes. One model that accounts for tumor suppressor gene silencing by epigenetic mechanisms invokes abnormal hypermethylation of the promoter CpG island followed by recruitment of MBD proteins, including complexes such as MeCP2 and MeCP1 that recruit HDACs to the area of hypermethylation and promote further transcriptional repression through histone modification. An alternate model proposes that transcriptionally repressive histone modifications are the first event in gene silencing and subsequently promote CpG methylation, resulting in further transcriptional repression. Experimental evidence supports both of these models and may be reflective of the variety of gene promoters and model systems used for study. Less equivocal is the fact that DNA methylation appears as the dominant silencing mechanism since inhibition of DNA methylation generally restores gene expression,

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while HDAC inhibitors generally exert more modest effects on gene silencing. DNA Hypomethylation While CpG islands associated with gene promoters are generally unmethylated in normal adult somatic cells, the majority of CpG dinucleotides elsewhere in the genome are generally methylated. Moreover, despite the fact that many CpG islands are subject to hypermethylation in cancer cells, it is equally well documented that tumor cells display an overall loss of methylated cytosines compared to normal tissue. This tumorassociated global DNA hypomethylation predominantly occurs in repetitive DNA sequences within the human genome although the molecular mechanisms responsible for this loss of DNA methylation are poorly understood. Recent work on a human genetic disorder has underscored an important role for DNA methylation in maintenance of genome stability. The immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by germline mutation of the DNMT3B gene. Loss of DNMT3B activity in ICF leads to hypomethylation of repetitive satellite DNA sequences within heterochromatin adjacent to the centromeric region of chromosomes. The loss of methylation is most prominent within the pericentric regions of human chromosomes 1 and 16 and leads to multiple chromosomal abnormalities including chromatin decondensation, chromosomal translocations and deletion, and multiradial chromosomal structures. These observations, as well as those made on cells with engineered disruption of DNMTs, clearly support the view that DNA methylation is critical in maintaining normal chromosome structure. Since cancer cells often show chromosomal rearrangements, it is likely that cancer-associated DNA hypomethylation allows for heightened rates of chromosomal instability. Retrotransposon sequences of the LINE (long interspersed nuclear element) and SINE (short interspersed nuclear element) classes as well as human endogenous retroviruses (HERVs) are major targets of tumor-associated DNA

Cancer Epigenetics

hypomethylation. Mobility of these DNA elements is kept in check in normal tissues owing, in part, to dense methylation of CpG dinucleotides within their genomic structure. It follows that increased mobility of these dormant mobile elements occurs as a result of cancer-associated DNA hypomethylation, and there have been reports of retrotransposition-like insertions involving LINE1 sequences in tumors although retrotransposition of endogenous elements seemingly occurs more often in rodents than humans. In addition to the hypomethylation of CpG dinucleotides present within repetitive DNA elements, cancer-associated hypomethylation also occurs in regions of the genome encoding single-copy genes. Dysregulation of allelespecific methylation will result in the loss of imprinting (LOI) and allow for both maternal and paternal gene expression. Perhaps the beststudied example of this is the insulin-like growth factor 2 (IGF2) gene where LOI occurs in primary tumors and in patients with the inherited, cancerprone ▶ Beckwith–Wiedemann. While not as well-studied as gene silencing due to DNA hypermethylation, it is likely that additional examples of cancer-promoting increased gene expression stemming from DNA hypomethylation will be uncovered in the future. Epigenetic Alterations as Targets for Diagnosis and Therapeutic Intervention Sequencing data obtained from the human genome project are currently undergoing analysis to construct a human epigenetic map based on CpG content. This knowledge coupled with cross-species comparisons of the epigenome will be invaluable in deciphering the epigenetic elements involved in gene regulation. Epigenetic alterations typically occur early during the oncogenic process, and detection of such early abnormalities may aid in early diagnosis and/or preventing cancer progression through dietary alterations or pharmacological intervention. With increasing awareness of the importance of epigenetics in tumorigenesis, and the advent of sensitive laboratory approaches to analyze epigenetic alterations, it is likely that epigenetic profiles will ultimately be used in the clinical setting to

Cancer Germline Antigens

provide information useful in predicting an individual’s predisposition to cancer, assisting in tumor staging, and guiding optimal therapeutic approaches. A promising feature of alterations in DNA methylation patterns and chromatin structure in cancer cells is their potential for reversibility, because these modifications occur without changing the primary nucleotide sequence. At present, two major pharmacological targets associated with these epigenetic changes are DNMTs and HDACs. The DNMT inhibitor 5-aza-deoxycytidine (5-azadC) and related compounds cause transcriptional reactivation of endogenous genes with hypermethylated promoters. This drug, also termed decitabine, is currently used to treat certain types of hematological malignancies, especially advanced ▶ myelodysplastic syndromes (MDS). HDAC inhibitors, such as trichostatin A and sodium butyrate, have been shown to increase the level of histone acetylation in cultured cells and to cause growth arrest, differentiation, and apoptosis. Based on these observations, the potent HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) is in clinical trials.

Cross-References ▶ Beckwith-Wiedemann Syndrome ▶ Cancer Epigenetics ▶ CpG Islands ▶ Epigenetic ▶ Epigenetic Gene Silencing ▶ Histone Modification ▶ Methylation ▶ Myelodysplastic Syndromes ▶ Tumor Suppressor Genes

See Also (2012) Acetyltransferase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 17. doi:10.1007/978-3-642-16483-5_27 (2012) DNA-methyl-transferases. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1147. doi:10.1007/978-3-642-164835_1681

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Cancer Etiology ▶ Cancer Causes and Control

C Cancer Germline Antigens Adam R. Karpf Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA

Synonyms Cancer-testis antigens; CG antigens

Definition CG antigens (cancer-testis (CT) antigens) are a class of immunogenic tumor antigens encoded by genes expressed in gametogenic cells of the testis and/or ovary and in human cancer.

Characteristics Identification The main criterion for the classification of a gene as a CG antigen pertains to its expression pattern in gametogenic, somatic, and tumor tissues. A gene is generally considered to be a CG antigen if it is expressed in the gametogenic cells of the testis or ovary (including fetal ovary) and in some proportion of human cancers, but is expressed in two or fewer normal somatic tissues. CG antigen genes are also commonly expressed in trophoblast tissue. CG antigens were originally identified from searches for autoantigens expressed in human cancer. The original method for antigen screening used autologous typing, in which T-cells (T-lymphocyte) from a ▶ melanoma patient were screened for reactivity with tumor cells from the same patient; this method led to the identification of MAGE-A (named for ▶ melanoma antigens)/CT1 genes. In later studies,

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another immunological assay was developed to identify tumor antigens and this method, ▶ SEREX (Serological analysis of recombinant cDNA expression libraries), was used to successfully identify a variety of important CG antigen genes, including NY-ESO-1/CT6 and SSX/CT5. Recognition of the unique expression pattern of CG antigen genes has led to the use of gene expression analyses (including EST or SAGE database searching) to identify CG antigen genes. This method has led to the identification of additional CG genes, including XAGE-1/CT12 and SCP-1/CT8. Although formally classified as CG antigen genes, genes identified by the latter nonimmunological method may not be antigenic in cancer patients. Nomenclature A nomenclature system for CG antigens has been devised, which is based on their chronology of discovery, and also accounts for the numerous family members that exist for certain CG antigens. In this system, CG antigen genes are referred to by their original given names and also are assigned a separate CT identifier or CT#. Currently, over 40 CG antigen gene families are recognized, comprising more than 89 distinct mRNA transcripts. CG antigen genes have been assigned into two groups on the basis of chromosomal localization. CG-X antigens: These genes reside on the X-chromosome where, interestingly, close to 10% of the total number of genes encode CG antigens. CG-X genes are typically members of large multigene families, e.g., MAGE-A/CT1, MAGE-B/CT3, and MAGE-C/CT7. In normal tissues, CG-X genes are often expressed in premeiotic spermatocytes in the testis. All of the current important targets of CG antigen ▶ cancer vaccines are members of this group, including MAGE-A1/CT1.1, MAGE-A3/CT1.3, and NY-ESO-1/CT6.1. Nonseminomatous gene cell tumor: A number of CG antigen genes are located on autosomal chromosomes. Unlike CG-X genes, these genes are highly dispersed in the genome and do not exist in multigene families. In normal tissues, non-X CG genes are often expressed during meiosis, where some members play roles in DNA recombination, including SCP-1/CT8 and

Cancer Germline Antigens

SPO11/CT35. The members of this gene group do not include any currently validated cancer antigens, although certain members are expressed at high levels in cancer. Regulation of Expression Certain cancer types appear to expresses CG antigen genes frequently, while others rarely express them. Tumor types that frequently express CG antigens include melanoma, lung, ovary, and ▶ bladder cancer; tumors that rarely express CG antigens include colon cancer, renal cancer, and leukemia/lymphoma. CG antigen genes show coordinate expression in human cancer. That is, the great majority of tumors either do not express CG antigen genes or express two or more CG antigen genes simultaneously, while relatively few tumors express only one CG antigen gene. Another characteristic of CG antigen gene expression in cancer (revealed by immunohistochemical staining) is that tumors that express CG antigens show heterogeneous expression within the tumor: often only focal staining is observed. The coordinate but heterogeneous expression of CG antigens in cancer has led to the intriguing hypothesis that CG antigen expression is indicative of the activation of a normally dormant gametogenic program in tumor cells (possibly corresponding to tumor stem cells). The observation of coordinate expression of CG antigen genes suggests that CG antigen gene activation may be controlled by a common molecular mechanism. Supporting this idea, a number of studies have suggested a key role for DNA methylation in regulating CG antigen gene expression. Promoter DNA hypermethylation has been observed to correlate with CG antigen gene repression in normal tissues and nonexpressing tumors, while treatment of tumor cell lines in vitro with DNA methyltransferase inhibitors such as ▶ 5-aza-20 -deoxycytidine (DAC) leads to CG antigen gene activation, coincident with promoter DNA hypomethylation. Conversely, tumor cell lines and tissues that endogenously express CG antigen genes often display promoter DNA hypomethylation. Many CG antigen genes have CpG-rich promoter regions that serve as targets for regulation by DNA methylation.

Cancer Germline Antigens

Other studies have shown that histone deacetylase (HDAC) inhibitors can either augment DAC-mediated CG antigen gene activation or activate CG antigen genes on their own. As DNA ▶ methylation and chromatin structure (in the form of histone modification status) are intimately linked, it is not surprising that both of these ▶ epigenetic mechanisms serve as important regulators of CG antigen gene expression. Consistent with the model that epigenetic mechanisms regulate CG antigen gene expression is the observation that DNA hypomethylation occurs during gametogenesis, which is the normal setting for CG antigen gene expression. Function CG antigens are a rare group of genes in that clinical studies designed to target these antigens for ▶ immunotherapy of cancer are more advanced than is our basic knowledge of the function of the gene products. However, some information about CG antigen gene function has come to light. As mentioned earlier, many non-X CG antigens have roles in germ cell maturation, including mediating the structure of synaptonemal complexes (SCP1/ CT8), facilitating DNA recombination during meiosis (SPO11/CT35), and contributing to spermatid function (ADAM2/CT15, OY-TES-1/CT23). In tumors, the function of CG antigen genes is less clear, but studies of the MAGE-type antigens, which share a region referred to as the MAGE homology domain (MHD), indicate that these proteins might serve as transcriptional repressors via interactions with other transcriptional regulatory proteins that themselves recruit corepressors such as HDACs. CG antigen genes have also been reported to play a role in the evolution of ▶ chemotherapy resistance in cancer cell lines, suggesting that the CG antigen gene products could serve as viable targets for anticancer therapy. A report appears to link these two observations by showing that MAGE-A2/CT1.2 disrupts p53 function by recruiting HDAC3 to p53, leading to chemotherapy resistance in cancer cells. Clinical Studies The identification of CG antigens as tumorspecific antigens has led to a great deal of interest

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in treating cancer by targeting CG antigens via vaccine-based immunotherapy. In particular, MAGE-A1/CT1.1, MAGE-A3/CT1.3, and NY-ESO-1/CT6.1 have been developed as targets for this approach. In early studies, the antigenic peptides from CG antigens that elicited T-cell dependent responses were mapped, and these peptides were utilized for vaccination. Because responses to peptide-based vaccine formulations are limited by patient HLA type, vaccination approaches targeting CG antigens have utilized full-length recombinant proteins. These recombinant proteins can be introduced using viral vectors, including vaccinia and fowlpox viruses. Alternatively, recombinant CG antigen proteins can be assembled with adjuvants such as ISOMATRIX, which further enhances immune responses. A common finding in CG antigen vaccine clinical studies is that the treatment is safe and elicits both antibody and T-cell mediated immune responses in vivo. In particular, NY-ESO-1/CT6.1 vaccine trials have shown encouraging results, with durable and multifaceted immune responses, as well as suggestive data indicating clinical benefit, in terms of disease stabilization and prolonged time to recurrence. Many of the patients targeted in these clinical trials have had malignant melanoma, and a proportion of these patients displayed evidence of immune recognition to the target antigen prior to vaccine therapy. In virtually all cases, patients have been selected for inclusion in CG antigen vaccine trials based on the expression of the antigenic target in tumor biopsies. To expand the patient population that would benefit from this ▶ immunotherapy approach, a number of investigators have proposed using DNA methyltransferase and/or HDAC inhibitors (which are FDA approved and known to augment CG antigen gene expression) in combination with CG antigen directed vaccines. The potential benefit of this multimodality approach awaits clinical testing.

Cross-References ▶ 5-aza-20 Deoxycytidine ▶ Bladder Cancer ▶ Cancer Vaccines

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▶ Chemotherapy ▶ Epigenetic ▶ Immunotherapy ▶ Melanoma Antigens ▶ Methylation ▶ SEREX ▶ T-Cell Response ▶ Testicular Cancer

Cancer of B-Lymphocytes (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi: 10.1007/978-3-642-16483-5_4331 (2012) Recombinant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3205. doi: 10.1007/978-3-642-16483-5_4991 (2012) T Cell. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3599. doi: 10.1007/978-3-642-16483-5_5645 (2012) Trophoblast. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3785. doi: 10.1007/978-3-642-16483-5_5989

References Davis ID, Chen W, Jackson H et al (2004) Recombinant NY-ESO-1 protein with ISOMATRIX adjuvant induces broad integrated antibody and CD4+ and CD8+ T cell responses in humans. Proc Natl Acad Sci U S A 101:10697–10702 Scanlan MJ, Gure AO, Jungbluth AA et al (2002) Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol Rev 188:22–32 Scanlan MJ, Simpson AJG, Old LJ (2004) The cancer/ testis genes: review, standardization, and commentary. Cancer Immun 4:1–15 Simpson AJG, Caballero OL, Jungbluth A et al (2005) Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 5:615–625

Cancer of B-Lymphocytes ▶ B-Cell Tumors

Cancer of the Large Intestine ▶ Colorectal Cancer Clinical Oncology

See Also (2012) Adjuvant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 75. doi: 10.1007/978-3-642-16483-5_107 (2012) Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 208. doi: 10.1007/978-3-642-16483-5_312 (2012) Antigen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 209. doi: 10.1007/978-3-642-16483-5_319 (2012) Autologous Typing. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 317. doi: 10.1007/978-3-642-16483-5_482 (2012) Chromatin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 825. doi: 10.1007/978-3-642-16483-5_1125 (2012) DNA Methylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1140. doi: 10.1007/978-3-642-164835_1682 (2012) Gametogenic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1493. doi: 10.1007/978-3-642-16483-5_2311 (2012) HLA. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1706. doi: 10.1007/978-3-642-16483-5_2765 (2012) Hypomethylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1791. doi: 10.1007/978-3-642-16483-5_2922

Cancer of the Lung ▶ Lung Cancer Clinical Oncology

Cancer Prevention ▶ Cancer Causes and Control

Cancer Prevention with Green Tea ▶ Green Tea Cancer Prevention

Cancer Process of the Large Intestine ▶ Colon Cancer Genomic Pathways

Cancer Stem Cells Targeted Drug Development

Cancer Stem Cell Therapies ▶ Targeting Cancer Stem Cells

Cancer Stem Cells ▶ Stem-Like Cancer Cells

Cancer Stem Cells Targeted Drug Development Galina I. Botchkina Department of Pathology, Stony Brook University, Stony Brook, NY, USA Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY, USA

Definition Cancer stem cells (CSCs) targeted drug development refers to the treatment strategy focused on the eradication or promotion of differentiation of CSCs and not only on tumor shrinkage.

Characteristics Cancer Stem Cells The consensus definition of a cancer stem cell (CSC) is “a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor,” as determined at the AACR workshop in 2006. Cancer stem cells are thus defined by functional test: their ability to generate physiologically relevant tumors after serial transplantation into immunodeficient animals. This central CSC feature is reflected in the alternative terms, such as “tumor-initiating cell” and “tumorigenic cell,” sometimes used in the literature to describe putative CSCs. Like normal tissue and embryonic

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stem cells, CSCs have the ability to undergo both symmetrical self-renewing cell division, producing identical daughter stem cells that retain self-renewal capacity, and asymmetrical selfrenewing cell division, resulting in one stem cell and one non-stem committed progenitor cell. A third type of possible stem cell division is symmetrical division producing two non-stem committed progenitor cells, leading to stem cell depletion. It is of great therapeutic value to promote this last form of stem cell division. Novel Concept of Carcinogenesis Suggests Alternative Paradigm of Cancer Treatment For the majority of cancer types, tumor regression induced by standard anticancer therapies can lead to only an insignificant increase in the overall survival of patients. The low effectiveness of standard treatment modalities has been attributed to the existence of relatively rare stemlike cells that are highly resistant to therapies and possess the ability to induce and maintain tumor growth and spread. After the first discoveries of CSCs in hematological malignancies and solid tumors, it became increasingly evident that the majority, if not all, of tumors exhibit a hierarchical organization similar to normal tissues: immature pluripotent cells occupy the top of the hierarchy and give rise to the heterogeneous majority of tumor cells at different stages of their maturation (differentiation). To identify novel molecular targets for the development of effective anticancer drugs, the tumor-initiating and drugresistant subset of cancer cells should be purified and characterized. CSC research is rapidly evolving and holds significant promise for the development of the next generation of anticancer therapeutics. Although presently there are no highly selective tools for the isolation of a pure population of CSCs, several cell surface markers or their combinations allow for initial enrichment and purification of the tumor-initiating cells. Thus, it was demonstrated that a single epithelial cell with a particular CSC-relevant phenotype can regenerate a whole tumor in an animal model. It is important to test the stemness state of all candidate cell subpopulations for their ability to serially induce tumors in immunodeficient animals after transplantation of a low number of cells of a particular phenotype.

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Numerous studies on different cancer types have demonstrated that the tumorigenic cells expressing common stemness markers, in particular CD133 and CD44, are not only exceptionally resistant to conventional anticancer drugs, such as 5-FU, oxaliplatin, irinotecan, docetaxel and others, but may also significantly increase in number after treatment. Such an enrichment of the immature stemlike cells is usually manifested as a more drug-resistant and more aggressive recurrence or metastatic disease. In many cancers, the ratio of CSCs correlates with tumor aggressiveness, histologic grade, poor prognosis, and distant metastasis. All of the above indicate that purified CSCs represent a critical source for the identification and validation of novel molecular targets and the development of effective anticancer therapies. Potential Molecular Targets Targeting CSC Signaling Pathways

Theoretically, any molecule that is in high demand in CSCs and any step in stemness-relevant signaling pathways can potentially provide an opportunity for therapeutic intervention. However, the network of mechanisms that regulate stem cells and CSC renewal and carcinogenesis is extremely complex and not completely known. In many types of human cancers, CSCs express the majority of genes and transcription factors characteristic of embryonic stem cells. Thus, they possess the upregulated levels of several key markers of pluripotent stem cells, including Sox2, Oct-4, and c-Myc, compared to differentiated cancer cells. In a variety of systems, such as embryonic stem cells, induced pluripotency cells, normal adult tissue stem cells, and CSCs, a number of critical pathways are conserved, including Wnt, Notch, Hedgehog (Hh), TGFb/BMP, JAK/STAT, FGF/MAPK/PI3K, and others. At least three of them, including Wnt, Notch, and Hh, are frequently deregulated in cancer (numerous reviews analyzed these pathways). Many signaling pathways have redundant functions and consist of multiple receptors, ligands, kinases, signal transducers, and transcription factors, which require complex multidisciplinary study. Nevertheless, several potential molecular targets have been

Cancer Stem Cells Targeted Drug Development

identified. In general, inhibition/modulation of signal transduction pathways can include (a) disruption of the receptor/ligand interaction; (b) abrogation of the cytosolic downstream cascade; and/or (c) abrogation of the nuclear signaling components using monoclonal antibodies, small molecules, and natural phytochemicals. Several CSC-targeted approaches to enhance responsiveness to systemic therapy are presently under development. They include: (i) CSC ablation using antitumor agents such as monoclonal antibodies, small molecules, engineered oncolytic viruses, or activated immune cells; (ii) blockade of CSC function; (iii) reversal of CSC resistance; (iv) CSC-directed differentiation therapy; and (v) targeting the CSC environment. Numerous preclinical therapeutics, as well as clinical trials involving Notch, Wnt and Hh pathway inhibitors, several kinase inhibitors, immunotherapy, and molecular chaperons, are currently under evaluation by many pharmaceutical companies and research laboratories. However, several major weaknesses limit their effectiveness, including poor penetration of solid tumors, inability to cross the blood–brain barrier, and systemic toxicities. Accumulated data indicate that successful normalization of the deregulated activities of multiple signaling pathways requires multiplex, combinatorial therapeutics involving protective natural phytochemicals with diverse, pleiotropic anticancer activities rather than single cytotoxic agents. Targeting CSC Resistance to Therapies

Stem cells in general are evolutionary predisposed to be resistant to any unfavorable conditions, including lack of oxygen, deficit of nutrients, and presence of cytotoxic agents. Induction of chemo and radioresistance is mediated, in part differentially, through many target genes regulated and orchestrated by multiple transcription factors. CSC resistance to conventional chemo and radiation therapies is associated with the deregulation or sustained activation of multiple developmental pathways, including increased Wnt/b-catenin and Notch signaling, upregulation of antiapoptotic Bcl-2 family members, downregulation of proapoptotic machinery, and

Cancer Stem Cells Targeted Drug Development

others. Resistance of CSCs to chemotherapy has been also attributed to the high levels of expression of multidrug ABC transporters family genes, resulting in a more efficient efflux of chemotherapeutic drugs, and the expression of multidrug resistance genes, including MDR1 and BRCP1. In addition, the expression of high cytoplasmic levels of aldehyde dehydrogenase (ALDH) enzyme activity, which inactivates the bioactive metabolic byproducts, as well as the relatively slow proliferation rate of CSCs (quiescence), allows them to escape toxicity by drugs in general and toxicity by cell cycle-dependent drugs targeting rapidly dividing cells in particular. CSC resistance to radiation is attributable to amplified DNA damage repair, although other common mechanisms of resistance can contribute significantly to the insensitivity of CSC to radiotherapy. Targeting Angiogenesis

The contribution of CSCs to angiogenesis is a relatively new but actively investigated field. It was found that CSCs in brain tumors closely interact with endothelial cells and can promote the growth of new vessels. Moreover, CSCs can directly differentiate into endothelial cells, thereby generating the necessary vasculature for secondary tumors. On the other hand, when CSCs are transplanted together with endothelial cells, it enhances their stemness state and accelerates tumor initiation and growth. Although targeting tumor vasculature with conventional anti-VEGF therapies seems reasonable, it has shown only limited survival benefit. This data suggests that a combination of VEGF production suppression with specific targeting of CSCs might be more effective. Targeting CSCs with Phytochemicals (Nutraceuticals)

Phytochemicals (also known as phytonutrients, plant compounds believed to have healthprotecting qualities) and nutraceuticals (natural foods or supplements with therapeutic effects) are becoming increasingly common as a form of alternative or complementary anticancer therapy. It is well known that cancer rates in Asia are

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significantly lower compared to those in Western countries, suggesting the existence of some epidemiological factors such as diet. Growing clinical and experimental data suggest that several natural phytochemicals, including curcumin, green tea, and soy isoflavones, which are consumed at high rates in Asia, exert multiple anticancer effects. As a rule, naturally derived compounds exert a fairly large spectrum of molecular mechanisms underlying their physiological activities. In particular, these agents induce antiproliferative, anti-invasive, anti-angiogenic, and proapoptotic effects on human cancer cells in vivo and in vitro without cytotoxic effects on healthy cells. For example, the pleiotropic effects of curcumin can be explained in part by its ability to scavenge, as well as to generate reactive oxygen species (ROS), induce apoptosis via rapid generation of ROS, and downregulate the expression of multiple anti-apoptotic proteins. Importantly, there is accumulating data that it can inhibit multiple stem cell-relevant signaling pathways, decrease the proliferative potential of CSCs, and/or induce their differentiation. Several clinical trials showed improved efficacy of conventional anticancer drugs, including 5-FU, dasatinib, and oxaliplatin, when used in combination with curcuminoids. Since curcumin has low biological availability and bioactivity, many laboratories are presently focused on the development of chemically modified or synthetic phytochemicals with improved characteristics. In this context, pan-inhibitory activities of the combination of new-generation toxoids (such as SBT-1214) with synthetic analogs of curcumin (such as CMC2.24) against multiple stemnessrelevant genes and transcription factors are highly promising. Other phytochemicals, such as resveratrol, cyclopamine, piperine, and the combination of piperine with curcuminoids, also exert pleiotropic anticancer and CSC-targeted activities. Cyclopamine is a naturally occurring teratogenic steroidal alkaloid isolated from the corn lily (Veratrum californicum). Cyclopamine inhibits Hedgehog signaling at the level of Smoothened. The effect of cyclopamine was uncovered when lambs fed to ewes grazing on Veratrum californicum were born with cyclopia (single

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eye). A similar phenotype results from mutations in Sonic hedgehog (Shh). Resveratrol, a natural polyphenol found in grapes, berries, red wine, and peanuts, may inhibit the proliferation and tumorigenicity of the CD133+ cells in vitro and in vivo and may enhance sensitivity to radiotherapy. In particular, the anticancer properties of resveratrol are realized by inhibiting the Notch pathway and induction of apoptosis via Fas-, p53-, and p21-mediated pathways. The steroidal alkaloid cyclopamine affects CSCs via downregulation of the Hh signaling. However, since each signaling pathway may contain multiple ligands with redundant functions, the combinatorial targeting of several pathways is more likely to successfully eliminate CSCs. Thus, neither inhibition of the Shh pathway with cyclopamine nor inhibition of mTOR signaling with rapamycin alone, but only a combination of inhibitors of these pathways, could deplete the CSCs pool. Importantly, in vivo studies have demonstrated that combined therapy with cyclopamine, rapamycin, and gemcitabine was well tolerated and resulted in tumor-free, long-time survival. CSC-Targeted Preclinical Evaluation of the Anticancer Agents Preclinical evaluation of the candidate anticancer agents is traditionally based on the use of two-dimensional cultures of the total, unselected cancer cells. However, this in vitro model ignores rare yet functionally significant, highly drugresistant tumor-initiating cells and thereby has low relevance to the complexity and pathophysiology of in vivo tumors. Monolayer cultures have unnatural cell-to-cell and cell-to-matrix contacts, which can significantly affect their phenotype, signal transduction pathways, and drug response. Since they are directly exposed to medium content and to oxygen, which is a key signal for many important biological processes including stem cell self-renewal, apoptosis, differentiation, and migration, studies on two-dimensional cancer cell cultures may lead to inaccurate conclusions. This represents one of the reasons for the high rate of attrition of candidate anticancer agents. Thus, only 5% of agents that have anticancer activity in preclinical development are licensed. In addition,

Cancer Stem Cells Targeted Drug Development

even highly purified CSCs can undergo relatively fast differentiation after being placed in standard adherent culturing conditions. An alternative three-dimensional model of floating cancer spheroids was established by Sutherland and colleagues long before the isolation and characterization of CSCs. This model is largely used in cancer research, since it is more closely related to original tumors than to the cancer cell monolayers with respect to cell morphology, metabolic and proliferative gradients, oxygen and drug penetration, cell–cell junctions, kinase activation, and other parameters. Floating cancer spheroids are organized hierarchically; can be passaged for many generations, which suggests that they contain a population of cells with extensive self-renewal capacity; coexpress multiple CSC markers, including CD133, CD166, CD44, CD24, CD29, Msi-1, Lgr5, and nuclear localization of b-catenin; and possess an increased resistance to chemo and radiotherapy. Therefore, it is conceivable that CSCs represent the most crucial target in the development of a new generation of anticancer drugs and that the search for effective therapeutic interventions should be focused on the evaluation of the status of cancer-specific tumor-initiating cells and not only on the bulk cancer cells or tumor shrinkage. However, the adequate cultivation and efficient propagation of CSCs in vitro and in vivo is critical for studying CSC biomolecular characteristics, as well as for high-throughput drug screening and rational drug development based on novel CSC markers and signaling pathways. Accumulated data indicate that early passage patient-derived mice tumor xenografts and early passage floating spheroids induced by purified patient-derived cancer-specific CSCs may be relatively appropriate models for CSC-targeted research and drug development. Conclusion Selective molecular targeting of cancer stem cells is a rapidly developing field in oncology and holds high promise for the efficient eradication of cancer. Progress in this direction depends on a better understanding of CSC biology and carcinogenesis, prompt and efficient translation of scientific

Cancer Stem-Like Cells

findings into clinical research, and adequately organized clinical trials. The extreme heterogeneity of human cancers requires the establishment of patient-derived low-passage CSC lines and CSC-relevant in vivo and in vitro models, critical for the identification of novel clinically relevant molecular targets. On the other hand, new criteria for the efficacy of anticancer therapies should be based on comparative, multidisciplinary analyses of the posttreatment status of CSCs and CSC-relevant signaling pathways, as well as long-term follow-up for possible tumor recurrence, and not exclusively on the achievement of temporary tumor shrinkage.

References Botchkina G (2013) Colon cancer stem cells–from basic to clinical application. Cancer Lett 338(1):127–140. doi: 10.1016/j.canlet.2012.04.006 Botchkina GI, Zuniga ES, Das M, Wang Y, Wang H, Zhu S, Savitt AG, Rowehl RA, Leyfman Y, Ju J, Shroyer K, Ojima I (2010) New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheriods induced by purified colon tumor-initiating cells. Mol Cancer 9:192–204 Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CHM, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells: perspectives on current status and future directions. AACR workshop on cancer stem cells. Cancer Res 66:9339 Harris PJ, Speranza G, Ullmann CD (2012) Targeting embryonic signaling pathways in cancer therapy. Expert Opin Ther Targets 16:49–66 Takebe N, Harris PJ, Warren RQ, Ivy SP (2011) Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol 8:97–106 Zhang Y, Gu Y, Lee H-M, Hambardjieva E, Vranková K, Golub LM, Johnson F (2012) Design, synthesis and biological activity of new polyenolic inhibitors of matrix metalloproteinases: a focus on chemicallymodified curcumins. Curr Med Chem 19:4348

See Also (2012) Smoothened. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3456. doi:10.1007/978-3-642-16483-5_5383 (2012) Sonic hedgehog. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3471. doi:10.1007/978-3-642-16483-5_5420 (2012) Teratogenic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3651. doi:10.1007/978-3-642-16483-5_5731

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Cancer Stem-Like Cells Gaetano Finocchiaro Unit of Experimental Neuro-Oncology, Istituto Nazionale Neurologico Besta, Milan, Italy

Synonyms Tumor-initiating cells

Definition Cancer stem(-like) cells are those cells that possess the capacity for self-renewal and for causing the heterogeneous lineages of cancer cells that comprise the tumor.

Characteristics The definition follows a consensus at a workshop on cancer stem(-like) cells (CSC) organized by the American Association for Cancer Research (AACR). There is considerable debate and some controversy on the CSC concept, so that a consensus definition is required. The importance of the debate is proportional to its relevance to the change in our perception of cancer, intrinsic to the CSC paradigm, implying that not all cancer cells are equal but that only a small fraction of them is endowed with the properties of perpetuating the disease. This hierarchical model has not only important biological consequences but also relevant therapeutic implications, as we discuss in this essay. The CSC paradigm fits in a model of cancer as a caricature of an organ that is already present in the literature as suggested by data published 30–40 years ago. In particular, Hamburger and Salmon established growth conditions for cancer cells in soft agar medium and found that tumor stem cell colonies, arising from different types of cancer with 0.001–0.1% efficiency, had differing growth characteristics and colony morphology. Studies by Dick and coworkers in the 1990s

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showed that in several forms of acute myeloid leukemia (AML) cells that could engraft in immunodeficient mice are restricted to a minority subpopulation defined as [CD34+/CD38neg]: these cells, therefore, shared a cell surface phenotype with normal human primitive hematopoietic progenitors, suggesting that they may have originated from normal stem cells rather than from committed progenitors. Also of interest was the observation that leukemic cells engrafted in NOD-SCID mice (nonobese diabetic-severe combined immunodeficiency: an immunodeficient mouse strain characterized by lack of B, T, and NK lymphocytes) showed similar phenotypic heterogeneity to the original donor: thus, [CD34+, CD38neg] retain the differentiating capacity necessary to give rise to CD38+ and Lin+ cells (lineage positive). The presence of CSC has also been demonstrated in chronic myeloid leukemia (CML). This disease has a chronic phase and a terminal stage; the blast crisis and molecular events underlying this evolution are not completely understood. In the chronic phase, the chromosomal translocation t(9:22)BCR-ABL, a diagnostic marker of CML, can be detected in most circulating mature lineages. In the blast crisis, however, highly undifferentiated BCR-ABL+ cells accumulate in the blood. In particular, an expansion of granulocyte-macrophage progenitors (GMP) is present in blast cells, showing aberrant acquisition of self-renewal properties and nuclear expression (i.e., activation) of beta-catenin, a key, positive regulator of stem cell self-renewal. These observations imply that during progression of CML the GMP subfraction of leukemic progenitors acquire stem cell characteristics. Thus, the functional hierarchy of CSC can be modified during the natural history of this tumor as a result of its progression. The requirement for a periodical renovation is not only present in blood but also in the skin and epithelia of the respiratory, gastrointestinal, reproductive, and genitourinary systems. Other tissues like brain, previously considered as exclusively post-mitotic, contain stem cells that can be mobilized and activated under conditions of stress, such as hypoxia. Thus the CSC model could also be applied to solid tumors, and a series of papers

Cancer Stem-Like Cells Cancer Stem-Like Cells, Table 1 Molecular markers of cancer stem-like cells Tumor Acute myeloid leukemia Breast cancer

Markers CD34+/CD38neg

References Bonnet and Dick (1997)

Glioblastoma

CD44+/CD24-/ neg CD133+

Myeloma

CD138neg

Prostate cancer

CD44+/ alpha2beta1 integrin high/ CD133+ CD20+ Sca-1+/CD45neg/ Pecam neg/CD34pos CD133+

Al-Hajj et al. (2003) Singh et al. (2003) Matsui et al. (2004) Collins et al. (2005)

Melanoma Lung cancer

Colon cancer

Pancreatic cancer

CD44+/CD24+/ ESA (epithelial specific antigen)+

Fang et al. (2005) Kim et al. (2005)

O’Brien et al. (2007), Ricci-Vitiani et al. (2007) Li et al. (2007)

report data supporting the identification of a stem cell population in different cancers (see Table 1). Initial data were gained in breast cancer where a small population of cells with a CD44+/CD24neglow phenotype appears exclusively capable of tumor initiation. The most malignant of brain tumors, glioblastoma multiforme (GBM), was also found to contain a fraction of neoplastic cells identified and selected on the basis of CD133 expression. Not only could CD133+ cells self-renew and differentiate into different neural lineages but also, in vivo, only the CD133+ cells were able to reinitiate malignant gliomas with phenotype similar to the original tumor. The CSC paradigm may also help explain intratumor heterogeneity, a frequent finding in most cancers: heterogeneity could be consequent to functional diversity of cells at different states of differentiation. On the other hand, the patterns of tumor heterogeneity and gene expression profiles can be highly similar in the original tumor and in distant metastasis.

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Cancer Stem-Like Cells, Table 2 Therapeutic potential of cancer stem-like cells Pathway/mechanism in CSC Angiogenesisincreased production of VEGF Increased resistance to radiation Specific patterns of expression Cell cycle deregulation Resistance to chemotherapy

Potential treatment Bevacizumab

Chk1 and Chk2 inhibitor Dendritic cell targeting Bone morphogenetic protein 4 BCRP1/ MGMT inhibition (?)

References Bao et al. (2006) Cancer Res Bao et al. (2006) Nature Pellegatta et al. (2006) Piccirillo et al. (2006) Liu et al. (2006)

It is conceivable that the existence of cancer stem cells may provide novel therapeutic targets of increased effectiveness in contrasting or even eliminating cancer. Brain tumors have provided a highly fertile ground to start verifying this hypothesis, as outlined in Table 2. Data are piling up indicating that CD133+ GBM CSC are highly proangiogenic, because of the high levels of VEGF expression, and have greater resistance to chemotherapy and radiotherapy. As a consequence, specific therapeutic strategies can be attempted and combined to overcome CSC. Upon radiotherapy CD133+ GBM CSC activates checkpoint kinases 1 and 2 and repair mechanisms more effectively than CD133neg cells. Resistance to chemotherapy can be linked to an intriguing aspect of the CSC phenotype, the side population (SP) phenotype. SP cells have the ability to extrude the DNA binding dye Hoechst 33342 via the drug transporter BCRP1/ABCG2. Interestingly, the BCRP1/ABCG2 pump can also effectively extrude chemotherapeutic drugs such as mitoxantrone. Also related, although of less immediate relevance in the clinical setting, are the observations reported by Pellegatta et al. using glioma neurospheres as a target for dendritic cell (DC, the most potent of antigen presenting cells) immunotherapy. Normal neural stem cells may grow as neurospheres (NS) in the absence of serum and in

the presence of two critical growth factors, EGF and bFGF. NS are enriched in neural stem cells but also contain partially committed progenitors as well as a differentiated progeny. Oncospheres with similar characteristics were obtained from GBM but also from other solid tumors like breast or colon carcinomas. Pellegatta et al. set up a murine model showing that DC loaded with GBM NS are much more effective in protecting mice against the GBM challenge than DC loaded with GBM cells where CSC are poorly represented. Thus, CSC targeting by immunotherapy is feasible and highly effective, opening new scenarios for clinical immunotherapy and supporting the idea that CSC are at the heart of malignant growth. Also of interest is the observation by Piccirillo et al. that treating GBM CSC with the differentiating factor BMP4 can block growth in vitro and avoid tumor formation in the majority of mice in vivo. Given the increasing number of observations supporting the CSC paradigm in different tumors, it is expected that more therapeutically relevant observations will be proposed in the near future. Together with therapeutic and clinical implications, the CSC concept seems to have important consequences for our understanding of tumor biology. Modern genetics and molecular biology have given a definition of cancer as a genetic disease in which a growing burden of mutations leads to a progressively more aggressive and ultimately lethal phenotype (Fig. 1). A Darwinian selection for these mutations, privileging those that can confer resistance to different challenges, like hypoxic stress or immune attack, appears to be the most plausible rationale for making sense of this evolutionary catastrophe. The hierarchical CSC model seems to introduce an element of rigidity in this highly flexible scenario, implying that only cells endowed with stem cell properties can afford tumor perpetuation (Fig. 1). Are these two models different or are they compatible? A convincing answer to this tough question will undoubtedly require a lot of robust science in the time to come but comments can be given on the basis of data that are already available. One important issue that the CSC model addresses is that of the cell of origin for cancer(s): stem cells,

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Cancer Stem-Like Cells

Darwinian

Hierarchical M1

M1 M2

M1

M1

CSC

M1 M4

M1 M2 M3

CC1

CSC

M1 M2

CSC

CC2

CC3

CC4

Integrated M1

M1 M2 CSC

M1

M1

M1 M4

M1 M2 M3

M1 M2 CSC

Cancer Stem-Like Cells, Fig. 1 Biological models for tumor evolution

because they are long-lived and self-renewing, are excellent candidates to play the “cell of origin” role. A stem cell hosting a critical mutation could be quiescent for years and then be engaged in a repair response requiring mobilization and proliferation. For example, hypoxic stress may activate the CXCR4 pathway that not only attracts stem cells but may also favor their proliferation, thus being the spark initiating the cancer fire. However, an initiating mutation could also arise in a more committed progenitor (see the integrated model in Fig. 1): acquisition of a stem-like phenotype could in this context be the consequence of environmental challenges; in vivo, for instance, hypoxia could play an important role in dedifferentiation; in vitro, the modification of growth factors could have similar consequences. Epigenetic changes could play important roles in mediating rapid and genome-wide changes that can substitute for genetic mutations and lead to dedifferentiation. In the Darwinian model, different mutations (M1 through M4) accumulate during evolution and confer heterogeneity. In the Hierachical model, tumor arises in a stem cell, thus becoming a cancer stem cell (CSC): heterogeneity is conferred by asymmetrical

divisions creating different types of cancer cells (CC1 through 4). In the Integrated model, a first mutation (M1) can arise in a progenitor or even a committed cell. During progression, though, external stimuli may give rise to a cancer stem cell that through asymmetric division will create other CSC as well as more differentiated tumor cells.

Cross-References ▶ Stem-Like Cancer Cells

References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100:3983–3988 Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, Hjelmeland AB, Shi Q, McLendon RE, Bigner DD, Rich JN (2006) Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66:7843–7848 Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737

Cancer Vaccines Clarke MF, Dick JE, Dirks PB et al (2006) Cancer stem cells – perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res 66:9339–9344 Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65:10946–10951 Dalerba P, Cho RW, Clarke MF (2007) Cancer stem cells: models and concepts. Annu Rev Med 58:267–284 Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65:9328–9337 Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33 Jamieson CH, Ailles LE, Dylla SJ et al (2004) Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 351:657–667 Kim CFB, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835 Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030–1037 Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, Lu L, Irvin D, Black KL, Yu JS (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:67 Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y, Smith BD, Civin CI, Jones RJ (2004) Characterization of clonogenic multiple myeloma cells. Blood 103:2332–2336 O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106–110 Pellegatta S, Poliani PL, Corno D, Menghi F, Ghielmetti F, Suarez-Merino B, Caldera V, Nava S, Ravanini M, Facchetti F et al (2006) Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas. Cancer Res 66:10247–10252 Piccirillo SGM, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G, Brem H, Olivi A, Dimeco F, Vescovi AL (2006) Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444:761–765 Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115 Sanai N, Alvarez-Buylla A, Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353:811–822 Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828

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Cancer Vaccines Malaya Bhattacharya-Chatterjee1, Sunil K. Chatterjee1, Asim Saha1 and Kenneth A. Foon2 1 University of Cincinnati and The Barrett Cancer Center, Cincinnati, OH, USA 2 The Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Definition A vaccine should activate a unique lymphocyte (B and/or T cell) response, which has an immediate antitumor effect as well as memory response against future tumor challenge (Fig. 1). The primary role of a cancer vaccine is the treatment of cancer or prevention of recurrence in a patient with surgically resected cancer, rather than “prevention” of cancer in a person who has never had cancer. Therefore, cancer vaccines are not thought of in the traditional sense of vaccines that are used for infectious diseases. If the current cancer vaccines prove to be useful in the above respects, then they may have a future role in preventing cancer in persons who have never had cancer but are at high risk for a particular type of cancer.

Characteristics The first and most obvious types of vaccines are prepared from autologous or allogeneic tumor cells. Autologous refers to a setting in which the donor and recipient are the same person, for instance in blood transfusion or transplantation. Alternatively, membrane preparations from tumor cells may be used. In some instances, tumor cell vaccines have been combined with cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-2 (IL-2). With advances in molecular biological approaches, gene modified-tumor cells expressing antigens designed to increase the immune response, or gene modified to secrete cytokines have been an additional tool used in vaccination. In addition,

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Cancer Vaccines Vaccine (cells, lysates, protein, peptide, cDNA) 14–25 mer peptide

CD4 T-cell

TCR CD4

Antigen presenting cell (APC)

MHC Class II

IL-2

TC CD R 6

Exogenous protein from lysed tumor

MHC 9–10 mer Class I peptide

CD8 T-cell

R TC 8 CD

Tumor cell undergoing lysis

IL-4 IL-5 IL-10

Tumor cell

B-cell

Cancer Vaccines, Fig. 1 T-cell activation. T-cells recognize antigens as fragments of proteins (peptides) presented with major histocompatibility complex (MHC) molecules on the surface of cells. The antigen presenting cell processes exogenous protein from the vaccine or from the lysed tumor cell in to a peptide and presents the 14/25 mer peptide to CD4 helper-T-cells on a class II molecule.

There is also data that suggests exogenous proteins can be processed into 9/10 mer peptides that may be presented on MHC class I molecules to CD8 cytotoxic T cells. Activated Th1 CD4 helper T-cells secrete Th1 cytokines such as IL-2 that upregulate CD8 cytotoxic T cells. Activated Th2 CD4 helper T-cells secrete Th2 cytokines such as IL-4, IL-5, and IL-10 that activate B cells

increase in our knowledge of tumor associated antigens (TAA) have led to the use of purified TAAs, DNA-encoding protein antigens, and/or protein derived peptides. All of these approaches are currently being tested in the clinic. Mechanistically, the ultimate aim of a vaccine is to activate a component of the immune system such as B lymphocytes, which produce antibodies or T lymphocytes, which directly kill tumor cells. Antibodies must recognize antigens in the native protein state on the cell’s surface. Once bound, these molecules can mediate antibody-dependent cellular cytotoxicity (ADCC) or complementmediated cytotoxicity, both mechanisms which are capable of destroying tumor cells. ADCC is a passive immune response in which the Fc fragment of a (therapeutic) monoclonal antibody binds or ligates activating immunoglobulin Fc receptors, e.g., Fc RI (CD64), Fc RIIa (CD32a), Fc RIIc (CD32c), or Fc RIII (CD16), present on monocytes, macrophages, granulocytes, and natural killer (NK) cells, driving cytotoxic effector functions to target membrane-associated antigens.

T lymphocytes, on the other hand, recognize proteins as fragments or peptides that vary in size, presented in the context of major histocompatibility (MHC) antigens on the surface of the cells recognized (Fig. 1). The proteins from which the peptides are derived may be cell surface or cytoplasmic proteins. MHC antigens are highly polymorphic, and different alleles have distinct peptide binding capabilities. The sequencing of peptides derived from MHC molecules have led to the discovery of allele-specific motifs that correspond to anchor residues that fit into specific pockets on MHC class I or II molecules. T Lymphocytes There are two types of T lymphocytes, helper T lymphocytes and cytotoxic T lymphocytes (CTLs), that recognize antigens through a specific T cell receptor (TCR) in close conjunction to the CD3 molecules, which is responsible for signaling. CD4 helper T cells recognize antigens in association with class II MHC gene products, and CD8 positive CTLs recognize antigens in

Cancer Vaccines

association with class I MHC gene products. CD4 helper T cells are activated by binding via their TCR to class II molecules that contain 14–25 amino acid peptides in their antigen-binding cleft. Specialized antigen presenting cells (APCs), such as dendritic cells (DCs), macrophages, and B lymphocytes, capture extracellular protein antigens, internalize and process them, and display class II-associated peptides to CD4 helper T cells. The CD8 positive CTLs are activated by binding via their TCR to class I molecules that contain 9–10 amino acid peptides in their antigen-binding cleft. All nucleated cells can present class I-associated peptides, derived from cytosolic proteins such as viral and tumor antigens, to CD8 positive T cells. There are two types of CD4 helper T cells capable of generating either antibody or cellmediated immune responses, based on the type of signaling they receive. Th1 CD4 helper T cells stimulate cell-mediated immunity by activating CTLs through the release of cytokines such as IL-2. Th2 CD4 helper T cells mediate an antibody response through the release of cytokines such as IL-4 and IL-10. Tumor Cells The most straightforward means of immunization is the use of whole tumor cell preparations (either autologous or allogeneic tumor cells). The advantage of this approach is that the potential TAAs are presented to the immune system for processing and presentation to the appropriate T cell precursors. The difficulty with this approach lies in the availability of fresh autologous tumor material and the scarcity of well-characterized long-term tumor cell lines. Regardless, whole tumor cell vaccines have been an area of intense interest. A variety of trials using autologous tumors for colon cancer and malignant melanoma have been reported. In one trial, freshly thawed autologous colon cancer cells were inactivated with radiation, mixed with BCG (bacille CalmetteGuerin) and injected into patients who had their primary colon cancer resected but were at risk for recurrence. This study did reveal disease-free survival and overall survival trends in favor of the vaccine arm. In a melanoma study, autologous

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tumor cells were mixed with dinitrophenyl (DNP) and mixed with BCG. Promising results were reported for patients with metastatic disease and for patients with locally resected melanoma. The weakness of autologous cell vaccines can be overcome with the allogeneic approach: First, an allogeneic vaccine is generic and developed from cell lines selected to provide multiple TAAs and a broad range of HLA expression. Second, allogeneic cells are more immunogeneic than autologous cells. Third, there is no requirement to obtain tumor tissue by surgical resection for a prolonged course of immunotherapy. A polyvalent melanoma cell vaccine called CancerVax developed for allogeneic viable melanoma cell lines has demonstrated promising results for patients with resected metastatic disease and for resected local disease. Randomized phase III studies are ongoing in the United States comparing CancerVax plus BCG versus BCG for patients with stage III melanoma. Another variation of cell vaccines is using “shed” antigen vaccines. These are vaccines that are prepared from the material shed by viable tumor cells into culture medium. The potential advantage is that it contains a broad range of antigens expressed on the surface of melanoma cells and the shed antigens are partially purified. Trials of such vaccines in melanoma patients have demonstrated specific humoral and cellular immune responses in patients and promising early clinical results. Another approach to tumor cell vaccines is the introduction of foreign genes encoding cytokines such as IL-2 and GM-CSF into tumor cells. Alternatively, molecules designed to increase the immunogenicity of the tumor cell such as CD80 and CD86. Gene transfer can be accomplished by transfection of plasmid constructs (electroporation) or transduction using a viral vehicle such as a retrovirus or an adenovirus. Another option tested for gene transfer is physical gene delivery in which a plasmid or “naked” DNA is delivered directly into tumor cells. There are a number of mechanisms to carry this out including liposomes as gene carriers, use of a “gene gun,” electroporation and calcium phosphate-mediated gene transfer. In one phase I trial, 21 patients with

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metastatic melanoma were vaccinated with irradiated autologous melanoma cells engineered to secrete human GM-CSF. Metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction. Peptides and Carbohydrates An advantage to peptide vaccines is that they can be synthetically generated in a reproducible fashion. The major disadvantage is that they are restricted to a single HLA molecule and are not of themselves very immunogenic. To increase their immunogenicity, peptides may be injected with adjuvants, cytokines or liposomes or presented on DCs. Whole proteins have the advantage over peptides in that they can be processed for a wider range of MHC class I and II antigens. Mucins such as MUC I are heavily glycosylated high molecular weight proteins abundantly expressed on human cancers of epithelial origin. The MUC I gene is over-expressed and aberrantly glycosylated in a variety of cancers including colorectal cancer. MUC 1 is being widely used as a focus for vaccine development. Using expression-cloning techniques, several groups have cloned the genes encoding melanoma antigens recognized by T cells and have identified the immunogenic epitopes presented on HLA molecules. Ten different melanoma antigens have been identified. Direct immunization using the immunodominant peptides from the tumor antigens or recombinant viruses such as adenovirus, fowlpox, and vaccinia virus encoding the relevant genes have been pursued to immunize patients with advanced melanoma. Initial results have demonstrated increased antitumor T cell reactivity in patients receiving peptide immunization. Immunization in melanoma patients with melanoma antigens has been reported. One study showed that immunization of melanoma patients with MAGE-1 peptide pulsed on DCs induced melanoma-reactive and peptide specific CTL responses at the vaccination sites and at distant tumor deposits. Administration of the gp-100 molecule in conjunction with high-dose bolus

Cancer Vaccines

IL-2 to 31 patients with metastatic melanoma revealed an objective response of 42%. This is compared with the typical response of high-dose systemic IL-2 without peptide of only 15%. Based on these data, a randomized trial was initiated to compare the peptide vaccine plus IL-2 versus IL-2 alone in metastatic melanoma patients. Immunization against tumor-associated carbohydrate antigens has also been attempted. Carbohydrate antigens typically bypass T cell help for B cell activation. Investigators demonstrated that some carbohydrates may activate an alternative T cell pathway. Vaccine studies have been reported using the GM-2 ganglioside vaccine. Patients were pretreated with low dose cyclophosphamide. After a minimum follow up of 72 months, there was a 23% increase in diseasefree interval and a 17% increase in overall survival in patients who produced antibody against GM-2. This suggested a benefit to the GM-2 ganglioside vaccine which has led to a current phase III trial. Recombinant Vaccines Expressing Tumor Antigens The ▶ carcinoembryonic antigen (CEA) is highly expressed on ▶ colorectal cancer and on a variety of other epithelial tumors and is thought to be involved in cell-cell interactions. A recombinant vaccinia virus expressing human CEA (rV-CEA) stimulates specific T cell responses in patients. This was the first vaccine to demonstrate human CTL responses to specific CEA epitopes and class I HLA-2 restricted T-cell mediated lysis, and demonstrated the ability of human tumor cells to endogenously process CEA to present a specific CEA peptide in the context of a MHC for T-cell mediated lysis. Anti-idiotype Vaccines The idiotype network offers an elegant approach to transforming epitope structures into idiotypic determinants expressed on the surface of antibodies. According to the network concept, immunization with a given TAA will generate production of antibodies against these TAA, which are termed Ab1; the Ab1 is then used to generate a series of anti-idiotype antibodies

Cancer Vaccines

against the Ab1, termed Ab2. Some of these Ab2 molecules can effectively mimic the threedimensional structure of the TAA identified by the Ab1. These Ab2 can induce specific immune responses similar to those induced by the original TAA and therefore can be used as surrogate TAAs. Immunization with Ab2 can lead to the generation of anti-anti-idiotypic antibodies (Ab3) that recognize the corresponding original tumor-associated antigen identified by Ab1. The anti-idiotype antibody represents an exogenous protein that should be endocytosed by APCs and degraded to 14–25 mer peptides to be presented by class II antigens to activate CD4-helper T cells. Activated Th2 CD4-helper T cells secrete cytokines such as IL-4 that stimulate B cells that have been directly activated by Ab2 to produce antibody that binds to the original antigen identified by Ab1. In addition, activation of Th1 CD4-helper T cells secrete cytokines that activate T cells, macrophages, and natural killer cells that directly lyse tumor cells and, in addition, contribute to ADCC. Th1 cytokines such IL-2 also contribute to the activation of a CD8-CTL response. This represents a putative pathway of endocytosed anti-idiotype antibody. The antiidiotype antibody may be degraded to 9/10 mer peptides to present in the context of class I antigens to activate CD8-cytotoxic T cells, which are also stimulated by IL-2 from Th1 CD4-helper T cells. Anti-idiotype antibodies that mimic distinct TAAs expressed by cancer cells of different histology have been used to implement active specific immunotherapy in patients with malignant diseases including colorectal carcinoma, malignant melanoma, breast cancer, B cell lymphoma and leukemia, ovarian cancer, or lung cancer. A murine monoclonal anti-idiotype antibody, 3H1 or CeaVac, which mimics CEA was developed by the authors and was used in a phase I clinical trial. Among 23 patients with advanced colorectal cancer, 17 patients generated anti-antiidiotypic Ab3 responses, and 13 of these responses were proven to be true anti-CEA responses. The median survival of 23 evaluable patients was 11.3 months, with 44% 1-year survival. Toxicity was limited to local swelling and

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minimal pain. In another clinical trial, 32 patients with resected colorectal cancer were randomized to treatment with CeaVac. All 32 patients entered into this trial generated high-titer IgG anti-CEA antibodies, and ~75% generated CEA specific T cell responses. These data demonstrated that 5fluorouracil based chemotherapy regimens did not have any adverse effect on the immune response developed by CeaVac. TriGem, an anti-idiotype monoclonal antibody that mimics the disialoganglioside GD2, was used as a vaccine in clinical trial consisting of 47 patients with stage IV melanoma. Forty of 47 patients developed high-titer IgG anti-GD2 antibodies. Seventeen patients were stable on the study from 8 to 34 months. Disease progression occurred in 27 patients on the study from 1 to 9 months. For the 26 patients with soft tissue disease, the median overall survival has not been reached. For 18 patients with visceral metastasis, the median overall survival was 15 months. These results exceed historical controls with stage IV melanoma. Another anti-idiotype monoclonal antibody, TriAb, which mimics the human milk fat globule (HMFG) membrane antigen, is highly overexpressed on breast cancer cells and a variety of other cancer cells, including ovarian cancer, non small-cell lung cancer, and colon cancer. Immunizations with this anti-idiotype antibody elicited both anti-HMFG antibodies and idiotype specific T cell responses in patients with breast cancer in the adjuvant setting as well as in patients with advanced disease following autologous bone marrow transplantation. Although these initial clinical data are promising, active specific immunotherapy with anti-idiotype antibodies need to be tested in combination with other conventional and experimental therapies to overcome the multiple mechanisms by which tumor cells escape immune recognition and destruction. The anti-idiotype vaccine therapy for patients with minimal residual disease might be curative in the adjuvant setting and may improve the quality of patients’ life. Dendritic Cell-based Vaccines DCs are the professional APCs of the immune system and are present in peripheral tissues, where they capture antigens. These antigens are

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subsequently processed into small peptides as the DCs mature and move toward the draining secondary lymphoid organs. There the DCs present the peptides to naïve T cells, thereby inducing a cellular immune response that involves both CD4 T helper 1 (Th1) cells and cytotoxic CD8 T cells. DCs are also important at inducing humoral immune response through their capacity to activate naïve and memory B cells. DCs can also activate natural killer (NK) cells and natural killer T (NKT) cells. Therefore, DCs can conduct all of the elements of the immune orchestra, and they are therefore a fundamental target and tool for vaccination. The development of ex vivo techniques for generating large numbers of DCs in vitro from mouse bone marrow cells supplemented with either GM-CSF alone or GM-CSF plus IL-4 allowed the approach of DC-based tumor vaccination to be fully exploited. Numerous studies in mouse tumor models have shown that DCs pulsed with tumor antigens can induce protective and therapeutic antitumor immunity. In 1996, Hsu et al. reported the first DC-based clinical trial of follicular B cell lymphoma patients who were treated with peripheral blood-derived DCs pulsed with a tumor-specific idiotype (Id) protein. Of these ten patients, eight developed a proliferative cellular response to Id and one patient developed an Id-specific CTL response. However, tumor regression was not reported in these DC-vaccinated patients. In several other trials, a correlation between immunological and clinical outcome has been demonstrated. However, the efficacy of therapeutic DC-based vaccination has been modest and these trials have had similar clinical outcome: mainly, immunized patients often demonstrate significant activation of adaptive immunity to the targeted tumor antigen(s) as shown by various methods such as tetramer analysis, IFN-g ELISPOT, and 51Cr-release assay; but only a limited number of immunized patients demonstrate significant tumor regression. The complexity of the DC system requires rational manipulation of DCs to achieve protective or therapeutic immunity. Further research is needed to analyze the immune responses induced in patients by distinct ex vivo generated DC

Cancer Vaccines

subsets that are activated through different pathways. These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs. Overall, we remain optimistic that improved cancer vaccines will ultimately yield favorable clinical results, particularly after these approaches have been modified in a manner that integrates progress related to the physiology of DCs and our improved understanding of how tumors and the host immune system interact with each other. Conclusion There exist several promising immunologic approaches to vaccine therapy of cancer. The challenge of immunotherapy research is to determine which combination of approaches leads to a favorable clinical response and outcome. Several studies have shown enhanced survival of patients receiving vaccines; however, a randomized phase III clinical trial has yet to show a statistically significant improvement in the survival of such patients.

Cross-References ▶ Carcinoembryonic Antigen ▶ Cancer Germline Antigens ▶ Colorectal Cancer ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶ T-Cell Response

References Bhattacharya-Chatterjee M, Chatterjee SK, Foon KA (2002) Anti-idiotype antibody vaccine therapy for cancer. Expert Opin Biol Ther 2:869–881 Dalgleish AG, Whelan MA (2006) Cancer vaccines as a therapeutic modality: the long trek. Cancer Immunol Immunother 55:1025–1032 Emens LA (2006) Roadmap to a better therapeutic tumor vaccine. Int Rev Immunol 25:415–443 Nestle FO, Farkas A, Conrad C (2005) Dendritic-cellbased therapeutic vaccination against cancer. Curr Opin Immunol 17:163–169 Saha A, Chatterjee SK, Mohanty K et al (2003) Dendritic cell based vaccines for immunotherapy of cancer. Cancer Ther 1:299–314

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See Also (2012) BCG. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 356. doi: 10.1007/978-3-642-16483-5_560 (2012) FcR. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1386. doi: 10.1007/978-3-642-16483-5_2135

Candidate of Metastasis 1 ▶ P8 Protein

C Canine Transmissible Tumor (CTVT) Cancer Without Disease

▶ Sticker Sarcoma

▶ Dormancy

Cannabinoids

Cancer/Testis Antigen1b ▶ NY-ESO-1

Guillermo Velasco and Manuel Guzmán Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain

Synonyms

Cancer-Mediated Bone Loss

Endocannabinoids; Marijuana; cannabinoids; Synthetic cannabinoids

Phyto-

▶ Bone Loss Cancer Mediated

Definition

Cancers of Hormone-Responsive Organs or Tissues ▶ Endocrine-Related Cancers

Cancer-Testis Antigen 1.11 ▶ MAGE-A11

Cannabinoids are a family of lipid molecules that comprises a series of metabolites produced by the hemp plant Cannabis sativa (the phytocannabinoids), several fatty-acid derivatives endogenously produced by most animals (the endogenous ligands for cannabinoid receptors), and different synthetic compounds structurally or functionally related with the natural cannabinoids. Activation of cannabinoid receptors by some of these molecules reduce the symptoms associated to cancer chemotherapy and inhibit the growth of tumor cells in culture and in animal models of tumor xenografts.

Cancer-Testis Antigens ▶ Cancer Germline Antigens

This entry was first published in the 2nd edition of the Encyclopedia of Cancer in 2009.

778 Cannabinoids, Fig. 1 Cannabinoids, cannabinoid receptors, and their mechanisms of action. (a) D9-tetrahydrocannabinol (THC), the main active component of marijuana, and the endocannabinoids anandamide and 2arachidonoylglycerol are ligands of cannabinoid receptors. (b) Both CB1 and CB2 receptors belong to the family of G-protein-coupled receptors. Binding of cannabinoids to cannabinoid receptors leads, among other actions and depending on the cell context, to: inhibition of adenylyl cyclase, modulation of the activity of several ion channels, modulation of phosphatidylinositol-3 kinase (PI3K) and of mitogen activated protein kinase cascades, or stimulation of ceramide generation

Cannabinoids

a Anandamide O OH OH

N

2-Arachidonoylglycerol THC

OH

O O

O

OH

b

K+

CB1 Ca2+

CB2

K+ Gi/o

Ca2+

Gi/o

AC

Ceramide P13K

Characteristics The hemp plant Cannabis sativa produces approximately 70 unique compounds known as cannabinoids, of which D9-tetrahydrocannabinol (THC) is the most important owing to its high potency and abundance in cannabis. THC exerts a wide variety of biological effects by mimicking endogenous substances – the endocannabinoids anandamide and 2-arachidonoylglycerol – that bind to and activate specific cannabinoid receptors (Fig. 1a, b). So far, two cannabinoidspecific G-protein-coupled receptors have been cloned and characterized from mammalian tissues: The CB1 receptor is particularly abundant in discrete areas of the brain, but is also expressed in peripheral nerve terminals and various extraneural sites. In contrast, the CB2 receptor

MAPKs

was initially described to be present in the immune system, although it has been shown that expression of this receptor also occurs in cells from other origins including many types of tumor cells. Signaling Pathways Modulated by Cannabinoid Receptors Most of the physiological, therapeutic, and psychotropic actions of cannabinoids rely on the activation of CB1 and CB2 receptors (Fig. 1a, b). Extensive molecular and pharmacological studies have demonstrated that cannabinoids inhibit adenylyl cyclase through CB1 and CB2 receptors. The CB1 receptor also modulates ion channels, inducing, for example, inhibition of Nand P/Q-type voltage-sensitive Ca2+ channels and activation of G protein-activated inwardly

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rectifying K+ channels. Besides these wellestablished signaling events that mediate – among others – the neuromodulatory actions of the endocannabinoids, cannabinoid receptors also modulate several pathways that are more directly involved in the control of cell proliferation and survival, including extracellular signalregulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and focal adhesion kinase. In addition, cannabinoids stimulate the generation of the bioactive lipid second messenger ceramide via two different pathways: sphingomyelin hydrolysis and ceramide synthesis de novo.

Mechanism Involved in Appetite Stimulation by Cannabinoids

Palliative Effects of Cannabinoids in Cancer Cannabinoids have been known for several decades to exert palliative effects in cancer patients, and nowadays capsules of THC (Marinol-TM) and its synthetic analog nabilone (Cesamet-TM) are approved to treat nausea and emesis associated with cancer chemotherapy. In addition, several clinical trials are testing other potential palliative properties of cannabinoids in oncology such as appetite stimulation and analgesia.

Mechanism Involved in the Analgesic Effect of Cannabinoids

Mechanism Involved in the Antiemetic Effect of Cannabinoids

One of the most important physiological functions of the cannabinoid system is to modulate synaptic transmission. Thus, activation of cannabinoid receptors at presynaptic locations leads to reduced neurotransmitter release. As the CB1 receptor is present in cholinergic nerve terminals of the myenteric and submucosal plexus of the stomach, duodenum and colon, it is likely that cannabinoid-induced inhibition of digestive tract motility is due to blockade of acetylcholine release in these areas. There is also evidence that cannabinoids act on CB1 receptors localized in the dorsal vagal complex of the brainstem – the region of the brain that controls the vomiting reflex. In addition, endocannabinoids and their inactivating enzymes are present in the gastrointestinal tract and may play a physiological role in the control of emesis.

The endogenous cannabinoid system may serve as a physiological regulator of feeding behavior. For example, endocannabinoids and CB1 receptors are present in the hypothalamus, the area of the brain that controls food intake; hypothalamic endocannabinoid levels are reduced by leptin, one of the most prominent anorexic hormones; and blockade of tonic endocannabinoid signaling with the CB1 antagonist rimonabant – inhibits appetite and induces weight loss. CB1 receptors present in nerve terminals and adipocytes also participate in the regulation of feeding behavior.

Cannabinoids inhibit pain in animal models of acute and chronic hyperalgesia, allodynia, and spontaneous pain. Cannabinoids produce antinociception by activating CB1 receptors in the brain (thalamus, periaqueductal gray matter, rostral ventromedial medulla), the spinal cord (dorsal horn), and nerve terminals (dorsal root ganglia, peripheral terminals of primary afferent neurons). Endocannabinoids serve naturally to suppress pain by inhibiting nociceptive neurotransmission. In addition, peripheral CB2 receptors might mediate local analgesia, possibly by inhibiting the release of various mediators of pain and inflammation, which could be important in the management of cancer pain. Antitumoral Effects of Cannabinoids Cannabinoids have been proposed as potential antitumoral agents on the basis of experiments performed both in cultured cells and in animal models of cancer. A number of plant-derived, synthetic, and endogenous cannabinoids are now known to exert antiproliferative actions on a wide spectrum of tumor cells in culture. More importantly, cannabinoid administration to nude mice curbs the growth of various types of tumor xenografts, including lung carcinoma, glioma, thyroid epithelioma, lymphoma, skin carcinoma, pancreatic carcinoma, and melanoma. The requirement of cannabinoid receptors for this antitumoral

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Cannabinoids

− Invasiveness

VEGF MMP2 CB1 CB2

Cannabinoids

ER stress MMP2

+ apoptosis − migration

VEGF − Angiogenesis

+ Apoptosis

Ceramide

Caspase 3

p8 ER stress-related Targets ? CHOP ATF-4 TRB3

ym

? Akt

Cannabinoids, Fig. 2 Mechanism of cannabinoid antitumoral action. (a) Cannabinoid administration decreases the growth of tumors by several mechanisms, including at least: (i) reduction of tumor angiogenesis, (ii) induction of tumor cell apoptosis, and perhaps (iii) inhibition of tumor cell migration and invasiveness. (b) Cannabinoid treatment induces apoptosis of several types of tumor cells via ceramide accumulation and activation of

an ER stress-related pathway. The stress-regulated protein p8 plays a key role in this effect by controlling the expression of ATF-4, CHOP, and TRB3. This cascade of events triggers the activation of the mitochondrial intrinsic apoptotic pathway through mechanisms that have not been unraveled as yet. Cannabinoids also decrease the expression of various tumor-progression molecules such as VEGF and MMP2

activity has been revealed by various biochemical and pharmacological approaches, in particular by determining cannabinoid receptor expression in the tumors and by using selective cannabinoid receptor agonists and antagonists. Although the downstream events by which cannabinoids exert their antitumoral action have not been completely unraveled, there is substantial evidence for the implication of at least two mechanisms: induction of apoptosis of tumor cells and inhibition of tumor angiogenesis (Fig. 2a).

triggers a cascade of events that involves the upregulation of several genes involved in the endoplasmic reticulum (ER) stress response including the activating transcription factor 4 (ATF-4) and the C/EBP-homologous protein (CHOP). These two transcription factors cooperate in the induction of the tribbles homologue 3 (TRB3), a pseudokinase that is involved in the induction of apoptosis (Fig. 2b). The processes downstream of ER stress activation involved in the execution of cannabinoidinduced apoptosis of tumor cells are not completely understood yet but include inhibition of the antiapoptotic kinase Akt and activation of the mitochondrial intrinsic pathway. Of interest, the proapoptotic effect of cannabinoids is selective of tumor cells. For instance, treatment of primary cultured astrocytes with these compounds does not trigger ceramide accumulation, induction of the aforementioned ER stress-related genes, or apoptosis. Furthermore, cannabinoids promote the survival of astrocytes, oligodendrocytes, and neurons in different models of injury, supporting the notion that cannabinoids activate opposite responses in transformed and nontransformed cells.

Induction of Apoptosis

Different studies have shown that the proapoptotic effect of cannabinoids on tumor cells relies on the stimulation of cannabinoid receptors and a subsequent activation of the proapoptotic mitochondrial intrinsic pathway. In glioma and pancreatic tumor cells, treatment with cannabinoids leads to accumulation of the proapoptotic sphingolipid ceramide which in turn leads to upregulation of the stress-regulated protein p8, which belongs to the family of HMG-I/Y transcription factors. The acute increase of p8 levels after cannabinoid treatment

Cannabinoids

Inhibition of Tumor Angiogenesis

To grow beyond minimal size, tumors must generate a new vascular supply (angiogenesis) for purposes of cell nutrition, gas exchange and waste disposal, and therefore blocking the angiogenic process constitutes one of the most promising antitumoral approaches currently available. Immunohistochemical analyses in mouse models of glioma, skin carcinoma, and melanoma have shown that cannabinoid administration turns the vascular hyperplasia characteristic of actively growing tumors to a pattern of blood vessels characterized by small, differentiated, and impermeable capillaries. This is associated with a reduced expression of vascular endothelial growth factor (VEGF) and other proangiogenic cytokines such as angiopoietin-2 and placental growth factor, as well as of type 1 and type 2 VEGF receptors, in cannabinoid-treated tumors. Pharmacological inhibition of ceramide synthesis de novo abrogates the antitumoral and antiangiogenic effect of cannabinoids in vivo and decreases VEGF production by glioma cells in vitro and by gliomas in vivo, indicating that ceramide plays a general role in cannabinoid antitumoral action. Other reported effects of cannabinoids might be related with the inhibition of tumor angiogenesis and invasiveness by these compounds (Fig. 2a, b). Thus, activation of cannabinoid receptors on vascular endothelial cells in culture inhibits cell migration and survival. In addition, cannabinoid administration to glioma-bearing mice decreases the activity and expression of matrix metalloproteinase-2, a proteolytic enzyme that allows tissue breakdown and remodeling during angiogenesis and metastasis. In line with this notion, cannabinoid intraperitoneal injection reduces the number of metastatic nodes produced from paw injection in lung, breast, and melanoma cancer cells in mice. Therapeutic Potential of Cannabinoids as Antitumoral Agents On the basis of these preclinical findings, a pilot clinical study of THC in patients with recurrent glioblastoma multiforme has been run. Cannabinoid delivery was safe and could be achieved without significant psychoactive effects. Also,

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although the limited number of patients involved in the trial did not permit the extraction of statistical conclusions, median survival of the cohort was similar to other studies performed in recurrent glioblastoma multiforme with temozolomide and carmustine, the drugs of reference for the treatment of these tumors. In addition, THC administration correlated with decreased tumor cell proliferation and increased tumor cell apoptosis. The significant antiproliferative action of cannabinoids, together with their low toxicity compared with other chemotherapeutic agents and their ability to reduce symptoms associated to standard chemotherapies, might make these compounds promising new antitumoral agents.

Cross-References ▶ Angiogenesis ▶ Apoptosis ▶ Ceramide ▶ Endoplasmic Reticulum Stress ▶ Matrix Metalloproteinases ▶ P8 Protein ▶ Vascular Endothelial Growth Factor

References Carracedo A, Lorente M, Egia A et al (2006) The stressregulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells. Cancer Cell 9:301–312 Guzman M (2003) Cannabinoids: potential anticancer agents. Nat Rev Cancer 3:745–755 Guzman M, Duarte MJ, Blazquez C et al (2006) A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme. Br J Cancer 95:197–203 Hall W, Christie M, Currow D (2005) Cannabinoids and cancer: causation, remediation, and palliation. Lancet Oncol 6:35–42 Mackie K (2006) Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol 46:101–122

See Also (2012) Allodynia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 138. doi:10.1007/978-3-642-16483-5_193

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782 (2012) G-protein couple receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1587. doi:10.1007/978-3-642-16483-5_2294 (2012) Hyperalgesia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1780. doi:10.1007/978-3-642-16483-5_2902 (2012) Mitochondrial intrinsic pathway. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2333. doi:10.1007/978-3-642-164835_3766 (2012) Pseudokinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3112. doi:10.1007/978-3-642-16483-5_4839 (2012) Tribbles homologue 3. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3783. doi:10.1007/978-3-642-164835_5971 (2012) Tumor xenografts. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3807. doi:10.1007/978-3-642-16483-5_6061

CAP20

Carbon Metabolism Nianli Sang and Chengqian Yin Department of Biology, Drexel University College of Arts and Sciences, Philadelphia, PA, USA

Definition Cells utilize reduced carbon sources including carbohydrates, lipids, and amino acids, to satisfy the basic needs for adenosine triphosphate (ATP), reducing power and building blocks, which are critical for cell survival, growth, and proliferation.

Characteristics

CAP20 ▶ p21

CAR ▶ Chimeric Antigen Receptor on T Cells ▶ Constitutive Androstane Receptor

8-Carbamoyl-3-methylimidazo (5,1-d)-1,2,3,5-tetrazin-4(3H)-one ▶ Temozolomide

Carbohydrate Part of Glycoconjugates ▶ Glycosylation

Three fundamental needs must be satisfied to support the robust proliferation of cancer cells: sufficient amount of ATP production to provide energy, rapid biosynthesis of biomolecules to support cell structure and function, and delicate maintenance of cellular redox status. Carbon metabolism plays an essential role in all three aspects (Yin et al. 2012; Cairns et al. 2011), and oncogenic signaling pathways activate the utilization of carbon sources to facilitate cell survival, growth, proliferation, and cancer progression. There are three major types of carbon sources for cell metabolism: carbohydrates, lipids, and amino acids. Glucose is the major product of carbohydrates after digestion and absorption and is a universal carbon source utilized in cancer cells. Cancer cells have an extraordinary dependence on glucose as they consume a large amount of glucose for glycolysis and lactate fermentation even in the presence of ample oxygen, which is well known as Warburg effect. Upon entering cells via a glucose transporter, glucose is first phosphorylated to glucose 6-phosphate (G6P) by hexokinases. G6P is the common starting point of multiple metabolic pathways. In glycolytic pathway, G6P is finally converted to pyruvate. Glycolysis provides ATP independent of mitochondria and molecular oxygen. Some

Carbon Metabolism

intermediates of the glycolytic pathway are important precursors for biosynthesis of other intermediary metabolites such as nonessential amino acids. Pyruvate can either be reduced to lactate by cytosolic nicotinamide adenine dinucleotide (NADH), reduced or enter the mitochondrion where it will be decarboxylated to acetylCoA or carboxylated to oxaloacetate and then enter the citric acid cycle. The citric acid cycle not only provides various precursors for the biosynthesis of many components such as heme and some amino acids but also transfers electrons to form reducing molecules NADH and flavin adenine dinucleotide (FADH2), reduced for ATP production via oxidative phosphorylation. In the pentose phosphate pathway (PPP), G6P is utilized to produce riboses and NADPH. Riboses are precursors for the biosynthesis of nucleotides and some cofactors, and NADPH is the most important reducing power for biosynthesis and maintenance of intracellular redox status. Triacylglycerol and free fatty acids collectively represent another type of carbon source from diet. Free fatty acids can be oxidized into acetyl-CoA through b oxidation. In addition, absorbed free fatty acids can be directly used as precursors of phospholipids for biomembrane construction. Generally, free fatty acids have little role in the production of NADPH. The carbon skeletons of amino acids also function as carbon source. Generally, carbon skeletons from proteinogenic amino acids can be converted to either ketone or glucose (by gluconeogenesis, except for lysine and leucine). Either way, they may end up in citric acid cycle and be used to produce ATP. Particularly, glutamine plays a vital role in cancer cell metabolism. Like glucose, glutamine is another nutrient which cancer cells have an extraordinary demand for. Through glutaminolysis, glutamine is degraded into glutamate and then a-ketoglutarate (a-KG). After entering the citric acid cycle, a-KG will be used either for energy production and anaplerosis or be converted to malate or isocitrate for NADPH generation. All anaplerotic amino acids may contribute to intracellular NADPH production via either the malate or isocitrate pathway.

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Finally, in carbon metabolism, a key molecule that links catabolism to anabolism is acetyl-CoA. Catabolism of carbohydrates, lipids, or amino acids generates acetyl-CoA, which can be used for the biosynthesis of fatty acids, ketones, mevalonate, isoprenes, and other indispensible biomolecules derived from them, such as cholesterol, heme, quinone and dolichol. Homeostatic Regulation of Carbon Metabolism Cancer cells require continuous and abundant energy supply for their survival, growth, and proliferation. Therefore, cancer cells need a delicate energy sensing and regulating system to maintain the energy homeostasis. The AMP-activated protein kinase (AMPK) plays a crucial role in the process. The decreasing of the ATP level leads to the increasing of AMP concentration, which activates AMPK through allosteric activation and protection against dephosphorylation. AMPK contributes to maintaining the energy level through two aspects: enhancement of ATP production and inhibition of ATP consumption. Activated AMPK activates a lot of catabolic enzymes participating in glycolysis and fatty acid oxidation to increase ATP generation. AMPK is also reported to promote the translocation of glucose transporter 4 (GLUT4) in short term and upregulate the expression of GLUT4 in long term to increase the glucose uptake. On the other hand, the activation of AMPK inhibits the synthesis of many molecules such as fatty acids, cholesterol, glycogen, and proteins. In addition, activated AMPK causes the G1-phase cell-cycle arrest where a large amount of energy is required or even promotes apoptosis through activating the tumor suppressor p53 (Hardie 2011). Besides the reduced biosynthesis of macromolecules, the reducing power is also indispensible for the maintenance of redox homeostasis in the cells. Various causes including normal metabolic processes, irradiation, and pharmaceutical agents result in the generation of reactive oxygen species (ROS). Although moderate levels of ROS are required and beneficial for certain cellular processes such as signal transduction, pathogen killing, and gene expression regulation, high levels of

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ROS damages biomolecules and cell structures. ROS can attack and damage macromolecules including DNA, proteins, and lipids, which is implicated in apoptosis, genetic instability, cancer, and many other diseases. Therefore, cells should maintain adequate levels of reducing power which is usually in the form of NADPH to balance the oxidative and reductive levels. The ratio of [NADPH]/[NADP+] is dynamically regulated by oxidizing reduced carbon sources. NADPH can be regenerated from NADP+ in several metabolic pathways: the pentose phosphate pathway which oxidizes G6P, the oxidation of glutamate catalyzed by glutamate dehydrogenase, the reaction converting isocitrate to a-KG facilitated by isocitrate dehydrogenase 1 and 2 (IDH 1 and 2), and the malate oxidation catalyzed by malic enzyme 1 (Yin et al. 2012). Another important function of carbon source is for the biosynthesis of building blocks. Particularly, biosynthesis of biomembranes has been found to be important for cancer progression. Important enzymes involved in the fatty acid synthesis are acetyl-CoA carboxylase and fatty acid synthase. The key rate-limiting enzyme of the cholesterol synthesis pathway is HMG-CoA reductase. The synthesis of fatty acids and cholesterol is regulated by sterol regulatory element (SRE) and SRE-binding proteins (SREBPs). Newly synthesized SREBP is inserted in the membranes of endoplasmic reticulum (ER), bound to the SREBP cleavage-activating protein (SCAP). When the intracellular sterol level is low, SREBP migrates to the Golgi apparatus, where SREBP is cleaved by site-1 and site-2 protease (S1P and S2P) activated by SCAP. The cleaved and activated SREBP then moves to the nucleus and upregulates more than 30 genes involved in the synthesis of cholesterol, fatty acids, and phospholipids, as well as the NADPH required for the reduced synthesis of these molecules (Horton et al. 2002). In addition to the biosynthesis of biomembrane, carbons are also needed for the generation of the skeletons of nonessential amino acids; some of them are expected to be actively synthesized as intermediary metabolites for the production of macromolecules such as proteins, DNA, and RNA.

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At the organismal level, glucose homeostasis is regulated by insulin, glucagon, and other hormones. At the cellular level, two transcription complexes have been reported to regulate gene expression in response to high glucose concentrations: MondoA/Mlx and MondoB/Mlx. When the intracellular G6P level increases, the two transcription complexes upregulate metabolic enzymes to utilize or store the carbon source. The cellular response to low G6P levels has not been well studied. Since low glucose levels usually result in ATP depletion, AMPK pathway has been considered to play a role in cell response to low-glucose conditions (Yin et al. 2012).

References Cairns RA, Harris IS, Mak TW (2011) Regulation of cancer cell metabolism. Nat Rev Cancer 11(2):85–95 Hardie DG (2011) AMP-activated protein kinase-an energy sensor that regulates all aspects of cell function. Genes Dev 25(18):1895–1908 Horton JD, Goldstein JL, Brown MS (2002) SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109(9):1125–1131 Yin C, Qie S, Sang N (2012) Carbon source metabolism and its regulation in cancer cells. Crit Rev Eukaryot Gene Expr 22(1):17–35

Carbonyl Metabolism Lakshmaiah Sreerama Department of Chemistry and Biochemistry, St. Cloud State University, St. Cloud, MN, USA Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar

Definition Carbonyl metabolism is a general term used to collectively describe the reactions in which either the carbonyl group is formed or carbonyl carbon is reduced and/or further oxidized (Fig. 1). These

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Carbonyl Metabolism, Fig. 1 Metabolism of compounds containing carbonyl groups

reactions are catalyzed by three distinct families of enzymes: • Alcohol dehydrogenases (ADHs) • Aldehyde dehydrogenases (ALDHs) • Aldo-keto reductases (AKRs) Each of these enzyme families catalyzes NAD (P)+- or NAD(P)H-dependent oxidation and/or reduction of the carbonyl carbon (Fig. 1) present in a wide variety of endogenous and exogenous compounds. Some of the endogenous compounds that are substrates for these enzymes are generated as a result of oxidative stress, metabolism of mono- and polyamines, prostaglandins, vitamins, sugars, and steroids. Exogenous compounds that are known to serve as substrates for these enzymes include anticancer drugs, alcohols, and carcinogens. Since these enzymes are involved in redox reactions, they are often referred to as phase I drug metabolizing enzymes. Certain cytochrome P450s and aldehyde oxidase are also capable of carbonyl metabolism; however, their role appears to be minimal in this process, accordingly are not considered here.

Characteristics Alcohol Dehydrogenases (ADHs) ADHs are ubiquitous. They are present in many organisms as well as in most tissue to varying levels. ADHs catalyze NAD+-dependent oxidation of alcohols to aldehydes as well as NADHdependent reduction of ketones and aldehydes to alcohols. In humans as well as animals, ADHs serve to bioactivate certain alcohols to their aldehydes, e.g., retinol àretinal, that are further metabolized to carboxylic acids which are important in cell growth and differentiation. They break down toxic alcohols and participate in the generation of useful aldehydes, ketones, or alcohol groups in various biosynthetic pathways. In certain organisms, including yeast, some plants, and many bacteria, ADHs catalyze the reduction of acetaldehyde to ethanol (part of fermentation process) to maintain a balance of NAD+/NADH ratio. The ADHs are a superfamily of isozymes. The human ADHs are coded for by at least seven different genes, and the isozymes are classified into five classes (I–V). The class I ADH (liver forms) in humans consists of a, b, and g subunits

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Carbonyl Metabolism, Fig. 2 ADH- and ALDH-catalyzed oxidation of ethanol

that are encoded by the genes ADH1A, ADH1B, and ADH1C. The class II, III, IV, and VADHs are encoded by ADH4, ADH5, ADH7, and ADH6, respectively. Each of the human ADH isozymes is a dimer, and each subunit has an active site with zinc ion (Zn2+) associated with it. The Zn2+ions are located at the catalytic site to aid in binding the hydroxyl group of an alcohol and are critical for its catalytic activity. Class I ADHs are primarily responsible for the oxidation of ethanol to acetaldehyde (Fig. 2). Although the purpose for the presence of these ADHs is most likely to break down alcohols naturally present in foods or those produced during metabolism, the reaction shown in Fig. 2 allows us to consume ethanol-containing beverages and other products. Another function of class I ADHs is to metabolize endogenous retinol that ultimately results in the formation of retinoic acid (Vitamin A) via carbonyl metabolism. It is also believed that ADHs primarily eliminate toxic levels of retinol. Class I ADHs are also responsible for bioactivation and toxicity associated with certain alcohols. For example, class I ADHs oxidize methanol, ethylene glycol, and many ethylene glycol ethers to their corresponding aldehydes. These aldehydes are known to cause various types of cancers in animal models. The ADH levels are gender, age, and race specific. For example, men generally have higher levels ADH activity as compared to women. Young women are unable to process alcohol at the same rate as young men because their ADH levels are lower. The ADH levels are also different among various populations. Polymorphism in these enzymes has clinical significance in alcoholism. For example, the expression of slower alcohol metabolizing isozymes ADH2 and AHD3 poses increased risk for alcoholism. ADH

polymorphism is also associated with drug dependence; however, this line of thought needs further investigations. Aldehyde Dehydrogenases (ALDHs) Like ADHs, ALDHs are also ubiquitous, present in most tissues as well as organisms. ALDHs function in conjunction with ADHs and catalyze NAD(P)-dependent oxidation (detoxification and/or bioactivation) of endogenous as well as exogenous aldehydes, Fig. 1. ALDHs exhibit relatively broad substrate specificity, and the substrates include straight- and branched-chain aliphatic and aromatic aldehydes. For example, the conversion of ethanol-derived acetaldehyde to acetic acid is considered detoxification (Fig. 2). The conversion of retinal to retinoic acid (physiologically active) and conversion of ethylene glycol ether-derived aldehydes to their corresponding acids (toxic and carcinogenic in certain cases) are considered bioactivation. Similar to ADHs, the ALDHs also belong to a superfamily of isozymes. According to the latest literature reports, the human genome contains 19 ALDH functional genes and three pseudogenes. The ALDH isozymes are classified into at least 12 classes, and each of these classes has multiple members. Human ALDHs are homotetrameric enzymes with the exception of class 3 isozymes which are homodimers. The most well-investigated ALDH isozymes include class 1 (ALDH1A members), class 2 (ALDH2), and class 3 (ALDH3A members). ALDH1A and ALDH2 isozymes are constitutive forms, whereas ALDH3A isozymes are inducible in response to oxidative stress. ALDH1A members are mainly responsible for retinal metabolism and thus play a significant role in vertebrate embryogenesis. ALDH1A members are expressed in stem cells and thus considered as

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markers in these cells. ALDH2 is mainly responsible for the detoxification of ethanol-derived acetaldehyde (Fig. 2). More than 40% of individuals from East Asian descent exhibit a functional polymorphism in ALDH2 gene (ALDH2*2; Glu487 has been replaced by a lysine) that leads to a partially inactive form of ALDH2. This results in acetaldehyde accumulation and an alcohol-induced flushing reaction, an increased sensitivity to alcohol and thus resulting in lower rates of alcoholism in this population. Polymorphism in ALDH2 in association with polymorphism in class I ADH isozymes is considered a risk factor for many cancers. ALDH2 has also been implicated in the bioactivation of nitroglycerin, a compound used to treat angina and heart failure. ALDH3A members are expressed in tumors, stomach, and cornea. They appear to be responsible for the maintenance of corneal transparency, protection of the lens crystallins by scavenging hydroxyl radicals, direct absorption of UV-light, and metabolism of cytotoxic aldehydes generated from UV-induced lipid peroxidation. ALDH1A1 and ALDH3A1 isozymes catalyze detoxification of certain anticancer drugs, e.g., oxazaphosphorines such as cyclophosphamide and ifosfamide. They also detoxify/bioactivate many biologically and environmentally important aldehydes such as acrolein, chloroacetaldehyde, and 2-butoxyacetaldehyde. The latter aldehydes are implicated in carcinogenesis. Polymorphisms in other ALDHs play a significant role in hyperprolinemia; neurological disorders including mental retardation, ataxia, and seizures; and stress management in vital organs such as the kidney. Aldo-Keto Reductases (AKRs) AKRs are also a superfamily of isozymes. Like ADHs and ALDHs, AKRs are present in prokaryotes as well as eukaryotes and are ubiquitously expressed in various tissues. They catalyze NAD (P)H-dependent reduction of aldehydes or ketones to primary or secondary alcohols, respectively (Fig. 1). AKRs, like other carbonylmetabolizing enzymes, exhibit broad substrate specificity. The compounds that serve as substrates for these enzymes include drugs, carcinogens, and reactive aldehydes. Many of the

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alcohols resulting from AKR-catalyzed reactions are further conjugated to sulfate or glucuronide for excretion (elimination reactions; detoxification) and some are bioactivated, e.g., tobacco carcinogens. AKRs are implicated in the metabolism of certain cancer chemotherapeutics leading to their detoxification, and thus, AKRs are associated with anticancer drug resistance. AKRs convert tobacco carcinogens such as polycyclic aromatic trans-dihydrodiols to reactive and redox-active o-quinones (bioactivation). They detoxify nicotine-derived nitrosoamino ketones. AKRs are also known to detoxify exogenous toxins such as aflatoxin and endogenous toxins such as lipid peroxides. More than 50 genes are known to code for AKR isozymes. AKR isozymes are mostly monomeric (~37 kDa) and cytosolic enzymes. They are categorized into 14 classes (families). Most of the human AKRs are placed into AKR1 class and are further subdivided into four subclasses, viz., (i) AKR1A (aldehyde reductases), (ii) AKR1B (aldose reductases), (iii) AKR1C (hydroxysteroid/dihydrodiol dehydrogenases), and (iv) AKR1D (steroid 5b-reductases). AKR1A and AKR1B members utilize sugars, glycation products (methylglyoxal), and lipid aldehydes (4-hydoxy-2-nonenal) as substrates. One of the members of AKR1B subclass, viz., AKR1B10, prefers retinals as substrates. AKR1B10 is primarily expressed in small intestine and colon. Its levels are elevated in some liver cancers suggesting it may be involved in liver pathogenesis. AKR1C1 (human 20a-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase 1) has been shown to be overexpressed (>50-fold) in non-small cell lung cancer (NSCLC). Elevated levels of AKR1C1 in NSCLC have been correlated with poor prognosis outcome in NSCLC, and it has been implicated in anticancer drug resistance.

References Moreb JS (2008) Aldehyde dehydrogenase as a marker for stem cells. Curr Stem Cell Res Ther 3:237–246

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788 Parkinson A (2001) Biotransformation of xenobiotics. McGraw-Hill, New York, pp 133–224 Penning TM (2015) The aldo-keto reductases (AKRs): Overview. Chemico-biological interactions, 234, 236–246 Sladek NE (2003) Human aldehyde dehydrogenases: potential pathological, pharmacological, and toxicological impact. J Biochem Mol Toxicol 17:7–23 Yokoyama A, Mizukami T, Yokoyama T (2015) Genetic polymorphisms of alcohol dehydrogense-1B and aldehyde dehydrogenase-2, Alcohol flushing, mean corpuscular Volume, and aerodigestive tract neoplasia in japanese drinkers. In: Biological basis of alcohol-induced cancer Springer international publishing, pp 265–279

Carbonyl Reductases ▶ Reductases

Carcinoembryonic Antigen Peter Thomas Departments of Surgery and Biomedical Sciences, Creighton University, Omaha, NE, USA

Synonyms CD66e; CEA; CEACAM5

Definition CEA is a glycoprotein of approximately 150–180 kDa. Its measurement in serum is used clinically as a biomarker for a number of cancers (pancreas, breast, stomach, ovary, lung, and medullary carcinoma of the thyroid), but its primary use is in monitoring cancers of the colon and rectum.

Characteristics Protein Structure CEA was discovered in 1965 in colon cancer and fetal tissue extracts and was described as an

Carbonyl Reductases

▶ oncofetal antigen. Many of the advances in tumor marker research lead directly back to the discovery of CEA. The protein component of CEA is 79 kDa in size, and the balance of 70–100 kDa is made from up to 28 complex N-linked multi-antennary carbohydrate structures containing N-acetyl-glucosamine, mannose, galactose, fucose, and sialic acid. Low-resolution X-ray studies have shown an elongated monomeric structure that could be described as a bottlebrush. The molecule is composed of a series of six disulfide-linked immunoglobulin-like domains (IgC2-like) of either 93 (type A) or 85 (type B) amino acids and a seventh N-domain of 108 amino acids which is an IgV (variable antigen recognition domain) structure without the stabilizing disulfide bridge. CEA can attach to the cell membrane, and this is achieved by posttranslational modification of a small (26 amino acids) hydrophobic C-terminal domain to a glycosylphosphatidylinositol linkage (see Fig. 1a). Cleavage of this linkage by phospholipases releases CEA into the lumen of the intestine or other extracellular compartments. The CEA Gene Family The complete gene for CEA has been cloned, and it includes a promoter region that confers cell type-specific expression. The ▶ CEA gene family comprises 29 genes or pseudogenes located between the q13.1 and q13.3 regions of chromosome 19. The family can be divided into three groups: The CEA group of 12 genes, the pregnancy-specific glycoprotein (PSG) group of 11 genes, and a third group composed of 6 pseudogenes. Only 16 of the 29 genes are expressed. Sequence data has shown that the CEA family is a subset of the immunoglobulin supergene family. Comparative sequence studies of the CEA gene family from various species suggest that the CEA family has a common ancestry and arose relatively recently in evolution. Function of CEA in Normal and Cancerous Tissue In general, members of the CEA family subgroup have a ubiquitous distribution in adult tissues. However, CEA itself has a more restricted

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Carcinoembryonic Antigen, Fig. 1 CEA structure. (a) Insertion of CEA into the plasma membrane (down arrow), the Ig domain structure, and the position of the N-linked sugar chains. An arrow marks the position of the PELPK receptor recognition sequence. (b) Homotypic binding between two CEA molecules with attachment between the N and A3 domains (Structures are modified from the CEA homepage http://cea.klinikum.uni-meunchen.de)

expression being found only in the colon, pyloric mucus cells, epithelial cells of the prostate, sweat glands, and squamous cells in the tongue, cervix, and esophagus. In the colon CEA is located at the apical surface of colonic enterocytes and is associated with the glycocalyx or fuzzy coat. In the normal colon CEA is maximally expressed on columnar cells at the level of the free luminal surface. CEA is also found in goblet cells in

association with mucins. The function of CEA in the normal individual is not well understood and has been the subject of much speculation. It has been estimated that the normal person can produce 70 mg or more of CEA a day and excrete it in the feces. CEA has been shown to bind to various fimbriated gut pathogens, and therefore, it has been suggested that it has a function in protecting the gut epithelia. In cancer cells CEA may perform a number of functions. Unlike the normal colonocyte where CEA expression is highly polarized in cancers, this polarity is lost and its expression occurs through the whole of the cell surface. It has been shown that CEA can act as a Ca2+-independent homotypic adhesion molecule binding with itself through an interaction between the N and A3 domains (Fig. 1b) and causing aggregation of tumor cells. This allows the malignant epithelium to adopt a multilayered structure and may disrupt the normal pattern of differentiation. CEA can also bind heterotypically to other members of the gene family including the nonspecific cross-reacting antigen (NCA, CD66c, CEACAM6) and the biliary glycoprotein (BGP, CD66a, CEACAM1), of which seven different forms have been identified. It is unlikely that CEA functions as a ▶ cell adhesion molecule in the normal colon because of its apical expression. CEA is cleared from the circulation by the hepatic ▶ macrophages (▶ Kupffer cells). A cell surface receptor identical to the heterogeneous nuclear RNA-binding protein hnRNP M4 recognizes a pentapeptide (Pro–Glu–Leu–Pro–Lys (PELPK)) located at the hinge region between the N and the first immunoglobulin loop domain (A1) of CEA. Patients with a mutation in the region coding for this peptide have extremely high circulating CEA levels presumably due to the inability of Kupffer cells to clear the protein from the blood. CEA has also been implicated in the development of hepatic ▶ metastasis from colorectal cancers by the induction of a localized inflammatory response that affects retention and implantation in the liver. Cytokines produced also protect the tumor cells against the toxic effects of hypoxia. CEA-producing cells therefore have a selective advantage for growth in the liver. Studies have also shown that CEA can protect cancer cells

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from a form of programmed cell death called ▶ anoikis, and this also seems to involve the PELPK motif and inhibition of Trail-R2 (DR5) signaling. CEA is also protective against other forms of ▶ apoptosis including drug- and UV light-induced programmed cell death. The related protein CEACAM-1, however, is a proapoptotic protein. Research has shown that CEA may be involved in promoting angiogenesis (growth of new blood vessels) in colorectal cancer, by interacting with endothelial cells directly through its receptor. This raises the possibility of alternate therapeutic approaches and further emphasizes CEA as a multi-functional glycoprotein. Clinical Aspects The main clinical use for CEA is as a tumor marker especially for cancers in the colon and rectum and approximately 90% of these cancers produce CEA. CEA has also been used as a marker for breast and small cell cancer of the lung. Approximately 50% of breast and 70% of small cell cancers express CEA. Accurate immunoassays are commercially available for its measurement in body fluids. Immunohistochemistry on biopsy or resection specimens is also often carried out, for example, the intensity of CEA staining has been associated with a worse prognosis for breast cancer. Normal serum levels are TT transitions, implicating ultraviolet radiation (UVR) as the causal somatic mutagen. Although CDKN2A is inactivated in the majority of melanoma cell lines examined, deletions and interstitial mutations of CDKN2A are much less common in uncultured melanoma tumors. Present studies indicate that only 5–10% of uncultured melanomas demonstrate mutations in CDKN2A, a surprisingly low figure given the obvious importance of CDKN2A in familial melanoma and the frequency of LOH seen at chromosome 9p21 in melanomas.

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CDKN2A, Fig. 2 Schematic representation of the protein interactions in the cyclin D/CDK4/p16/pRb pathway. Through a complex system of signal transduction, growth factors lead to the assembly of cyclin D and CDK4. This complex is then activated through phosphorylation by the CDK-activating kinase (CAK), and cyclin D/CDK4 in turn phosphorylates pRb, leading to the release of transcription factors of the E2F family. These are then capable of transactivating the genes necessary for entry into S phase, and p16 has been shown to inhibit this process in several ways, by binding to the complex and inhibiting the kinase activity of CDK4, inhibiting CAK-dependent phosphorylation of CDK4, or inhibiting the assembly of the cyclin D/CDK4 complex, with the latter being the principal

mechanism of inhibition in vivo. The scheme provided is necessarily simplistic; however, it appears that p16 may also inhibit the phosphorylation of pRb by indirectly inactivating other CDKs, e.g., CDK2, as a consequence of the redistribution of other CDK inhibitors, e.g., p27 and p21. There is also a feedback loop whereby the release of the E2F transcription factor results in the activation of p16 expression, although the absence of E2F binding sites in the CDKN2A promoter precludes direct transactivation by E2F. Aberration of this pathway through either deletion or mutation of pRb, the binding of viral oncogenes to pRb, overexpression or activation of CDK4 or cyclin D, or deletion or mutation of CDKN2A all can result in constitutive transactivation of S phase genes by E2F transcription factors

P16 Is a CDK Inhibitor P16 is the archetype member of the ▶ INK4 (inhibitor of CDK4) family of CDK inhibitors, which is comprised of p16INK4A, p15INK4B, p18INK4C, and p19INK4D, encoded by CDKN2A, CDKN2B, CDKN2C, and CDKN2D, respectively. Each of the proteins inhibits CDK4or CDK6-mediated phosphorylation of the ▶ retinoblastoma susceptibility gene product, pRb, thereby providing a powerful negative signal, or “brake,” to progression through the cell cycle. The ▶ cyclin D1/CDK4/p16/pRb signaling pathway is the major growth control pathway for entry into the cell cycle. For cells to progress through G1 into S phase they must pass the late G1 restriction point, which controls entry into S phase. For progression past this restriction

point, cyclin D/CDK4 must phosphorylate the ▶ retinoblastoma protein pRb. During G0/G1 the Rb protein exists in a DNA-bound protein complex, where it is bound to the transactivation domain of E2F transcription factors, preventing transactivation of E2F target genes. The phosphorylation of pRb results in the disassociation of this protein complex and the release of E2F such that it can transactivate genes required for entry into S phase. Overexpression of p16 inhibits progression of cells through the G1 phase of the cell cycle by binding to CDK4/cyclin D complexes (or CDK6/cyclin D) and blocking the kinase activity of the holoenzyme. Given that p16 normally functions to inhibit CDK4, it is easy to understand how inactivation of this gene could result in uncontrolled cellular growth leading to

CDKN2A CDKN2A, Fig. 3 Schematic representation of the role of ARF in p53 activation by DNA damage and oncogenic stimuli. ARF functions to sequester MDM2 in the nucleus preventing nucleocytoplasmic shuttling of the MDM2/p53 complex; however, the details have not yet been fully elucidated and results suggest the mechanism may differ between humans and mice

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cancer. In many tumor types, an inverse correlation between mutations of p16 and pRb has been observed. Since p16 lies upstream of pRb, inactivation of both proteins would be redundant. Role of the Alternative Reading Frame (ARF) Product The ARF protein also regulates the G1/S phase transition via a distinct pathway involving the ▶ TP53 ▶ tumor suppressor gene product p53 and MDM2, which function upstream of p21 (a cyclin-dependent kinase inhibitor closely related to p16) and the CDK2/cyclin E complex (Fig. 3). p53 is a transcription factor that plays a major role in monitoring the integrity of the genome and can be activated to inhibit cell cycle progression or initiate apoptosis through two distinct pathways: (i) in response to a variety of cellular stresses including ▶ DNA damage and ▶ hypoxia and (ii) via overexpression of viral or cellular oncoproteins such as E1A and c-myc. In this way, cells prevent the repair of mutations in successive generations by inducing apoptosis in incipient cancer cells. ARF plays a crucial role in p53-induced apoptosis. Murine p19ARF is capable of inducing a p53-dependent G1 cell cycle arrest that is not mediated through the direct inhibition of known CDKs. Ectopic expression of ARF leads to stabilization of p53 in multiple cell types, but unlike other known upstream effectors of p53, this activation is not through

phosphorylation. Instead, ARF binds to MDM2 and blocks both MDM2-mediated p53 degradation and the transactivational silencing of p53. MDM2 continuously shuttles between the nucleus and the cytoplasm. This shuttling is essential for its ability to promote p53 degradation, indicating that MDM2 must export p53 from the nucleus to the cytoplasm to target p53 to the cytoplasmic proteosome. ARF activates p53 by binding to MDM2 in the nucleus and blocking the transport of the MDM2/p53 complex out of this organelle. Results obtained with murine and human ARF are somewhat different. In murine cells results indicate that p19ARF sequesters MDM2 away from p53 into the nucleolus. In human cells p14ARF moves out from the nucleolus to form discrete nuclear bodies in conjunction with MDM2 and p53, thereby blocking their nuclear export and leading to p53 stabilization. The discovery that ARF transcription is induced by the overexpression of a variety of cellular and viral oncoproteins including c-myc, E1A, and E2F has provided the link by which hyperproliferative signals result in p53-dependent apoptosis. To determine whether mutations in CDKN2A contribute to tumorigenesis via p19ARF in addition to p16, cDNAs carrying a variety of exon 2 mutations have been transfected into cell lines and cell cycle arrest monitored. These mutations have included several that are silent in p16 but caused missense mutations in p19ARF, as well as

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several deletion mutants that removed either exon 1b or various portions of exon 2. Results indicate that the majority of p19ARF activity is encoded by the exon 1b sequences, as all missense mutations in exon 2 of p19ARF remained fully active in blocking cell cycle progression, and removal of exon 2 sequences only marginally reduced the ability to induce arrest. In contrast, deletion of exon 1b resulted in a transcript that was incapable of inhibiting cell cycle progression. Missense mutations in exon 2 of the human p14ARF transcript similarly did not reduce the growth suppressive function of p14ARF. Senescence p16 is not normally expressed at detectable levels in most cycling cells; however, CDKN2A mRNA and p16 protein accumulate in late-passage non-immortalized cells, implicating a role for p16 in cellular ▶ senescence. This is supported by studies revealing that loss of p16 expression is a critical event in ▶ immortalization (the flip side to senescence) of a range of cell types. This conclusion was initially alluded to by finding that the frequency of deletions and intragenic mutations of CDKN2A in uncultured tumors was considerably lower than in immortalized cell lines. Growth and survival experiments using cells with impaired CDKN2A function suggest that a p16/pRbdependent form of senescence may be particularly important in melanocytes. Individuals with defective p16INK4a have been found to have increased numbers of naevi, and it has been speculated that naevi are senescent clones of melanocytes. Mouse Models The generation of a CDKN2A “knockout” mouse, carrying a germline homozygous deletion encompassing exons 2 and 3 of the gene, revealed that p16 and p19ARF (since both proteins are eliminated by deletion of exon 2) were not essential for viability or organomorphogenesis. However, the mice did demonstrate abnormal extramedullary hematopoiesis, suggesting that p16 or p19ARF may regulate the proliferation of some hematopoietic lineages. In addition, the mice developed spontaneous tumors at an early age, specifically fibrosarcomas and B cell

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lymphomas, and were highly sensitive to carcinogens. In contrast to wild-type mouse embryonic fibroblasts (MEFs), cultured MEFs from Cdkn2a nullizygous mice (Cdkn2a/) failed to undergo senescence crisis and could be transformed by oncogenic ras alleles. Although Cdkn2a/ mice did not develop melanomas, transformation of Cdkn2a/ MEFs by activated ras prompted experiments to cross the Cdkn2a/ mice with a previously generated transgenic mouse in which an activated ras allele was targeted exclusively to melanocytes under the control of the tyrosinase promoter. These mice spontaneously developed melanomas at high frequency and with short latency. To determine whether p16 or p19ARF was the principal mediator of the above effects, knockout mice strains with targeted deletions of p16 and p19ARF were generated. In general, p19ARF null animals were observed to develop a tumor spectrum more closely related to p53 null rather than p16 null mice. Tumors observed in p19ARF null mice included lymphomas and an increased incidence of soft tissue sarcomas, carcinomas, and osteosarcomas. Mice lacking p16 were found to develop soft tissue sarcomas, osteosarcomas, and melanomas. Mouse strains with specific inactivation of either p16 or p19ARF were tumor prone, but neither was as severely affected as animals lacking both p16 and p19ARF, suggesting cooperation between p16 and p19ARF loss in tumorigenesis. Clinical Aspects CDKN2A Mutations and Melanoma

Germline CDKN2A mutations have been observed in approximately 20–40% of melanoma families worldwide. However, melanoma appears to segregate with chromosome 9p markers in a far greater proportion of families than have been shown to carry mutations of CDKN2A. This suggests that melanoma predisposition in some of these families is caused by: (i) another gene in the vicinity of CDKN2A, (ii) mutations outside of the p16 coding region, and (iii) another gene somewhere else in the genome, with linkage to this region occurring simply by chance. The most

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parsimonious explanation is that a combination of all these possibilities is likely. Overall, approximately 40% of pedigrees with three or more cases of melanoma have been found to harbor mutations in the CDKN2A gene. This figure varies with location and is lowest in regions of high ▶ UV radiation (UVR), e.g., Australia (20%), and higher in regions with low incident UVR, e.g., Europe (57%). There is a significant increase in the yield of CDKN2A mutations with increasing number of affected cases in families with melanoma. In addition, an early age of diagnosis and the presence of family members with multiple primary melanomas or with ▶ pancreatic cancer have also been shown to be significantly associated with an increased likelihood of finding a CDKN2A mutation. The population-based frequency of CDKN2A mutations in melanoma cases is of the order of 1–2%, even in those individuals that had developed multiple primary tumors, much lower than observed in families selected for multiple cases of melanoma. Disease-associated mutations are distributed along the entire length of the p16 coding region. At least one mutation has been described in the promoter of the gene, and several putative mutations have been identified in the intronic sequences. The most frequent CDKN2A mutations identified to date are c.255_243del19 (also known as p16 Leiden), p.M53I, p.G101W, c.331_332insGTC (p.R112_L113insR) (all in exon 2), c.-34G > T (promoter), and c.IVS2105A > G (intron). There are considerable differences in the frequencies and distribution of CDKN2A mutations across the world. Many mutations have been shown to arise from a common founder and are more frequent in particular geographic locations. For example, Sweden and the Netherlands have single predominant founder mutations (p.R112_L113insR and p16 Leiden, respectively) involving over 90% of families tested. The G101W mutation, common in Italy, France, and Spain, has been calculated to arise from a single genetic event approximately 93 generations ago. Many additional mutations have been repeatedly reported, and where analysis has

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been performed these have invariably been shown to be due to common founders. The only exception to this appears to be a 24 bp insertion in exon 1a, that has arisen multiple times, presumably because of DNA slippage over a 24 bp repeat region. Mutation of ARF Germline mutations affecting ARF but not p16INK4a have been reported in a small number (3%) of melanoma families. Whereas the distribution of p16 mutation types (approximately 70% missense or nonsense, 23% insertion or deletion, 5% splicing, and 2% regulatory) is consistent with that observed in the Human Genome Mutation Database, the reported ARF-specific mutations are almost all either splicing mutations (affecting the 30 splice site of exon 1b) or large deletions. Penetrance The pattern of susceptibility in melanoma pedigrees is consistent with the inheritance of autosomal dominant genes with incomplete penetrance. The overall penetrance of CDKN2A mutations in melanoma families has been estimated to be 0.30 by the age of 50 years and 0.67 by the age of 80 years. There is significant variation in the penetrance of CDKN2A mutations with geographical location. By the age of 50 years, penetrance was estimated to be 0.13 in Europe, 0.5 in the United States, and 0.32 in Australia and by the age of 80 years 0.58 in Europe, 0.76 in the United States, and 0.91 in Australia (Fig. 4). This indicates that the CDKN2A mutation penetrance varies with melanoma population incidence rates, thus the same factors that effect population incidence of melanoma may also mediate CDKN2A penetrance. Multiple Primary Melanoma

General characteristics of inherited susceptibility to many types of cancer are early age of onset and the development of multiple primary tumors. Hence the presence of multiple primary melanomas (MPM) in an individual may be a sign of them being a CDKN2A mutation carrier. This is the case for a small proportion (13/133, 10%) of MPM cases without a family history of the disease. In contrast, analysis of MPM cases with a

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family history of disease yields CDKN2A mutations in 55/139 (40%) of samples tested. The proportion of CDKN2A mutations in sporadic MPM cases increases with increasing number of melanomas (10/119 (8.5%) of cases with two primary melanomas, compared to 11/83 (33%) cases with three or more primary tumors).

Modifiers of Penetrance of CDKN2A Mutations The MC1R gene (16q24) which encodes for the melanocyte-stimulating hormone has been shown to be a risk factor in families with segregating CDKN2A mutations. MC1R variants have been shown to act as modifier alleles, increasing the penetrance of CDKN2A mutations and reducing the age of onset of melanoma.

CDKN2A Mutations and Nonmelanoma Cancers

Since CDKN2A is a tumor suppressor found to be inactivated in a wide range of different tumors, one might expect individuals carrying germline mutations of CDKN2A to be prone to cancers other than melanoma. ▶ Brms1, prostate, colon, and ▶ lung cancers have been suggested to be associated with CDKN2A mutations; however, these common cancers may occur in CDKN2Apositive pedigrees by chance. Convincing evidence for susceptibility to another tumor type has been shown only for pancreatic cancer, which has been shown to be significantly associated with CDKN2A mutations in all regions except Australia, the reason for this is not yet understood. There appears to be no evidence of an association between neural system tumors (NSTs) and CDKN2A mutations involving p16. However, there is marginal evidence for the association of NSTs with ARF-specific mutations.

CDKN2a Polymorphisms as Low-Risk Factors The A148T variant, located in exon 2 of the CDKN2A gene, has no observed effect on p16 function and does not segregate with disease in melanoma pedigrees. The contribution of this polymorphism to melanoma risk remains unclear; an association with increase in risk has been seen in some populations, but not in others. The 500 C > G and the 540 C > T polymorphisms in the 30 untranslated region of the CDKN2A gene have been shown to be associated with melanoma risk. The frequencies of the rare alleles at these loci have been shown to be higher in melanoma cases than in controls. It is possible that these variants might alter the stability of the CDKN2A transcript or the level of transcription, or that they may be in linkage disequilibrium with an unidentified variant which is directly responsible for melanoma predisposition. The contribution of these polymorphisms to melanoma risk is

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likely to be small in comparison to that of CDKN2A inactivating mutations. CDKN2A and the Atypical Mole Syndrome Since the description of the “B-K mole syndrome,” much debate has ensued regarding the association between melanoma and the atypical mole syndrome (AMS). Several authors have concluded that atypical moles segregate independently of CDKN2A mutations, although individuals with high numbers of naevi in melanoma-prone families are three times more likely to be CDKN2A mutation carriers than those with a low number of naevi. Support for the notion that CDKN2A is naevogenic comes from a study of a large series of 12-year-old twins in which total naevus count was found to be tightly linked to CDKN2A. This finding has been corroborated by two independent genome wide association studies that have mapped loci responsible for naevi in twin cohorts. Both studies showed peaks of high linkage scores at 9p21 directly over the CDKN2A gene.

References Bishop JN, Harland M, Randerson-Moor J et al (2007) Management of familial melanoma. Lancet Oncol 8(1):46–54 Goldstein AM, Chan M, Harland M et al (2007) Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents. J Med Genet 44(2):99–106 Hayward NK (2003) Genetics of melanoma predisposition. Oncogene 22(20):3053–3056 Sharpless NE (2005) INK4a/ARF: a multifunctional tumor suppressor locus. Mutat Res 576(1–2):22–38 Sharpless E, Chin L (2003) The INK4a/ARF locus and melanoma. Oncogene 22(20):3092–3098

CDKN4 ▶ p27

cDNA Chips ▶ Microarray (cDNA) Technology

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CDX2 Isabelle Gross and Isabelle Hinkel INSERM U1113, Université de Strasbourg, Strasbourg, France

Synonyms Caudal type homeobox 2; CDX3; CDX-3

Definition CDX2 is a member of the caudal-related homeobox transcription factor gene family. As a determinant of cell fate, CDX2 is critical for various aspects of embryonic development, including intestinal morphogenesis. In the adult, CDX2 expression is restricted to the gut and is required to maintain intestinal homeostasis. Altered CDX2 expression is associated with several types of cancer, namely, colon cancer and acute myeloid leukemia.

Characteristics Structure CDX1, CDX2, and CDX4 are the three members of the mammalian homeobox transcription factor gene family related to the Drosophila gene caudal and belong to the ▶ ParaHox gene cluster, a paralogue of the Hox gene cluster. The human CDX2 gene is located on chromosome 13 at band q12.3 and consists of three exons encoding a 313 amino acid protein. The central region of the CDX proteins is the most conserved and corresponds to the homeodomain, a 60 amino acid sequence arranged in three alpha-helices, which binds to DNA. The N-terminal region of CDX2 acts as a transcriptional activator domain and together with the C-terminal region modulates its activity. Alternative splicing of the CDX2 gene can also generate miniCDX2 in which the N-terminal transactivation domain is replaced by a specific 13 amino acid extension.

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Expression, Activity, and Mechanisms of Regulation Nuclear CDX2 expression is detected at E3.5 in the murine ▶ trophectoderm and around E8.5 in several developing tissues of the embryo itself (posterior gut, tail bud, neural tube, etc.). By E12.5 onwards, CDX2 expression is restricted to the intestinal ▶ epithelium where it is maintained throughout life. Species- and stage-specific gradients of expression along the anteroposterior and dorsoventral axes have been described: for instance, CDX2 expression generally increases with differentiation in the small intestine but not in the colon. The regulation of CDX2 transcription is highly dynamic, involving stage-specific promoter elements and possibly various transcription factors such as HNF4alpha, GATA6, TCF4/beta-catenin, NF-kappaB, SMAD, or CDX2 itself. The transcription of CDX2 can be modified by multiple extracellular factors (collagen I, Laminin 1, Wnt5A, sodium butyrate, etc.) and is highly sensitive to the cellular microenvironment. CDX2 levels are also regulated by posttranslational modifications affecting the half-life of the protein. Indeed, phosphorylation of CDX2 by kinases implicated in cell cycle progression, such as ERK1/2 and CDK2, leads to its polyubiquitination and degradation by the ▶ proteasome. Conversely, in intestinal cells that start to differentiate, the ▶ cyclin-dependent kinase inhibitor p27Kip1stabilizes CDX2 by preventing its phosphorylation by CDK2. Posttranslational modifications are not only involved in the regulation of CDX2 protein levels but can also modulate the transcriptional activity of CDX2. For instance, the MAPK p38alpha phosphorylates CDX2 on a not yet identified residue in differentiated cells and this leads to enhanced transcription of CDX2 target genes. On the opposite, high levels of S60-phosphorylated CDX2 are detected in the proliferative crypt cells, and this phosphorylation actually inhibits CDX2 transcriptional activity: this might explain why CDX2 target genes are mainly activated in the upper third of the crypt, although no CDX2 expression gradient is observed in colonic ▶ crypts.

CDX2

Finally, another way of regulating CDX2 activity was revealed with the detection in the proliferative ▶ crypt cells of a dominant negative isoform of CDX2 (miniCDX2), that lacks the transcription activator domain and whose fixation on the CDX2 binding sites inhibits transcription by fulllength CDX2. Structure Physiological Functions The existence of a large panel of mice models provides us with considerable information about the biological functions of CDX2. Ubiquitous and homozygous gene invalidation of CDX2 is lethal before gastrulation as CDX2 is required for ▶ trophectoderm maturation and consequently blastocyst implantation. In contrast, heterozygous CDX2/+ mice are viable and fertile and present no major dysfunctions despite morphological defects. Indeed, these CDX2/+ mice display anterior homeotic shifts of their axial skeleton, tail abnormalities, or stunted growth, illustrating the role of CDX2 in anteroposterior patterning and posterior axis elongation. In addition, these mice totally lose CDX2 expression in some regions of the proximal colon, which allows intercalary growth of more anterior gastrointestinal tissue types (esophageal, gastric), highlighting the role of CDX2 in intestinal identity. Accordingly, ectopic expression of CDX2 in the stomach of transgenic mice induces the conversion of gastric epithelial cells into enterocytelike cells. To circumvent the problem of embryonic lethality induced by complete CDX2 depletion, conditional inactivation of CDX2 was performed to study the consequences of CDX2 loss at different stages of development and in the adult. Because CDX1 and CDX2 can be functionally redundant, double knockout mice for CDX1 and CDX2 were sometimes analyzed using CDX1/ mice, which are viable and only show alterations of the skeleton. For instance, ubiquitous inactivation of CDX2 post-implantation at E5.5 in CDX1/ mice is lethal at E10: the mice present abnormal axis elongation, neural tube closure defects, and ▶ somite patterning alterations, demonstrating that the CDX genes are crucial for these events in early embryonic development.

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CDX2 expression was also specifically suppressed in the developing intestine: strikingly, none of these mice survived longer than 2 days after birth because of severe abnormalities in the morphology and function of the gut. For instance, mice in which CDX2 is invalidated at E9.5 in the early endoderm fail to form a colon. In addition, the small intestine lacks most of the ▶ villi critical for nutrient absorption and displays more cycling cells, and many of the mutant cells resemble more to keratinocytes that constitute the esophageal ▶ epithelium than to differentiated intestinal cells. If ablation of CDX2 in the developing intestine is performed later at E13.5 or E15.5, colon formation occurs, but the ▶ epithelium of mutant mice is highly disorganized and ▶ villi are smaller. Inactivation of CDX2 at E13.5 leads to an upregulation of gastric markers (H+/K+ATPase, ghrelin) and a downregulation of intestinal markers (I-FABP). Ablation of CDX2 at E15.5 generates enterocytes that display profound defects in their typical microvilli and disrupted apicobasal polarity, but no features of gastric/ esophageal transdifferentiation. Finally, specific ablation of CDX2 in the adult intestinal ▶ epithelium is also lethal, indicating that CDX2 expression is required throughout life to maintain a functional intestine. Indeed, mutant mice lose weight, have chronic diarrhea, and die of starvation (malabsorption) at the latest 3 weeks after CDX2 inactivation. The ▶ villi of these mice are smaller and the microvilli on absorptive cells are shorter, less dense, and disorganized compared to those of their wild-type littermates. Although conversion into stomach-like tissue is not observed, analysis of the gene expression profiles of CDX2/ mice shows upregulation of stomach-specific markers. Mode of Action at the Cellular Level In line with the spectacular consequences of CDX2 depletion on intestinal cell differentiation in mice, numerous reports show that overexpression of CDX2 can induce various degrees of intestinal differentiation in vitro. For instance, undifferentiated colorectal cell lines can acquire a polarized, columnar shape with apical microvilli, produce various digestive enzymes,

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and form tight, adherens, and desmosomal junctions upon CDX2 expression. The effect of CDX2 on apicobasal polarity was demonstrated using a 3D culture system and was associated with defective apical transport. This effect is consistent with the formation of large cytoplasmic vacuoles and downregulation of genes involved in endolysosomal function in intestinal cells of conditional CDX2 knockout mice. CDX2 expression can also reduce anchoragedependent or anchorage-independent growth of normal, ▶ adenoma, and carcinoma epithelial cells. This may be achieved through reduced cell proliferation as CDX2 can block the G0/G1-S progression in intestinal cell lines. However, a proapoptotic effect of CDX2 can also be observed in various intestinal contexts and thus may also contribute to reduced cell numbers. Of note, the activity of CDX2 on cell growth appears to be dependent on the context and cell type: for instance, somatic knockout of CDX2 reduces anchorage-independent growth of LoVo intestinal cells, and shRNA silencing of CDX2 expression inhibits the proliferation of various human leukemia cell lines. CDX2 can inhibit intestinal cell ▶ migration and ▶ invasion in Boyden chambers coated or not with Matrigel. These are hollow plastic chambers sealed at one end with a porous membrane and suspended in a well containing chemoattractants. Cells are placed inside the chamber and allowed to migrate through the pores to the other side of the membrane. CDX2 expression appears to influence chromosome segregation, as well as DNA damage repair in intestinal cells. Mode of Action at the Molecular Level As a bona fide transcription factor, the main function of CDX2 is to activate specific gene expression in the embryo and later in the intestinal ▶ epithelium. The consensus binding site of CDX2 is (C/TATAAAG/T), an AT-rich sequence typical of homeobox proteins, but CDX2 can also bind to sequences that are slightly different. During early development, CDX2 regulates anteroposterior patterning by stimulating the expression of various HOX genes such as HOXA5. Later, in the developing of mature

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intestinal ▶ epithelium, CDX2 regulates a large number of genes involved in intestinal identity and in various intestinal functions. Indeed, CDX2 regulates the transcription of genes implicated in cell-fate decision, such as the Notch ligand DLL1, the transcription factors Math1 or KFL4, and even itself. Since CDX2 is critical for enterocyte maturation, the first direct target genes identified encoded digestive enzymes like sucrase-isomaltase, lactase, or phospholipase A/lysophospholipase. Many transporters, necessary for the absorption and secretion of nutrients by enterocytes, are also CDX2 target genes, for instance, the iron transporter hephaestin, the multidrug resistance 1 (MDR1/P-glycoprotein/ ABCB1), or the solute carrier family 5, member 8 (SLC5A8). Other direct CDX2 target genes are involved in the modeling of the intestinal mucuscovered brush border: they encode, for example, the actin-binding protein villin 1 (important for microvilli architecture) and mucus constituents such as MUC2 and MUC4. Some CDX2 target genes encode adhesion molecules, potentially involved in intestinal barrier function and cell polarization: several members of the cadherin superfamily such as LI-cadherin, Mucdhl, and Desmocollin 2, but also claudin-2 and claudin-1. Finally, the transcription of the cyclin-dependent kinase inhibitor p21Cip1 can be stimulated by CDX2 and thus may contribute to the antiproliferative effect of CDX2. CDX2 does not necessarily bind to DNA and use its properties of transcriptional activator to modulate gene expression. For instance, CDX2 affects the ▶ Wnt signaling pathway by direct interaction with beta-catenin, thereby inhibiting the formation of the beta-catenin/TCF4 complex and consequently Wnt target gene activation. Another example is the binding of CDX2 to the p65 subunit of NF-кB, which prevents its binding and activation of the COX-2 promoter. More unexpectedly, CDX2 can modulate the activity of proteins that are not involved in gene transcription: as an example, CDX2 interacts by its homeodomain with the protein complex Ku70/ Ku80 and inhibits its activity of DNA repair by the nonhomologous end joining process. Furthermore, CDX2 can affect proteins without direct

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interaction: indeed, CDX2 can stabilize the cyclin-dependent kinase inhibitor p27Kip1 by inhibiting its polyubiquitination and thereby reduce cell proliferation. In some cases, the exact mechanism of CDX2 activity is not yet understood but might be important for intestinal homeostasis. One example is the potential repression of the mTOR pathway by CDX2, which might oppose cell cycle progression and chromosomal segregation defects. Another example is the enhanced trafficking of ▶ E-cadherin to the membrane of colon cancer cells, which strengthens Ca2+-dependent adhesion and might be linked to the fact that CDX2 can reduce the phosphorylation of beta-catenin and p120 catenin. Clinical Relevance for Colon Cancer In colon ▶ adenocarcinomas, nuclear CDX2 expression is generally reduced, becoming sometimes diffuse and cytoplasmic, but there is a lot of heterogeneity in the level of reduction between different tumors or even between different areas within a tumor, which might explain why conflicting results have been obtained in separate studies. Reduced expression of CDX2 can be associated with high ▶ microsatellite instability (MSI) status, advanced tumor stage, higher tumor grade, lymph node metastasis, and reduced survival. In addition, CDX2 expression is more systematically decreased in cells located at the tumor front or disseminated in the adjacent stroma compared to the cells of the tumor center. Strikingly, most of the time, ▶ metastases (lymph nodes, liver) exhibit a similar level of CDX2 expression than the primary tumor, suggesting a dynamic expression pattern of CDX2 during tumor progression, with a specific but transient reduction in invasive cells. Deletions or mutations at the CDX2 locus occur very rarely in colon tumors. Actually, most (chromosomal instability) CIN tumors present a gain of CDX2 copy number, but this gene amplification does not correlate with CDX2 expression. On the other hand, somatic cell hybrid experiments indicate that silencing of CDX2 expression was transferable upon cell fusion, suggesting a dominant repression mechanism. Since no

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epigenetic modifications of the CDX2 promoter have been detected in colon cancer cell lines, the existence of a transcriptional repression pathway is likely. Of note, such a regulatory mechanism would be consistent with a transient change of CDX2 expression in invasive cells. Several oncogenic signaling pathways (PI3K, Raf-MEK-ERK1/ 2) that are aberrantly activated in a large fraction of colon tumors can repress CDX2 expression in colon cancer cell lines. Transcriptional repressors inducing EMT (Slug, Snail, and Zeb1) can repress CDX2 transcription in vitro and may be involved in the systematic decrease of CDX2 expression in invasive cells. Several microenvironmental factors linked to tumor progression (▶ hypoxia, extracellular matrix, protein changes) can modify CDX2 transcription in colon cancer cell lines, and nude mice grafting experiments highlight the plasticity of CDX2 expression. However, all of the above data obtained with cell lines still await confrontation with cohorts of human colon tumors. Given that CDX2 expression is downregulated in colon tumors and impacts on cell proliferation and ▶ migration, it is hypothesized that CDX2 acts as a tumor suppressor in the colon. Heterozygous CDX2/+ mice do not develop spontaneous tumors (the initially described “intestinal polyps” turned out to be nonneoplastic; see above), suggesting that the loss of CDX2 alone is not sufficient to initiate tumor formation, but only one allele is invalidated in these mice to allow survival. In contrast, upon tumor initiation, the tumor suppressor activity of CDX2 becomes obvious. Indeed, CDX2+/mice treated with a colon carcinogen (azoxymethane) develop numerous ▶ adenocarcinomas in the distal colon much faster than their wild-type littermates. Similarly, when CDX2+/ mice are crossed with mice that spontaneously develop adenomatous polyps in the small intestine (APC+/D716 mice), they form six times more adenomatous polyps, and these are now located in the distal colon. Finally, forced expression of CDX2 in colon cancer cells injected in nude mice correlates not only with reduced tumor size, but also with decreased metastasis incidence, suggesting that CDX2 opposes metastatic dissemination. Thus, even if CDX2 cannot be considered as a classic

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▶ tumor suppressor gene (no genomic alteration, no spontaneous tumor), it impacts on various cellular processes (proliferation, ▶ adhesion, polarity, ▶ migration; see above) involved in tumor growth and dissemination, and experimental evidences in mice indicate that reduced expression of CDX2 has important consequences for colon tumor (speed, number, location) and ▶ metastasis formation. Clinical Relevance for Other Types of Cancer Ectopic CDX2 expression is described in various types of ▶ adenocarcinomas, especially in those arising in the stomach, esophagus, and ovary. More surprisingly, leukemia patients, and above all 90% of patients with ▶ acute myeloid leukemia (AML), exhibit ectopic CDX2 expression. The mechanism involved in this aberrant expression of CDX2 is not yet elucidated. Nevertheless, CDX2 expression represents a marker of bad prognosis and reduced survival for leukemia patients. In contrast to most intestinal cell lines, CDX2 stimulates the proliferation and the ability to form colonies of hematopoietic cells in vitro. In addition, ectopic CDX2 expression in transplanted hematopoietic cells was sufficient to induce AML in mice by perturbing the expression of HOX genes. The pro-oncogenic role of CDX2 in leukemia may be linked to the involvement of CDX genes in embryonic hematopoiesis described in zebrafish or murine pluripotent stem cells but awaits further investigation.

References Aoki K et al (2011) Suppression of colonic polyposis by homeoprotein CDX2 through its nontranscriptional function that stabilizes p27Kip1. Cancer Res 71(2):593–602 Beck F, Stringer EJ (2010) The role of Cdx genes in the gut and in axial development. Biochem Soc Trans 38(2):353–357 Gao N, White P, Kaestner KH (2009) Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2. Dev Cell 16(4):588–599 Lengerke C, Daley GQ (2012) Caudal genes in blood development and leukemia. Ann N Y Acad Sci 1266:47–54 Subtil C et al (2007) Frequent rearrangements and amplification of the CDX2 homeobox gene in human sporadic colorectal cancers with chromosomal instability. Cancer Lett 247(2):197–203

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CDX3 ▶ CDX2

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and differentiation and their overexpression (CEA and CEACAM6) or their downregulation (CEACAM1 and CEACAM7) contributes to progression of many epithelial cancers and immune dysfunctions.

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Characteristics

▶ CDX2

The CEA gene family encodes a set of 22 genes and 11 pseudogenes clustered in a 1.8 Mb region on human chromosome 19q13.2 between the CY2A and D19S15 marker genes. The CEA genes encompass an N-terminal Ig variable domain followed by one to six Ig constant-like domains. A striking characteristic of these proteins is their extensive ▶ glycosylation on asparagine residues with multiantennary carbohydrate chains. CEA and CEACAM1 are further modified by the addition of Lewis and sialyl-Lewisx highmannose residues. The proteins differ, however, in their C-terminal regions producing either secreted entities such as the pregnancy-specific glycoproteins (PSG1–11) or others, tethered to the cell surface by either a glycosyl phosphatidylinositol linkage (CEA, CEACAM6–8) or a bona fide transmembrane domain (CEACAM1, CEACAM3, CEACAM4, CEACAM18–21) (Fig. 1). The CEACAM1 gene is unique in this family in that it produces 12 different splicing variants. More information on the structural features of the CEA gene family members is available at http://www.carcinoembryonic-antigen.de/. CEA is a monomeric protein adopting a b-barrel cylindrical shape resembling a “bottle brush,” whereas CEACAM1 is present as both a monomeric and dimeric protein.

CEA ▶ Carcinoembryonic Antigen

CEA Gene Family Nicole Beauchemin Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada

Synonyms C-CAM; CD66a; CD66b; CD66c; CD66e; CEACAM1 = BGP; CEACAM5 = CEA; CEACAM6 = NCA; CEACAM7 = CGM2; CEACAM8 = CGM6

Definition The carcinoembryonic antigen (CEA) gene family comprises 33 genes, 22 of which are expressed. All family members share similar structural features encompassing immunoglobulin (Ig) variable and/or constant domains and therefore constitute members of the large immunoglobulin superfamily. These proteins are either secreted or membrane bound. Several CEACAMs function as homophilic or heterophilic intercellular ▶ cell adhesion molecules. CEA, CEACAM1, CEACAM6, and CEACAM7 also play a significant role as regulators of tumor cell proliferation

Expression and Functions of CEA Family Members in Normal and Tumor Tissues Although not ubiquitous, CEA family members exhibit a wide tissue distribution. CEA and CEACAM6 are found mainly in columnar epithelial and goblet cells of the colon in the early fetal period and are maintained in adult life. In the colonic brush border, CEA, CEACAM1, 6 and 7 demonstrate maximal expression at the free luminal surface, although CEACAM1 and 7 are

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CEA or CEACAM1-4L CEACAM5 CEACAM6 CEACAM7 CEACAM8 CEA Gene Family, Fig. 1 Schematic representation of some members of the CEA family. Most CEA family members, except the pregnancy-specific glycoproteins (PSG) that are secreted proteins, are associated with the cell membrane (depicted in grey). The immunoglobulin variable-like domains (the N domain) are shown in blue and the immunoglobulin constant-like domains are

represented in orange. The N-linked glycosylation sites are indicated by sticks and balls, colored in dark orange. The glycosylphosphatidylinositol membrane anchors are represented by arrows. The CEACAM1 gene expresses many splice variants. However, only the CEACAM-4L isoform containing four Ig domains and the longer cytoplasmic tail is shown here

also found at the lateral membrane. In addition to its expression in epithelia, CEACAM1 is located on granulocytes, lymphocytes, and endothelial cells, whereas CEACAM6 is also expressed on granulocytes and monocytes. CEACAM3 and 8 are found exclusively on granulocytes. CEA, CEACAM1, and CEACAM6 are recognized as cell adhesion molecules contacting each other by antiparallel self-binding (homophilic). Some associations are exclusive, such as CEACAM8-CEACAM6. The first Ig domain is crucial in these interactions. Various CEA family members also act as heterophilic partners for E-selectin and galectin-3. Another striking feature of CEA family members is their ability to act as pathogen receptors binding to outer membrane proteins of Neisseria gonococci and Haemophilus influenzae as well as fimbriae of Salmonella typhimurium and Escherichia coli. In addition, CEACAM1 is the receptor for the mouse hepatitis viruses. The bacterial and viral adhesin functions of the CEA family members confer strong immunosuppressive activity in T and B lymphocytes, whereas they enhance integrin-dependent cell adhesion in epithelial cells with concomitant

increase of the TGF-b1 receptor CD105. Other functions for CEA and CEACAM6 include the inhibition of cellular differentiation as demonstrated in a number of cellular systems and inhibition of the apoptotic process of ▶ anoikis by activation of b1 integrins. PSG1–11 are mainly expressed in syncytiotrophoblast during the first trimester of pregnancy where they act as immunomodulators and inhibit cell-matrix interactions. CEA is abundantly expressed in tumors of epithelial origin such as colorectal, lung, mucinous ovarian, and endometrial adenocarcinomas. For these reasons, CEA has a long history as a marker of colonic, intestinal, ovarian, and breast tumor progression and its high expression is associated with poor prognostic and recurrence of disease postsurgically. High preoperative CEA levels are indicative of a poor prognosis whereas low levels are associated with increased survival of the patients. The tumorigenic potential of CEA and CEACAM6 was clarified by transgenic overexpression of a bacterial artificial chromosome fragment of 187 kb encoding the full CEA, CEACAM6, and CEACAM7 genes. When the

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CEABAC transgenic mice were treated with the azoxymethane carcinogen to induce colon cancers, expression of CEA and CEACAM6 was increased by 2–20 fold, a situation reminiscent to that observed in the human cancer. Information on CEACAM7 expression in tumors is more limited. It is downregulated in colorectal cancers, but increased in gastric tumors. CEACAM6, however, exhibits a broader distribution than in the cancers described above, as it is additionally found in gastric and breast carcinomas and ▶ acute lymphoblastic leukemias. In fact, overexpression of CEACAM6 in ▶ pancreatic cancer confers increased resistance to anoikis and increased metastasis. It also modulates chemoresistance to the ▶ gemcitabine agent, thereby suggesting that CEACAM6 determines cellular susceptibility to apoptosis. Expression and Functions of CEACAM1 CEACAM1 expression is more complex. It is downregulated in colon, prostate, hepatocellular, bladder, endometrial, renal cell, and 30% of breast carcinomas, but overexpressed in gastric and squamous lung cell carcinomas, bladder cancer and ▶ melanomas. In thyroid carcinomas, CEACAM1 was shown to restrict tumor cell growth. However, it increases the thyroid cancer metastatic potential. Manipulation of CEACAM1 expression levels in colonic, prostatic, and bladder tumor cell lines, negative for CEACAM1, has indeed confirmed that expression of the longer variant, CEACAM1-4L, produces reduction of tumorigenic potential in vitro and inhibition of tumor growth in xenograft mouse models. The importance of cell surface CEACAM1 expression for maintenance of normal epithelial cellular behavior has been confirmed in vivo; a Ceacam1-null mouse exhibits a significantly increased colon tumor load compared to the wild-type littermates upon carcinogenic induction of colorectal cancer. CEACAM1’s role as a modulator of tumor progression depends on the involvement of its cytoplasmic domain in signaling via its tyrosine and serine phosphorylation. Two Tyr residues are positioned within immunoreceptor tyrosine-based inhibition motifs (ITIM). The

CEA Gene Family

membrane-proximal Tyr488 is a phosphorylation substrate of Src-like kinases as well as of the insulin and epidermal growth factor receptors. Upon Tyr phosphorylation, CEACAM1-L associates with the tyrosine phosphatases SHP-1 and SHP-2. The SHP-1-CEACAM1-L protein complex regulates its function in various tissues such as inhibition of epithelial cell growth, CD4+ T cell activation, and insulin clearance from hepatocytes. CEACAM1-L tyrosine phosphorylation also stimulates its association with the cytoskeletal proteins G-actin, tropomyosin, and paxillin, thereby influencing cell adhesion, and with the b3 integrin, hypothesized to influence cell motility. The CEACAM1-L cytoplasmic domain also carries 17 serine residues most of which lie in consensus sequences recognized by serine kinases. However, little is known about their functional implications apart from the CEACAM1-S Thr/Ser452 and Ser456, shown to modulate direct binding to G- and F-actin, tropomyosin, and calmodulin, and CEACAM1-L’s Ser503 whose mutation to an Ala residue enhances colonic or prostatic tumor development in xenograph models. Additionally, Ser503 renders permissive Tyr488 phosphorylation by the insulin receptor. Transgenic mice overexpressing a Ser503Ala CEACAM1-L mutant in the liver developed hyperinsulinemia, secondary insulin resistance, and defective insulin clearance. As a consequence of the decreased insulin receptor endocytosis and altered insulin signaling, the transgenic mice became obese demonstrating increased visceral adiposity, elevated serum free fatty acids and plasma and hepatic triglyceride levels. CEACAM1-L also contributes to important functions in the immune system. It functions as an inhibitory coreceptor in T lymphocytes. Its conditional deletion in these cells amplified TCR-CD3 signaling, whereas overexpression in T cells was responsible for decreased proliferation, allogeneic reactivity, and cytokine production in vitro, with delayed type hypersensitivity and inflammatory bowel disease in vivo. Regulation of this function involves the ITIM motifs and the SHP-1 tyrosine phosphatase. A similar function and mechanism have been described in B lymphocytes and natural killer cells. Indeed,

CEA Gene Family

CEACAM1-mediated intercellular adhesion between melanomas with increased CEACAM1 expression and NK cells allows inhibition of NK-cell-elicited killing, thereby conferring upon CEACAM1 a role in tumor immunosurveillance. Similarly, heterophilic engagement of CEACAM1 with CEA, overexpressed in many tumors, also inhibits lymphocyte-mediated and NK-cell-mediated killing having therefore detrimental effects on immune surveillance. In addition, increased expression of CEACAM1 on endothelial cells present in tumors in response to VEGF activation and/or hypoxia provokes a proangiogenic switch with increased endothelial tube formation and invasion. Therefore CEACAM1’s contribution to cancer progression most likely depends on its positive or negative expression and signaling in epithelial tumor cells, on its systemic effects on metabolism and adiposity, on its role in immunosurveillance, and most probably on endothelial proliferation and invasion. Transcriptional Regulation The upstream promoters of the CEA and CEACAM1 genes have been dissected to identify important binding sites responsible for their transcriptional regulation. These two genes do not encompass classical TATA and CAAT boxes and are considered members of the housekeeping gene family. Their distal promoter regions (> 500 bp) contain highly repetitive elements, whereas their proximal promoter regions are rich in GC boxes and SP1 binding sites. Five footprinted regions have been identified in the CEA promoter, the first three binding respectively, to the upstream stimulatory factor (USF) and SP1 and SP1-like factors. Similarly, the human CEACAM6 promoter is regulated by the USF1 and USF2 as well as SP1 and SP3 transcription factors. A silencer element has also been located in its first intron. In contrast, the human CEACAM1 promoter does not bind the SP1 factors, but associates with an AP-2-like factor and the USF and HFN-4 transcription factors. The gene is additionally controlled by the hormonal changes (estrogens and androgens) and can be induced by cAMP, retinoids, glucocorticoids, and insulin. Moreover, many genes of this

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large family are triggered by inflammation via interferons, tumor necrosis factors, and interleukins. It has been reported that expression of the CEACAM1 gene is influenced by TPA and calcium ionophore in endometrial cancers, the expression of BCR/ABL in leukemias, the expression of the b3 integrin in melanomas, and VEGF and hypoxia in angiogenic situations. In prostate cancer, there is an inverse correlation between the downregulation of CEACAM1 and the increased expression of the transcriptional repressor Sp2 that acts to recruit histone deacetylase to the CEACAM1 promoter. The Next Frontier The diversity of functions of the members of the CEA gene family and their dynamic expression patterns in normal and tumor tissues has slowed the development of effective targeted therapies. Effective strategies have been devised using vaccination with CEA peptide-loaded mature dendritic cells that induced potent CEA-specific T cell responses in advanced colorectal cancer patients. Effective protection from tumor development have also been seen with delivery of adenoviral vectors encoding CEA fused to immunoenhancing agents such as tetanus toxin or the Fc portion of IgG1. Likewise, targeting of CEACAM6 in pancreatic cancer may result in decreased tumor load. The therapeutic and selective targeting of CEACAM1 in melanomas, gastric and lung carcinomas as well as its location in tumor endothelia may prove to be a favorable avenue of future interventions.

References Beauchemin N, Arabzadeh A (2013) Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer and Mets Rev 32:643–671 Beauchemin N, Draber P, Dveksler G, Gold P, Gray-Owen S, Grunert F, Hammarstrom S, Holmes KV, Karlsson A, Kuroki M, et al (1999) Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res 252:243–249 Gray-Owen SD, Blumberg RS (2006) CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 6:433–446

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874 Hammarström S (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 9:67–81 Horst A, Wagener C (2004) CEA-related CAMs. Handb Exp Pharmacol 165:283–341 Kuespert K, Pils S, Hauck CR (2006) CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol 18:1–7 Leung N, Turbide C, Marcus V et al (2006) Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) contributes to progression of colon tumors. Oncogene 25:5527–5536

CEACAM1

CEACAM1

molecules. Additionally, a number of pseudogenes have been identified. To date, 29 genes are known, which are clustered on human chromosome 19 (19q13.1-19q13.2). The CEA-related members of the CEA family display a complex expression pattern on human healthy and malignant tissues. They are linked to the cell membrane via GPI anchors, or they are transmembrane proteins with a cytoplasmatic tail. The PSG-related molecules are soluble glycoproteins; their expression is restricted to the placenta, more specifically, to the syncytiotrophoblast, which is the outermost fetal component of the placenta. CEACAM1 has been structurally and functionally conserved in humans and rodents.

▶ CEACAM1 Adhesion Molecule

Characteristics

CEACAM1 Adhesion Molecule Andrea Kristina Horst1 and Christoph Wagener2 1 Inst. Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Synonyms BGP; Biliary glycoprotein; CD66a; CEACAM1; CEA-related cell adhesion molecule 1; Cluster of differentiation antigen 66 a; NCA-160; Nonspecific cross-reacting antigen with a Mw of 160kD

Definition CEACAM1 (CEA-related cell adhesion molecule 1) belongs to the CEA (▶ carcinoembryonic antigen, ▶ CEA gene family) family of cell surface glycoproteins, a subfamily of the immunoglobulin gene superfamily. The CEA family comprises two major groups, the CEA-related molecules and the PSG (pregnancy-specific glycoprotein)-related

Properties of CEACAM1 Human CEACAM1 has been originally identified in human bile due to its crossreactivity with CEA-antisera. It was therefore named biliary glycoprotein I or nonspecific cross-reacting antigen at first. Amongst the cluster of differentiation antigens on human leukocytes, CEACAM1 used to be referred as CD66a. However, with the latest revision of the nomenclature for the CEA family, CD66a, BGP, or NCA-160 became CEACAM1. Its structural similarities to CEA and the immunoglobulin superfamily proteins became apparent, once the cDNA sequence for CEACAM1 became available. CEACAM1 displays the broadest expression pattern amongst CEA family members; it has first been described as a cell–cell adhesion molecule on rat hepatocytes. CEACAM1 is expressed on epithelia, endothelia, and leukocytes. CEACAM1 is a heavily glycosylated molecule that exists in 11 known isoforms emerging from differential splicing and proteolytic processing. The two major isoforms of CEACAM1 consist of four extracellular Ig-like domains, a transmembrane domain, and either a long or a short cytoplasmic tail, referred to as the long (CEACAM1-4L) and the short isoform (CEACAM1-4S), respectively. In addition to these transmembrane isoforms, soluble

CEACAM1 Adhesion Molecule

CEACAM1 isoforms are found in body fluids, for example, in saliva, serum, seminal fluid, and bile. Glycans on the extracellular domains of CEACAM1 are linked to the protein backbone via N-glycosidic linkages. It is presently unknown whether all of the 19 motifs that may render N-linked ▶ glycosylation actually harbor sugar moieties. On human granulocytes, CEACAM1 is a major carrier of Lewisx glycans that are implicated in cellular adhesion to cognate lectins on blood vessels, within the extracellular matrix, or antigen presenting cells. CEACAM1 also elicits cell–cell adhesion via self-association in a homomeric fashion or via formation of heteromers with other CEA-family members and different adhesion molecules that are either located on the same cell or on neighboring cells. The resulting adhesive properties are modulated by differential expression ratios between the long and short CEACAM1 isoform, respectively. Through its long and short cytoplasmic tail, CEACAM1 mediates molecular interactions with cytoskeletal components or adapter proteins, which are integral parts of various key signal transduction pathways (signal transduction, cell biology). These interactions are in part dependent on differential phosphorylation of the CEACAM1-4L cytoplasmic domain on tyrosine and serine residues. The overall phosphorylation status of the CEACAM1-4L cytoplasmic domain relays signals, which contribute to cellular motility and differentiation, and thus determine cell fate by promoting proliferation or cell death. Phosphorylation of CEACAM1-4L cytoplasmic tyrosines that are part of an imperfect ITIM (immune receptor tyrosine-based inhibition motif) and serine residues regulate the interaction with kinases, phosphatases, cellular receptors for insulin (▶ Insulin receptor), the epidermal growth factor (epidermal growth factor receptor ligand, epidermal growth factor receptor inhibitor), and other cellular adhesion molecules, for example, integrin avb3 (integrin signaling and cancer). These qualities make CEACAM1 an important tool for cellular communication and they illustrate why so many different biological functions have been attributed to CEACAM1 in different biological contexts (Fig. 1).

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CEACAM1 in Cancer The first report on CEACAM1, in the context of human pathological conditions, was on elevated serum levels of a biliary glycoprotein in patients with liver or biliary tract disease. Later, aberrant CEACAM1 expression in a broad variety of human malignancies has been reported. In the progression of malignant diseases, two general patterns in the changes of CEACAM1 expression levels have emerged. In the first group of tumors, CEACAM1 expression is downregulated in the course of progressing disease. In the second group of tumors, CEACAM1 expression appears to be upregulated; often, this upregulation of CEACAM1 expression is observed in the context with increased invasiveness (▶ invasion) of the primary tumor or is found on microvessels in progressing (▶ progression) tumor areas (Fig. 2). Loss of CEACAM1 Expression in Tumorigenesis and Tumor Progression Human cancers that show the downregulation of CEACAM1 expression in the course of tumor progression are carcinomas of the liver (▶ hepatocellular carcinoma), colon (colon cancer, colorectal premalignant lesions), kidney (renal cell carcinoma, renal carcinoma), urinary bladder (bladder cancer, bladder tumors), prostate (prostate cancer, clinical oncology), mammary gland (▶ breast cancer), and the endometrium (▶ endometrial cancer). In general, downregulation and subsequent loss of CEACAM1 expression is more frequent in high-grade tumors that are poorly differentiated and often associated with a larger tumor size. On epithelia, especially those that form a lumen, CEACAM1 exhibits a pronounced apical expression, like in the entire gastrointestinal tract, breast, liver, prostate, bladder, and kidney. CEACAM1 expression has been implicated in morphogenesis of lumen formation. In the process of building an asymmetrical epithelium, lateral CEACAM1 expression on neighboring cells is lost and often becomes entirely apical once a lumen or a duct has been formed. The loss of CEACAM1 expression in the context of tumorigenesis has been studied most extensively in the context of breast, colonic, and prostate carcinomas.

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CEACAM1 Adhesion Molecule

B1

A2

S-S

A1

A1

B1

S-S

Integrin ανβ3

A2

S-S

N

S-S

N

N. meningitidis N. gonorrhoe M. catarrhalis Murine hepatitis virus

S-S

CEACAM1-4S

S-S

CEACAM1-4L

Galectin-3 DC-SIGN S. typhimurium E. coli

src, SHP1, SHP2, caspase-3, paxillin, filamin, calmodulin

Tyr488

Actin, tropomyosin Tyr515

Ser503

CEACAM1 Adhesion Molecule, Fig. 1 Schematic representation of CEACAM1-4L and CEACAM1–4S and their participation in extracellular and intracellular communication. The two major CEACAM1 isoforms consist of four extracellular immunoglobulin-like domains, a transmembrane domain and either a long or a short cytoplasmic tail. The N-terminal domain (N) resembles a variable-like Ig domain but lacks the cystin bond usually found in Ig members. The A1, B1, and A2 domain resemble constant I-type-like Ig domains. Motifs for N-linked glycosylation are represented by lollipops. With its extracellular domains, CEACAM1 mediates recognition of various pathogens, such as Escherichia coli, Salmonella typhimurium, Moraxella catarrhalis, Neisseria gonorrhoeae, and Neisseria meningitidis. The murine homologue of CEACAM1 is the receptor for the murine

hepatitis virus: Additionally, CEACAM1 binds to galectin3, DC-SIGN (dendritic cell ICAM3-grabbing nonintegrin), and integrin avb3. Tyrosine and serine residues involved in relaying CEACAM1-4L-mediated signal transduction are indicated by red and grey circles, respectively. Through its long cytoplasmic tail, CEACAM1-4L interacts with intracellular kinases of the SRC-family (▶ SRC), the tyrosine phosphatases SHP-1 and SHP-2, caspase-3 as well as with paxillin, filamin, and calmodulin. Differential phosphorylation of the CEACAM1-4L cytoplasmic domain is required for its interaction with the insulin receptor, regulating insulin receptor internalization and recycling, and for modulating immune responses elicited by lymphocytes, for example. The short cytoplasmic domain of CEACAM1–4S binds to actin and tropomyosin

A hallmark of carcinomatous lesions is the loss of polarity of their epithelial structures. In colonic epithelium, for example, loss of polarity is accompanied by the loss of apical CEACAM1 expression that occurs in early adenomas and carcinomas. In these tumors, the presence and absence of CEACAM1 correlate with normal and reduced apoptosis (apoptosis, apoptosis signals), respectively. Furthermore, the naturally occurring process of ▶ anoikis, once cells lose contact to their substratum, is compromised.

This observation and the fact that the CEACAM1 gene is silenced in the course of aberrant cell growth prompted the hypothesis that CEACAM1 acts as a tumor suppressor. In intestinal cells, the presence of the long CEACAM1 isoform is required to suppress tumor growth, and the lack of CEACAM1-4L expression is accompanied by a decrease in proteins that inhibit cell cycle progression. In human mammary epithelial cells, CEACAM1 expression is causally related to

CEACAM1 Adhesion Molecule

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Brain

CEACAM1

Thyroid

Breast Lung

Liver

Pancreas Colon

Kidney

Skin

Endometrium Bladder Prostate

CEACAM1

CEACAM1 Adhesion Molecule, Fig. 2 Dysregulation of CEACAM1 expression in human cancers. Changes of epithelial CEACAM1 expression in the course tumor progressison: In mammary carcinomas and carcinomas of the liver, colon, endometrium, kidney, bladder, and prostate, CEACAM1 expression is downregulated on tumor epithelium (epithelial cancers). Downregulation of CEACAM1 levels often correlates with dedifferentiation of the tumor and loss of tissue architecture. In carcinomas of the thyroid, ▶ non-small cell lung cancer (▶ lung cancer), pancreatic tumors (pancreas cancer, clinical oncology), and malignant melanomas, CEACAM1 is induced or upregulated in the course of tumor growth. Here, CEACAM1 expression is found on the invasive front of the tumors and is related to development of metastatic disease (▶ metastasis) and poor prognosis. In pancreatic cancer, CEACAM1 has been identified as a novel biomarker (biomarker, clinical cancer biomarker) that indicates the presence of malignant disease

lumen formation and differentiation. In mammary glands, CEACAM1-4S is the predominating isoform, and only the short cytoplasmic tail induces apoptosis of the central cells and subsequently leads to lumen formation in mammary morphogenesis. During tumor progression, CEACAM14S expression is lost and acinar polarity no longer can be observed.

However, since particular mutations or allelic loss of the CEACAM1 gene in human cancers has not been described so far, it is likely that the dysregulation of CEACAM1 expression rather than irreversible loss of the CEACAM1 gene are linked to tumorigenesis and tumor progression in vivo. Hence, gene silencing may attribute to the loss of the tumor suppressive qualities of CEACAM1. Though there are no changes in promoter ▶ methylation of the CEACAM1 gene linked to tumor progression, CEACAM1 promoter activity appears to be regulated by binding of the transcription factor Sp2. In high-grade prostate carcinomas, Sp2 is highly abundant, whereas CEACAM1 expression is lost. Sp2 localizes to the CEACAM1 promoter and imposes repression of gene transcription by recruiting histone deacetylase. Upregulation of CEACAM1 Expression in Malignant Diseases Opposed to its tumor suppressive functions, certain tumors gain CEACAM1 expression in the course of cancer development. In the case of malignant melanomas and thyroid carcinomas, expression of CEACAM1 correlates with an increase of tumor invasiveness and development of metastatic disease. In primary cutaneous malignant melanomas, for example, CEACAM1 expression is found at the invasive front of the tumors, and its coexpression with integrin avb3 indicates that CEACAM1 may directly promote on cellular invasion. In a follow-up study, CEACAM1 was identified as an independent prognostic marker, predicting the development of metastatic disease and poor survival. In this context, it is noteworthy that CEACAM1 on melanoma cells forms homophilic cell–cell contacts with CEACAM1 molecules on tumorinfiltrating lymphocytes and leads to the inhibition of their cytolytic function. Similarly, in human non-small cell lung cancer, CEACAM1 expression correlates with advanced disease, whereas it is not expressed on the normal bronchiolar epithelium; this CEACAM1 neoexpression was identified as an independent prognostic marker, indicating lower incidence of relapse-free survival.

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In pancreatic carcinomas, CEACAM1 has been identified as a novel serum biomarker, with an increased CEACAM1 expression on neoplastic cells of pancreatic adenocarcinomas and elevation of serum levels at the same time. Additionally, significant differences in CEACAM1 serum levels were found in patients with either pancreatic cancer or chronic pancreatitis. Opposed to the classical pancreatic tumor marker CA19-9, CEACAM1 was confirmed as an independent marker to distinguish between the presence of malignant disease and pancreatitis. CEACAM1 and Tumor Angiogenesis CEACAM1 expression on human blood vessels is restricted to newly formed vessels, and usually, no CEACAM1 is found on mature, large vessels. The first indication that CEACAM1 is related to ▶ angiogenesis was the description of CEACAM1 neoexpression on newly formed vessels in the human placenta. Furthermore, CEACAM1 is expressed on vessels in wound healing tissues and on tumor vessels of human bladder carcinomas, the prostate, hemangiomas, and ▶ neuroblastomas. CEACAM1 expression in endothelia is induced by VEGF (▶ vascular endothelial growth factor)-dependent pathways and appears to favor vessel maturation. In human prostate carcinomas, CEACAM1 shows divergent expression on tumoral blood vessels and the tumor epithelium. The presence of epithelial CEACAM1 is observed in the context of poor tumoral blood vessel growth and loss of epithelial CEACAM1 expression parallels enhanced tumor angiogenesis. Especially in high-grade prostate carcinomas, tumor proximal vessels are expressing CEACAM1. Contrary to prostate carcinomas, microvessels in human neuroblastomas are CEACAM1-positive only during tumor maturation, but absent in undifferentiated, high-grade tumors. In ▶ Kaposi sarcomas, CEACAM1 upregulation is observed, indicating that CEACAM1 might be related to lymphatic reprogramming of the vasculature in these tumors. Studying CEACAM1 in Cancer: Animal Models In animal models investigating CEACAM1 function in tumorigenesis in vivo, the observations

CEACAM1 = BGP

from human diseases could be confirmed. The focus of the mouse and rat models (▶ Mouse model) studied to date was set largely on the tumor-suppressive effects or enhancement of metastatic disease of CEACAM1-4L on the progression of colonic cancer, prostate cancer, hepatocellular carcinomas, and malignant melanomas. In CEACAM1-knockout mice, chemically induced colonic tumor growth was significantly increased in terms of tumor numbers and size opposed to CEACAM1-expressing wild type littermates. In syngeneic and xenotypic transplantation of tumor cells of the colon, prostate, and hepatocellular carcinomas, the tumor-suppressive effects of CEACAM1-4L expression could also be validated. After xenotransplantation of human CEACAM1-expressing melanoma cell lines into immune-deficient mice, enhanced metastasis was observed when compared to transplantation of CEACAM1-negative cell lines.

References Beauchemin N, Draber P, Dveksler G et al (1999) Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res 252:243–249 Gray-Owen SD, Blumberg RS (2006) CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 6:433–446 Kuespert K, Pils S, Hauck CR (2006) CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol 18:565–571 Prall F, Nollau P, Neumaier M et al (1996) CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues. J Histochem Cytochem 44:35–41 Singer BB, Lucka LK (2005) CEACAM1. UCSD-nature molecule pages. Nat Publ Group. doi:10.1038/mp. a003597.01

CEACAM1 = BGP ▶ CEA Gene Family

Celastrol

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CEACAM5

Celastrol

▶ Carcinoembryonic Antigen

Qing Ping Dou1 and Xiao Yuan2 1 The Prevention Program, Barbara Ann Karmanos Cancer Institute and Department of Pathology, School of Medicine, Wayne State University, Detroit, MI, USA 2 Research and Development Center, Wuhan Botanical Garden, Chinese Academy of Science, Wuhan, Hubei, People’s Republic of China

CEACAM5 = CEA ▶ CEA Gene Family

Synonyms

CEACAM6 = NCA ▶ CEA Gene Family

CEACAM7 = CGM2 ▶ CEA Gene Family

CEACAM8 = CGM6 ▶ CEA Gene Family

CEA-Related Cell Adhesion Molecule 1 ▶ CEACAM1 Adhesion Molecule

CED ▶ Convection-Enhanced Delivery

Quinone methide friedelane tripterene (2R,4aS,6a S,12bR,14aS,14bR)-10-hydroxy-2,4a,6a ,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a ,11,12b,13,14,14a,14b-tetradecahydropicene-2carboxylic acid; Tripterine

Definition Celastrol is a natural quinone methide friedelane tripterene, widely found in the plant genera Celastrus, Maytenus, and Tripterygium, all of which are present in China. For example, celastrol is one of the active components extracted from Tripterygium wilfordii Hook F, an ivy-like vine also known as “Thunder of God Vine,” which belongs to the family of Celastraceae and has been used as a natural medicine in China for hundreds of years (Fig. 1).

Characteristics Biological Properties Celastrol has strong antifungal, antiinflammatory, and antioxidant effects. It has been shown that celastrol isolated from the roots of Celastrus hypoleucus (Oliv) Warb f argutior Loes exhibited inhibitory effects against diverse phytopathogenic fungi. Celastrol was also found to inhibit the mycelial growth of Rhizoctonia solani Kuhn and Glomerella cingulata (Stonem) Spauld and Schrenk in vitro. Furthermore,

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Celastrol

Celastrol, Fig. 1 The chemical structure and nucleophilic susceptibility of celastrol. (a) The chemical structure of celastrol is shown. (b) Nucleophilic susceptibility of

celastrol analyzed using CAChe software. Higher susceptibility was shown at the C2 and C6 positions of celastrol

celastrol has good preventive effect and curative effect against wheat powdery mildew in vivo. Celastrol in low nanomolar concentrations suppresses the production of the proinflammatory cytokines tumor necrosis factoralpha (TNF-a) and interleukin-1 beta (IL-1b) by human monocytes and macrophages. Celastrol also decreases the induction of class II major histocompatibility complex (MHC) expression by microglia. In macrophage lineage cells and endothelial cells, celastrol decreases induction of nitric oxide (NO) production. Celastrol also suppresses adjuvant arthritis in the rat, demonstrating in vivo anti-inflammatory activity. Low doses of celastrol administered to rats could significantly improve the performance of these animals in memory, learning, and psychomotor activity. In an isolated rat liver assay of lipid peroxidation, the antioxidant potency of celastrol (IC50 7 mM) is 15 times stronger than that of a-tocopherol or vitamin E. Under in vitro conditions, celastrol was found to inhibit ▶ cancer cell proliferation and induce programmed cell death (or ▶ apoptosis) in a broad range of tumor cell lines, including 60 National Cancer Institute (NCI) human cancer cell lines. As a ▶ topoisomerase II inhibitor, celastrol was fivefold more potent than the well-known topoisomerase inhibitor etoposide to induce apoptosis in HL-60 leukemia cells. Celastrol was also found to be a

tumor ▶ angiogenesis inhibitor. In a sharp comparison, celastrol can block neuronal cell death in cultured cells and in animal models. These unique features of celastrol suggest potential use for treatment of cancer and neurodegenerative diseases accompanied by inflammation, such as Alzheimer disease. Potential Molecular Targets Celastrol is a naturally occurring potent inhibitor of the ▶ proteasome and nuclear factor kappa B (NFkB). Proteasome, or 26S proteasome, is a multicatalytic protease complex consisting of a 20S catalytic particle capped by two 19S regulatory particles. The ubiquitin-proteasome pathway is responsible for the degradation of most endogenous proteins involved in gene transcription, cell cycle progression, differentiation, senescence, and apoptosis. Inhibition of the proteasomal chymotrypsinlike but not trypsin-like activity is associated with induction of apoptosis in tumor cells. Both computational and experimental data support the hypothesis that celastrol is a natural proteasome inhibitor. Atomic orbital energy analysis demonstrates high susceptibility of C2 on A-ring and C6 on B-ring of celastrol toward a nucleophilic attack. Computational modeling shows that celastrol binds to the proteasomal chymotrypsin site (b5 subunit) in an orientation and conformation that is suitable for a nucleophilic attack by the hydroxyl (OH) group of N-terminal

Celastrol

Celastrol, Fig. 2 Docking solution of celastrol. Celastrol was docked to S1 pocket of b5 subunit of 20S proteasome. Celastrol was shown in pink while b5 subunit was shown in purple. The selected conformation with 92% possibility showed the distances to the OH group of N-Thr from C6 and C2 were 2.96 Å and 4.16 Å, respectively

threonine of b5 subunit. The distances to the OH of N-terminal threonine of b5 from the electrophilic C6 and C2 of celastrol are measured as 2.96 Å and 4.16 Å, respectively. Both carbons, more probably C6, of celastrol potentially interact with N-terminal threonine of b5 subunit and inhibit the proteasomal chymotrypsin-like activity (Fig. 2). Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value 2.5 mM. Celastrol at 1–5 mM inhibits the proteasomal activity in intact human prostate cancer cells. The inhibition of the cellular proteasome activity by celastrol results in accumulation of ubiquitinated proteins and three natural proteasome substrates, IkB-a, Bax, and p27, leading to induction of apoptosis in ▶ androgen receptor (AR)-negative PC-3 cells. In AR-positive LNCaP cells, celastrol-mediated proteasome inhibition was accompanied by suppression of AR protein, probably by inhibiting ATP-binding activity of heat shock protein 90 (Hsp90) that is responsible for AR folding. Treatment of PC-3 tumor-bearing nude mice with celastrol (1–3 mg/kg/day, i.p., for 1–31 days) resulted in significant inhibition (65–93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and

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end time points demonstrated in vivo inhibition of the proteasomal activity and induction of apoptosis after celastrol treatment. Antitumor activity of celastrol was also observed in a breast cancer mouse model. Celastrol inhibited 60% tumor growth in breast cancer xenograft through NFkB inhibition. NFkB inhibition by celastrol includes inhibition of its DNA-binding activity and inhibition of IkBa degradation induced by TNF-a or phorbol myristyl acetate. Further investigation showed that the cysteine-179 in the IkBa kinase was a potential target of celastrolsuppressed IkBa degradation. Since the proteasome is required for the activation of NFkB by degrading IkBa, the proteasome inhibition may also contribute to the NFkB inhibition by celastrol. TNF could send both anti-apoptotic and pro-apoptotic signals. The effects of celastrol on cellular responses activated by the potent pro-inflammatory cytokine TNF have also been investigated. Celastrol was able to potentiate the apoptosis induced by TNF and chemotherapeutic agents and inhibited invasion, both regulated by NFkB activation. TNF induced the expression of gene products involved in anti-apoptosis (IAP1, IAP2, ▶ Bcl2, Bcl-XL, c-FLIP, and survivin), proliferation (cyclin D1 and COX-2), invasion (MMP-9), and angiogenesis (VEGF), and celastrol treatment suppressed the expression of these genes. Celastrol also suppressed both inducible and constitutive NFkB activation. Furthermore, celastrol was found to inhibit the TNF-induced activation of IkBa kinase, IkBa phosphorylation, IkBa degradation, p65 nuclear translocation and phosphorylation, and NFkBmediated reporter gene expression. Therefore, celastrol potentiates TNF-induced apoptosis and inhibits invasion through suppression of the NFkB pathway. Clinical Relevance Due to its antioxidant or anti-inflammatory effects, celastrol has been effectively used in the treatment of autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus), asthma, chronic inflammation, and neurodegenerative diseases. As a bioactive component in Chinese traditional medicinal products from the extract of the roots of Tripterygium

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wilfordii Hook F, celastrol has been used since the 1960s in China for autoimmune diseases but has showed some side effects such as nausea, vomiting, etc. Celastrol has not been used solely as a medication product. Celastrol has antitumor activities via inhibition of the proteasome and NFkB activation, indicating that celastrol has a great potential to be used for cancer prevention and treatment. This finding can be applied to various human cancers and diseases in which the proteasome is involved and on which celastrol has an effect.

Celebra

Celecoxib Numsen Hail1 and Reuben Lotan2 1 Department of Pharmaceutical Sciences, The University of Colorado at Denver and Health Sciences Center, Denver, CO, USA 2 Department of Thoracic Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Cross-References ▶ Topoisomerases

Celebra; Celebrex; 4-[5-(4-Methylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide

References Hieronymus H, Lamb J, Ross KN et al (2006) Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. Cancer Cell 10:321–330 Sassa H, Takaishi Y, Terada H (1990) The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria. Biochem Biophys Res Commun 172:890–897 Sethi G, Ahn KS, Pandey MK et al (2006) Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-?B-regulated gene products and TAK1-mediated NF-?B activation. Blood 109:2727–2735 Setty AR, Sigal LH (2005) Herbal medications commonly used in the practice of rheumatology: mechanisms of action, efficacy, and side effects. Semin Arthritis Rheum 34:773–784 Yang HJ, Chen D, Cui QZC et al (2006) Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine”, is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res 66:4758–4765

Celebra ▶ Celecoxib

Celebrex ▶ Celecoxib

Characteristics Celecoxib, a diaryl-substituted pyrazole drug, was developed by G. D. Searle & Company and is currently marketed by Pfizer Incorporated under the brand names Celebrex and Celebra. Celecoxib is a member of the class of agents known as ▶ non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are the most commonly used therapeutic agents for the treatment of acute pain, fever, menstrual symptoms, osteoarthritis, and rheumatoid arthritis. Because of their ability to reduce tissue ▶ inflammation, which is often associated with tumorigenesis at various sites in the body (e.g., gastrointestinal tract and lung), celecoxib and certain other NSAIDs are also considered to have a potential in cancer chemoprevention as exemplified by their ability to prevent the formation and decrease the size of polyps in familial adenomatous polyposis (FAP) patients. Orally administered celecoxib exhibits good systemic bioavailability and tissue distribution with an estimated plasma half-life of approximately 11 h. Celecoxib binds to plasma albumin and is metabolized primarily by hepatic enzymes prior to excretion. In humans, long-term exposures to celecoxib taken for arthritis pain relief at 100 mg twice daily caused no biologically significant adverse reactions. However, higher doses of

Celecoxib

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H3C

CF3

N N O H2N S O

Celecoxib, Fig. 1 The chemical structure of celecoxib

400 mg twice daily recommended for patients with FAP resulted in threefold increased risk of cardiovascular events (Fig. 1). ▶ Cyclooxygenase Dependent Mechanisms for Cancer Chemoprevention by Celecoxib. Cyclooxygenases are enzymes that are indispensable for the synthesis of ▶ prostaglandins. Prostaglandins are ▶ hormones generated from arachidonic acid, and they are found in virtually all tissues and organs. Prostaglandins typically act as short-lived local cell signaling intermediates that regulate processes associated with inflammation. In the early 1990s, cyclooxygenases were demonstrated to exist as two isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX-1 is characterized as a constitutively expressed housekeeping enzyme that mediates physiological responses like platelet aggregation, gastric cytoprotection, and the regulation of renal blood flow. In contrast, COX-2 is recognized as the inducible cyclooxygenase isoform that is primarily responsible for the synthesis of the prostaglandins that are involved in pathological processes (e.g., chronic inflammation) in cells that mediate inflammation (e.g., macrophages and monocytes). COX-2 is inducible by oncogenes (e.g., RAS and ▶ SRC), interleukin-1, ▶ hypoxia, benzo[a]pyrene, ultraviolet light, epidermal growth factor, ▶ transforming growth factor b, and tumor necrosis factor a. Many of these inducers activate

nuclear factor kappa B (NF-kB), which controls COX-2 expression and has been associated with tumorigenesis in various cell types. The COX-2 isoenzyme is frequently unregulated in cancer cells, as well as cells that constitute premalignant lesions, which are important targets for cancer chemoprevention. The expression of the inducible COX-2 is enhanced in 50% of colon adenomas and in the majority of human colorectal cancers, as opposed to COX-1, which typically remains unchanged. Thus, the increase in COX-2 expression, which is an early event in colon carcinogenesis, is believed to be necessary for tumor promotion. Aberrant COX-2 expression has also been implicated in tumorigenesis in the lung, prostate, esophagus, ▶ Brms1, liver, pancreas, and skin. The activity of COX-2 to produce arachidonic acid metabolites appears to enhance the proliferation of transformed cells and/or increases their survival through the suppression of ▶ apoptosis. Furthermore, COX-2 expression by tumor cells can stimulate ▶ angiogenesis at the tumor site and alter tumor cell adhesion to promote ▶ metastasis. Celecoxib is a highly selective inhibitor of COX-2. Traditional NSAIDs (e.g., aspirin) inhibit both COX-1 and COX-2 isozymes. In contrast, celecoxib is approximately 20 times more selective for COX-2 inhibition compared to its inhibition of COX-1. This specificity allows celecoxib, and other selective COX-2 inhibitors, to reduce inflammation while minimizing adverse drug reactions (e.g., stomach ulcers and reduced platelet aggregation) that are common with non-selective NSAIDs. This selectivity for COX-2 is also intimately associated with the putative cancer chemopreventive activity of celecoxib, which has been demonstrated in colorectal cancer prevention. Epidemiological studies have shown that persons who regularly take aspirin have about a 50% lower risk of developing colorectal cancer. Celecoxib was the most effective NSAID in reducing the incidence and multiplicity of colon tumors in a rat colon carcinogenesis model. Moreover, in a clinical setting celecoxib has been used effectively to suppress the development and/or reduce the number of colorectal polyps in patients with FAP. This

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inflammatory disease often predisposes individuals to the development of ▶ colorectal cancers. The anti-inflammatory mediated anticancer effects of celecoxib may be tissue-specific considering that celecoxib reduced lung inflammation in mice, but failed to inhibit the formation of chemically induced lung tumors in these animals. Cyclooxygenase Independent Mechanisms for Cancer Chemoprevention by Celecoxib. The results of several in vitro and animal studies suggest the celecoxib may suppress tumorigenesis through several COX-2-independent mechanisms, which may account, at least in part, for celecoxib’s anti-cancer effects in humans. For example, celecoxib inhibited the proliferation of various cancer cell types in vitro irrespective of their expression of COX-2, including transformed haematopoietic cells and immortalized and transformed human bronchial epithelial cells that were deficient in COX-2 expression. Celecoxib also inhibited the growth of human COX-2deficient colon cancer cells that were transplanted as xenografts in nude mice. Thus, the chemopreventive effect of COX-2-specific inhibitors like celecoxib may be due to their effect on COX-2 as well as targets other than COX-2. One putative COX-2 independent target for celecoxib is the phosphatidylinositol 3-kinase (PI3K) pathway, which is often deregulated in tumor cells. Celecoxib appears to directly inhibit the phosphoinositide-dependent kinase-1 (PDK1), and its downstream substrate protein kinase B/AKT, in the PI3K pathway. Protein kinase B/AKT inhibits apoptosis through the phosphorylation, and thus inactivation, of the proapoptotic ▶ BCL-2 family protein BAD. During apoptotic stimuli, BAD antagonizes BCL-2 and BCL-XL activity, which can promote mitochondrial membrane permeabilization and cell death. The inhibition of the PI3K pathway by celecoxib is believed to be specific in its ability to promote apoptosis in transformed cells. For example, rofecoxib, another specific COX-2 inhibitor, had only marginal protein kinase B/AKT inhibitory activity in tumor cells during apoptosis induction. Another presumed COX-2 independent target of celecoxib in tumor cells is sphingolipid

Celecoxib

metabolism. Celecoxib treatment increases the level of the sphingolipid ceramide in murine mammary tumor cells irrespective of COX-2 expression. This increase in ▶ ceramide was considered essential to apoptosis induction in these cells. Ceramide has been shown to mediate apoptosis in response to inflammatory cytokines like Fas and tumor necrosis factor a, and/or conditions associated with ▶ oxidative stress. During conditions of cell stress, the deregulation of ceramide generating and/or utilizing processes are believed to cause a net increase in cellular ceramide that is sufficient to trigger apoptosis induction via a mitochondrial membrane permeabilization mechanism. Celecoxib treatment has also been shown to suppress the activity of the Ca ATPase located in the endoplasmic reticulum of human prostate cancer cells. The inhibition of the Ca2 ATPase by celecoxib disrupted Ca2+ homeostasis in the prostate cancer cells. This activity was highly specific for celecoxib and was not associated with the exposure to other COX-2 inhibitors, including rofecoxib. Microsome and plasma membrane preparations from the human prostate cancer cells showed that only the Ca2 ATPases located in the endoplasmic reticulum were the direct targets of celecoxib. The disruption of Ca2+ homeostasis played a central role in apoptosis induction in the prostate cancer cells because it was required for the activation of Ca2+-dependent hydrolyses that carried out cellular degradation. Moreover, mitochondrial membrane permeabilization, which releases cytochrome c to activate cell death, is sensitive to elevations in intracellular free Ca2+. Consequently, the celecoxib-induced inhibition Ca2 ATPases located in the endoplasmic reticulum may provide a link to mitochondrial membrane permeabilization for apoptosis induction much in the same way that celecoxib inhibition of the PI3K pathway can regulate BAD phosphorylation to trigger mitochondrial-mediated cell death. It is apparent that the central hypothesis of a dominant role for COX-2 inhibition in cancer prevention by celecoxib may need re-examination. Furthermore, the COX-2 dependent and independent action of celecoxib in cancer prevention may be tissue specific. Since the aberrant expression of COX-2 is implicated in the

Cell Adhesion Molecules

pathogenesis of various types of human cancers, perhaps this inducible enzyme may be a useful surrogate biomarker of the anticancer activity of celecoxib when evaluating the chemoprevention of cancer at various sites in the body. Although the precise molecular mechanism for its chemopreventive effects are still fairly unknown, celecoxib may be still useful as a chemopreventive agent for a variety of malignancies, especially since it triggers less toxicity and adverse side effects during longtern use when compared to traditional NSAIDs. Celecoxib may be useful when combined with other cancer chemopreventive/therapeutic agents to control the process of tumorigenesis.

References Chun KS, Surh JY (2006) Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol 68:1089–1100 Grosch S, Maier TJ, Schiffmann S et al (2006) Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 98:736–747 Kismet K, Akay MT, Abbasoglu O et al (2004) Celecoxib: a potent cyclooxygenase-2 inhibitor in cancer prevention. Cancer Detect Prev 28:127–142 Psaty BM, Potter JD (2006) Risks and benefits of celecoxib to prevent recurrent adenomas. N Engl J Med 355:950–952 Schroeder CP, Kadara H, Lotan D et al (2006) Involvement of mitochondrial and akt signaling pathways in augmented apoptosis induced by a combination of low doses of celecoxib and N-(4-hydroxyphenyl) retinamide in premalignant human bronchial epithelial cells. Cancer Res 66:9762–9770

Cell Adhesion Molecules Kris Vleminckx Department of Biomedical Molecular Biology and Center for Medical Genetics, Ghent University, Ghent, Belgium

Synonyms Adhesion molecules; CAMs

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Definition Cell ▶ adhesion molecules are transmembrane or membrane-linked glycoproteins that mediate the connections between cells or the attachment of cells to substrate (such as stroma or basement membrane). Dynamic cell-cell and cell-substrate adhesion is a major morphogenetic factor in developing multicellular organisms. In adult animals, adhesive mechanisms underlie the maintenance of tissue architecture, allow the generation of force and movement, and guarantee the functionality of the organs (e.g., to create barriers in secreting organs, intestines, and blood vessels) as well as the generation and maintenance of neuronal connections. Cell adhesion is also an integrated component of the immune system and wound healing. At the cellular level, cell adhesion molecules do not function just as molecular glue. Several signaling functions have been attributed to adhesion molecules, and cell adhesion is involved in processes such as contact inhibition, growth, and ▶ apoptosis. Deficiencies in the function of cell adhesion molecules underlie a wide variety of human diseases including cancer. By their adhesive activities and their dialogue with the ▶ cytoskeleton, adhesion molecules directly influence the invasive and metastatic behavior of tumor cells and by their signaling function they can be involved in the initiation of tumorigenesis.

Characteristics At the molecular level, cell adhesion is mediated by molecules that are exposed on the external surface of the cell and are somehow physically linked to the cell membrane. In essence, there are three possible mechanisms by which such membrane-attached adhesion molecules link cells to each other (Fig. 1a). Firstly, molecules on one cell bind directly to similar molecules on the other cell (homophilic adhesion). Secondly, adhesion molecules on one cell bind to other adhesion receptors on the other cell (heterophilic adhesion). Finally, two different adhesion molecules on two cells may both bind to a shared secreted multivalent ligand in the extracellular

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Cell Adhesion Molecules

a Cell-cell adhesion Homophilic

Heterotypic Heterophilic

Homotypic Linker-mediated

Cell-substrate adhesion

b Cytoskeletal strengthening

Cell Adhesion Molecules, Fig. 1 Different modes of cellcell and cell-substrate adhesion and the mechanism of cytoskeletal strengthening. (a) Three possible mechanisms by which cell adhesion molecules mediate intercellular adhesion. A cell surface molecule can bind to an identical molecule (homophilic adhesion) on the opposing cell or can interact with another adhesion receptor (heterophilic adhesion). Alternatively, cell adhesion receptors on two neighboring cells can bind to the same multivalent, secreted ligand (linkermediated adhesion). Intercellular adhesion can take place between

identical cell types (homotypic adhesion) or between cells of different origin (heterotypic adhesion), independently of the involved adhesion molecules. Cell-substrate adhesion molecules attach cells to specific compounds of the extracellular matrix. Cell-cell and cell-substrate adhesion can occur simultaneously. (b) Intercellular and cell-substrate adhesion can be strengthened by indirect intracellular linkage of the cytoplasmic tail of the adhesion molecules to the cytoskeleton and by lateral clustering in the membrane

Cell Adhesion Molecules

space. Also, cell-cell adhesion between two identical cells is called homotypic (cell) adhesion, while heterotypic (cell) adhesion takes place between two different cell types. In the case of cell-substrate adhesion, the adhesion molecules bind to the extracellular matrix (ECM). Cell Adhesion Molecules and the Cytoskeleton Adhesion molecules can be associated with the cell membrane either by a glycosylphosphatidylinositol (GPI) anchor or by a membrane-spanning region. In the latter case, the cytoplasmic part of the molecule often associates indirectly with components of the cytoskeleton (e.g., actin, intermediate filaments, or submembranous cortex). This implies that adhesion molecules, which by themselves establish extracellular contacts, can be structurally integrated with the intracellular cytoskeleton, and they are often clustered in specific restricted areas in the membrane, the so-called junctional complex (Fig. 1b). This combined behavior of linkage to the cytoskeleton and clustering, considerably strengthens the adhesive force of the adhesion molecules. In some cases, exposed adhesion molecules can be in a conformational configuration that does not support binding to its adhesion receptor. A signal within the cell can induce a conformational change that activates the adhesion molecule. Dynamic adhesion can also be mediated via regulated endocytosis of the adhesion molecules. These mechanisms of regulation allow for a dynamic process of cell adhesion that, amongst others, is required for morphogenesis during development and for efficient immunological defense. Classification of Cell Adhesion Molecules Based on their molecular structure and mode of interaction, five classes of adhesion molecules are generally distinguished; the cadherins, integrins, immunoglobulin (Ig) superfamily, selectins, and proteoglycans (Fig. 2). Cadherins

Cadherins and protocadherins form a large and diverse group of adhesion receptors. They are Ca2 + -dependent adhesion molecules, involved in a

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variety of adhesive interactions both in the embryo and the adult. Cadherins play a fundamental role in metazoan embryos, from the earliest gross morphogenetic events (e.g., separation of germ layers during gastrulation) to the most delicate tunings later in development (e.g., molecular wiring of the neural network). The extracellular part of vertebrate classical cadherins consists of a number of cadherin repeats whose conformation is highly dependent on the presence or absence of calcium ions. Homophilic interactions can only be realized in the presence of calcium, usually by the most distal cadherin repeat. Classical cadherins are generally exposed as homodimers and their cytoplasmic domain can be structurally or functionally associated with the actin cytoskeleton. Cadherins are the major adhesion molecules in tissues that are subject to high mechanical stress such as epithelia (▶ Ecadherin) and endothelia (VE-cadherin). However, finer and more elegant intercellular interactions, such as synaptic contacts, also involve cadherins. Integrins

Integrins are another group of major players in the field of cell adhesion. They are involved in various processes such as morphogenesis and tissue integrity, homeostasis, immune response, and inflammation. Integrins are a special class of adhesion molecules not only because they mediate both cellcell and cell-substrate interactions (with components in the ECM such as laminin, fibronectin and collagen) but also because they function as heterodimers consisting of an a- and b-subunit. To date, at least 16 a-subunits and 8 b-subunits have been indentified. Of the theoretical 128 heterodimeric pairings, at least 21 are known to exist. While most integrin heterodimers bind to ECM components, some of them, more particularly those expressed on leukocytes, are heterophilic adhesion molecules binding to members of the Ig superfamily. The a-subunit mostly contains a ligand-binding domain and requires the binding of divalent cations (Mg2+, Ca2+, and Mn2+, depending on the integrin) for its function. Interestingly, integrins may be present on the cell-surface in a nonfunctional and functional configuration. The cytoplasmic domain appears to be responsible for the conformational change that activates the integrin.

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Cell Adhesion Molecules Binding partner

Adhesion molecule

Cadherins

Ca2+

Ca2+

Ca2+

Cadherins

Ca2+

a Lg-like, ECM

Integrins b

Lg-like

FnIII FnIII

s-s

s-s

s-s

s-s

s-s

Ca2−

Selectins

Lg-like, integrins

Carbohydrates

– – – – – – – – – – – – – – – – – – – – –

Proteoglycans –

– – – – – – – – – – – – – – – – – – –

Miscellanious

Cell Adhesion Molecules, Fig. 2 The five major classes of cell adhesion molecules and their binding partners. Cadherins are Ca2+-dependent adhesion molecules that consist of a varying number of cadherin repeats (five in case of the classical cadherins). The conformation and activity of cadherins is highly dependent on the presence of Ca2+-ions. In general, cadherin binding is homophilic. Integrins are functional as heterodimers and consist of an a- and b-subunit. They interact with members of the immunoglobulin superfamily or with compounds of the extracellular matrix (e.g., fibronectin, laminin). Members of the immunoglobulin superfamily (Ig-like proteins) are characterized by a various number of immunoglobulin-

like domains (open circles). Membrane-proximal, fibronectin type III repeats are often observed (gray boxes). They can either bind to other members of the Ig-family (homophilic) or to integrins. Selectins contain an N-terminal Ca2+-dependent lectin domain (circle) that binds carbohydrates, a single EGF-like repeat (gray box) and a number of repeats that are related to those present in complement-binding proteins (ovals). Proteoglycans are huge molecules that consist of a relatively small protein core to which long side chains of negatively charged glycosaminoglycans are covalently attached. They bind various molecules, including components of the extracellular matrix

The Ig Superfamily

heterophilic interactions that play a central role in regulation and organization of neural networks, specifically in neuron-target interactions and fasciculation. The basic extracellular structure consists of a number of Ig domains, which are responsible for homophilic interaction, followed by a discrete number of fibronectin type III repeats. This structure is linked to the membrane either by a GPI anchor or a transmembrane

Among the classes of adhesion molecules discussed here, the Ig superfamily is probably the most diverse. The main representatives are the neural cell adhesion molecules (NCAMs) and V(ascular)CAMs. As the name suggests, the members of this family all contain an extracellular domain consisting of different immunoglobulinlike domains. NCAMs sustain homophilic and

Cell Adhesion Molecules

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Secondary tumor

Step II

C Step IV

Step I

Step III Primary tumor

Cell Adhesion Molecules, Fig. 3 Cell adhesion processes involved in the metastatic cascade. A subset of cells (gray) growing in a primary tumor will reduce cellcell contacts (Step I) and migrate in the surrounding stroma by increasing specific cell-substrate adhesion (Step II). These invasive tumor cells can extravasate into the

circulation and, at distant sites, attach to the endothelial blood vessel wall through specific cell-cell interactions (Step III). Once these cells have extravasated through the vessel wall they use cell-substrate adhesion molecules to invade the surrounding stroma (Step IV). See text for details

domain. The VCAM subgroup, including I (ntercellular)CAMs and the mucosal vascular addressin adhesion molecule (MAdCAM), is involved in leukocyte trafficking (or homing) and extravasation. They consist of membranelinked Ig domains that make heterophilic contacts with integrins. Other members of this family that are associated with cancer are carcinoembryonic antigen (CEA), “deleted in colon cancer” (DCC) and platelet endothelial (PE)CAM-1.

proteoglycans may bind to each other or may be the attachment site for other adhesion molecules.

Selectins

These types of adhesion molecules depend on carbohydrate structures for their adhesive interactions. Selectins have a C-type lectin domain that specifically binds to discrete carbohydrate structures present on cell-surface proteins. Intercellular interactions mediated by selectins are of particular interest in the immune system, where they play a fundamental role in trafficking and homing of leukocytes.

Role of Adhesion Molecules in Cancer The Metastatic Cascade

Cell adhesion molecules play an important role during the progression of tumors, more particularly in the metastatic cascade (Fig. 3). When a benign tumor becomes malignant, cells at the periphery of the tumor will lose cell-cell contact (step I) and invade the surrounding stroma (step II) (see also ▶ invasion). Cells then extravasate and enter the vasculature or lymphatic system, where they are further transported. A fraction of the circulating tumor cells survives and is arrested at a distant site, attaches to the endothelium (step III), and extravasates through the blood vessel wall and into the surrounding tissue (step IV). Here the tumor cells grow, attract blood vessels, and develop to a secondary tumor (▶ metastasis).

Proteoglycans

Proteoglycans are large consisting of a relatively which long chains of are attached. Although

extracellular proteins small protein core to glycosaminoglycans poorly documented,

Adhesive Events in Metastasis

All the classes of cell adhesion molecules play a role in the metastatic cascade. During the first step, tumor cells need to disrupt intercellular

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junctions in order to detach from the primary tumor. This step often involves the suppression of cadherin function. The second step of ▶ migration through the stroma and into the blood or lymphatic vessels requires dynamic cell-substrate adhesion, mostly mediated by integrins. In the third step, where cells arrest in the circulation by aggregation with each other or attachment to platelets, leukocytes, and endothelial cells, critical roles have been attributed to cell adhesion molecules of the Ig superfamily, selectins, integrins, and specific membrane-associated carbohydrates. The fourth step is similar to step II and mostly involves integrins. Details on the adhesive events associated with metastasis are outlined below. • In benign epithelial tumors, cells maintain firm intercellular adhesive contacts, mostly by formation of a junctional complex (including tight junctions, ▶ adherens junctions, and desmosomes). Establishment and maintenance of such a strong junctional complex requires expression and function of cadherins (more particularly E-cadherin). Loss of E-cadherin expression or function appears to be a hallmark of progression of a benign epithelial tumor (adenoma) to a malignant one (carcinoma). Epithelial tumor cells often acquire invasive properties by mutational inactivation of E-cadherin or one of its cytoplasmic binding partners (catenins). It is important to keep in mind that cadherin-mediated adhesion is a dynamic process and that E-cadherin can be temporarily inactivated at the functional level, for example by phosphorylation or other posttranslational modifications. E-cadherin and other molecules of the junctional complex are very often suppressed or functionally modulated in the epithelial-mesenchymal transitions (EMT), a hallmark of malignant tumor progression. EMT can be a tumor-intrinsic feature or can be induced by their microenvironment. Paracrine factors such as scatter factor or juxtacrine signaling via Ephrin/Eph receptor or via ▶ semaphorins/plexins can affect adhesion via direct activity on the cell adhesion molecules or via regulation of the cytoskeleton. • Dynamic cell-substrate adhesion is a critical factor in the migration of invasive tumor cells into

Cell Adhesion Molecules

the surrounding stroma. Integrins are instrumental in this process. Several studies have correlated the migratory behavior of tumor cells either with an increased or decreased expression of particular integrins. This apparent paradox may be explained by the fact that firm but temporary cell-substrate contacts are required for cells to migrate on a substrate. In order to crawl directionally through the stroma, a cell needs to “grab” the ECM, release after pulling itself forward and then has to establish the next contact. Both inhibiting adhesion and preventing release of the substrate contacts “locks” the cell in its position and prevents migration. It should be remembered that integrins may exist in two functional states and that signals passed through the cytoplasm determine whether membraneexposed integrins are functional or not. • In the third step of the metastatic cascade, cellcell interactions are again the most determining. Homotypic interactions between circulating tumor cells promote formation of aggregates that are preferentially retained in the capillary network. PECAM-1 is a cell adhesion molecule potentially involved in this process. It should be pointed out that (re) expression of the invasion-suppressor molecule E-cadherin would actually promote metastasis formation. Besides these homotypic interactions, heterotypic interactions are also of major importance in the metastatic process. Tumor cells can attach to the blood-vessel wall either directly or indirectly through platelets and leukocytes. The adhesion molecules involved in this process are similar to those involved in the “multistep adhesion cascade” observed during homing and extravasation of leukocytes or trafficking of lymphocytes. Cell adhesion events include interactions of tumorassociated lectins with selectins expressed on platelets, leukocytes, and endothelium (P-, L-, and E-selectins, respectively). These adhesion molecules are also involved in the initial transient low-affinity interactions (rolling) of circulating leukocytes (and probably tumor cells) with the endothelium. Other and more stringent heterotypic heterophilic interactions in this metastatic stage include the binding of

Cell Adhesion Molecules

integrins on tumor cells to ICAMs expressed on the surface of the endothelial cells. • The fourth step in the metastatic cascade is extravasation and invasion at a distant site. This process is very similar to step 2 and the same adhesion molecules are likely to be involved. Specific interactions of the tumor cells with molecules present on the endothelial cells (e.g., N-cadherin) will facilitate the extravasation process. Other Cancer-Related Functions of Cell Adhesion Molecules

It has become clear that some cell adhesion molecules are involved in signaling processes that are relevant to cancer. Germline mutations in E-cadherin predispose patients to the development of diffuse gastric carcinomas, and in lobular breast carcinoma, E-cadherin seems to act as a tumor suppressor. Interestingly, b-catenin, a protein cytoplasmically linked to cadherins, has a central role in ▶ Wnt signaling and has oncogenic properties that are counteracted by the adenomatous polyposis coli (APC) gene product. Signaling by integrins can also be an important factor that prevents cells from undergoing apoptosis (apoptosis upon loss of cell adhesion is called ▶ anoikis), which might be critical when tumor cells are traveling in the circulation. Interdisciplinary research has revealed new unexpected functions for known cell adhesion molecules. The suspected tumor suppressor DCC, a member of the Ig superfamily of adhesion molecules, turned out to be the receptor for netrin-1, an axonal chemoattractant crucial in neuronal development. Other molecules known to have adhesive or repulsive activities in the axonal growth cone or in migrating neural crest cells, turn out to have similar activities in tumor cells (see also the chapters on ▶ EPH receptors, Ephrin signaling in cancer, ▶ semaphorins, and ▶ plexins).

Cross-References ▶ Adherens Junctions ▶ Adhesion ▶ Anoikis

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▶ Apoptosis ▶ Carcinoembryonic Antigen ▶ Cytoskeleton ▶ E-Cadherin ▶ Eph Receptors ▶ Invasion ▶ Metastasis ▶ Migration ▶ Plexins ▶ Semaphorin ▶ Wnt Signaling

References Cavallaro U, Christofori G (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4:118–132 Chothia C, Jones EY (1997) The molecular structure of cell adhesion molecules. Annu Rev Biochem 66:823–862 Hynes RO (2000) Cell adhesion: old and new questions. Trends Cell Biol 9:M33–M37 Mizejewski GJ (1999) Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 222:124–138 Sanderson RD (2001) Heparan sulfate proteoglycans in invasion and metastasis. Semin Cell Dev Biol 12:89–98

See Also (2012) Cadherins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 581– 582. doi:10.1007/978-3-642-16483-5_770 (2012) Contact Inhibition. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 973–974. doi:10.1007/978-3-642-16483-5_1323 (2012) E-Selectin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1317. doi:10.1007/978-3-642-16483-5_1780 (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Homophilic and Heterophilic Adhesion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1729. doi:10.1007/9783-642-16483-5_2804 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Junctional Complex. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1929. doi:10.1007/978-3-642-16483-5_3188 (2012) Lectin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1999. doi:10.1007/978-3-642-16483-5_3303 (2012) Proteoglycans. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3100. doi:10.1007/978-3-642-16483-5_4816

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Cell Biology Filippo Acconcia1 and Rakesh Kumar2 1 Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Biochemistry and Molecular Medicine, George Washington University, Washington, DC, USA

Definition Cell biology deals with all aspects of the normal and of the tumor cell, their normal and abnormal multiplication, their differentiation, their stem origins, and their regulated cell death.

Characteristics The Cell The intracellular environment is separated from the external environment by a lipid bilayer called plasma membrane. The plasma membrane controls the movement of substances in and out of the cell and it is important for the cell to sense the surrounding environment. Within the cell the nucleus occupies most of the space. The cell nucleus contains genes, which drive all cellular activities and processes. Genes are organized in chromosomes (i.e., genome) and are made of DNA. The genetic information is used to produce proteins, which are the critical effectors required for all cellular processes. The nucleus is separated from the rest of the cellular content by the nuclear membrane, which remains in contact with the cytoplasm as well as the nucleoplasm. In the cytoplasm, proteins are organized into specific functional structures and also connected with the structural network referred to as cytoskeleton network, which physically sustains the cell. Moreover several intracellular organelles are located in the cytoplasm (e.g., mitochondria, Golgi apparatus) and allow the cells to self sustain. To continuously adjust the intracellular processes and to promptly respond to the demands of the

Cell Biology

extracellular environment, cells need to exchange matter, energy, and information with the external milieu. Cell Division and Reproduction One of the unique features of cell is its ability to divide and produce two daughter cells that are an exact copy of their parental cell, by a process called “mitosis.” However, some differentiated cells undergo the process of meiosis. For simplicity, meiotic division can be considered as the sum of two successive mitotic divisions, which result in four daughter cells with half the number of chromosomes and rearranged genes. These specialized cells (i.e., gametes) serve as reproductive cells. The fusion of the female and male gametes (eggs and spermatozoa, respectively) results in a new cell called zygote. The zygote, by definition, is a stem cell. Following mitotic division, it becomes an embryo and, at the end of the embryonic development, results in a new organism. Cell Proliferation The physiological functions of an organ require maintenance of homeostasis, a process of regulated balance between cell proliferation and cell death (also known as ▶ apoptosis), in the differentiated tissue. Indeed, a variety of extracellular stimuli activate specific ▶ signal transduction pathways that affect the expression and activity of molecules involved in the control of cell proliferation or cell death. Thus, the balance between cell cycle progression and apoptosis defines the cell fate, and this process depends on genetic factors as well as the kinetics of signal transduction pathways in exponentially growing cells. Cell Cycle In mammalian cells, one cell cycle takes about 24 h in most cell types and can be schematically divided into two stages: mitosis and interphase. Mitosis (M phase) consists of a series of molecular processes that result in cell division. On the other hand, the interphase can be subdivided into three major gaps (G1, S, and G2 phase). The G1 phase of the cell cycle separates the M and S phases. In G1 phase, cells express a specific pattern of gene products required for the DNA

Cell Biology

synthesis; the G2 phase of the cell cycle resides in between the S and M phases and is important for the completion of processes that are necessary for mitosis. The G0 phase of the cell cycle is entered by the cells from the G1. In the G0 phase, cells are out of the cell cycle and into a quiescent state where they do not proliferate. Regulation of Cell Cycle Progression Cell cycle progression is achieved through a series of coordinated molecular events that allow the cells to transit across the restriction points, also known as cell cycle checkpoints. There are three main restriction points in the cell cycle (G2/M, M/G1, and G1/S, respectively). Broadly, these checkpoints are defined as points after which the cell is committed to progress to the next phase in a nonreversible manner. Therefore, the transition between the phases of the cell cycle is strictly regulated by a specific set of proteins. ▶ Cyclindependent kinases (CDK) act in various phases of the cell cycle by binding to its activating proteins called cyclins. For example, both ▶ cyclin D/ CDK4 and cyclin E/CDK2 complexes regulate transition of the cells through G1/S phase whereas cyclin A/CDK1, cyclin A/CDK2, and cyclin B/CDK1 complexes are active during the rest of the cell cycle. On the other hand, another class of regulatory proteins, the cyclin-dependent kinase inhibitors (CKI) (e.g., p21Cip/Kip; p19Ink4d) antagonizes the activation of CDK activity, thus impeding the progression of the cell cycle. Programmed Cell Death Programmed cell death (PCD) is a physiological process of eliminating a living cell. The PCD involves activation of specific intracellular programs that commit cells to a “suicidal route.” The process of PCD plays an important role in a variety of biological events, including morphogenesis, maintenance of tissue homeostasis, and elimination of harmful cells. To date, different forms of PCD have been described among which apoptosis, necrosis, and ▶ autophagy are the most common. Apoptosis One of the critical events in apoptosis is the activation of cystein proteases, called caspases, upon

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a given signal. The initiator caspases (▶ Caspase 8 and 9) are the first enzymes involved in the activation of the apoptotic cascade. Caspase 8 and 9 activate the downstream effector caspases (caspase 3, 6, and 7) by proteolytic cleavage which in turn results in the hydrolysis and inactivation of the enzymes involved in the processes of DNA repair such as by poly-ADP-ribose polymerase (PARP). Upon stimulation of apoptotic cascade, cells display a specific set of characters, which constitute the hallmark of apoptosis (DNA fragmentation, cell shrinkage, cytoplasmic budding, and fragmentation). The activation of caspases is achieved through two principle pathways – an extrinsic pathway that transduces signals from the plasma membrane directly to the caspases, and an intrinsic pathway that involves activation of caspases through a series of biochemical events leading to permeabilization of the mitochondrial membrane and release of cytochrome c (▶ Cytochrome P450) in the cytoplasm. Apoptotic cells are eventually eliminated by the immune system without the activation of inflammatory reactions (▶ Inflammation). Necrosis Necrosis results from a severe physical, mechanical, or metabolic cellular damage. The necrotic phenotype is very different from those of an apoptotic cells. Overall, the cell switches off its metabolic pathways and the DNA condenses at the margins of the nucleus and the cellular constituents start to degrade. In general, necrosis consists in a general swelling of the cell before it disintegrates. Furthermore, upon leakage of the intracellular content, necrotic cells stimulate an inflammatory response that usually damages the surrounding tissue. Autophagy Autophagy, i.e., autophagic cell death, occurs by sequestration of intracellular organelles in a double membrane structure termed autophagosome. Subsequently, the autophagosomes are delivered to the lysosomes and degraded. Autophagy is responsible for the turnover of dysfunctional organelles and cytoplasmic proteins and thus, contributes to cytosolic homeostasis. Autophagy

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can occur either in the absence of detectable signs of apoptosis or concomitantly with apoptosis. Indeed, autophagy is activated by signaling pathways that also control apoptosis. Signal Transduction Extracellular signals are transduced by the activation of a series of phosphorylation-dependent intracellular pathways initiated by cell surface receptors. Eventually, such signals feed into the nucleus, stimulate transcription factors, and regulate gene transcription. Signaling Targets Signaling pathways regulate gene transcription by triggering the promoter activity of the target gene. For example, regulation of cyclin D is critical for cell cycle progression. The extracellular signalmediated activation of specific signal transduction pathways stimulates the activity of transcription factors such as AP-1, SP-1, and NF-kB, which coordinate the activation of the cyclin D1 promoter and thus lead to cyclin D1 expression. On the other hand, signaling molecules can also change the activity of a preexisting protein. For example, activation of p21-activated kinase (PAK) induces the phosphorylation of phosphoglucomutase (PGM) that stimulates its enzyme activity and the phosphorylation of ▶ estrogen receptor alpha (ERa) thus inducing its transcriptional activity. One of the most studied signaling pathways is the extracellular-regulated kinase (ERK) (▶ MAP kinase) cascade. It consists of three steps of sequential phosphorylations that impact on diverse cellular effectors. The ERK cascade is activated by mitogenic stimuli (e.g., growth factors (▶ Fibroblast growth factors)) and plays a critical role both in cell proliferation and cell survival. Indeed, activation of ERK induces the activation of AP-1 transcription factor, which, in turn, regulates cyclin D1 expression in addition to many of other proliferative molecules. Further, ERK activity leads to an increased expression of the antiapoptotic protein ▶ BCL-2 and inactivation of the proapoptotic protein Bad. Conversely, the JNK/SAPK (▶ JNK Subfamily) and the p38/MAPK (MAP kinase) pathways mediate stress and apoptotic stimuli (e.g., UV, ischemic-reperfusion

Cell Biology

damage). Activation of JNK/SAPK and p38/ MAPK often results in an increased expression of proapoptotic proteins (e.g., Bax), and in the activation of the caspase cascade and cytochrome c release from the mitochondria. Systems Biology Systems biology represents a new analytical tool that has begun to emerge for balanced comprehensive analyses of cellular pathways at the level of genes and proteins. Signal transduction pathways often cross-talk and influence each other, and the functionality of the effector molecule is influenced by the overall outcome of a set of signaling pathways. Thus, cells form a web of intracellular interactions that are critical for a timely and dynamic response. The intracellular signaling network is considered a complex system rapidly adapting to extracellular challenges. Therefore, an additional level of complication is the evaluation of the network as a whole, rather than the individual pathway. Cell Motility and Migration ▶ Motility and ▶ migration are important components for the functionality of a variety of cell types and are involved in physiologic processes such as embryonic development, immune response, as well as in pathologic processes such as ▶ invasion and ▶ metastasis. Cell motility and migration are coordinated physiological processes that allow the cells to move or to invade the surrounding tissues, respectively. They occur as a result of a complex interplay between the focal ▶ adhesion sites (cell-to-substrate contacts) and the extracellular matrix (ECM) (substrate). Phenotypically, migratory cells develop motile structures such as pseudopodia, lamellipodia, and filopodia. An ordered sequence of events (protrusion of motile structures, formation and disruption of focal contacts) generate the traction forces that drive the cell movement. Moreover, when migration is required, cells secrete specific proteolytic enzymes (matrix metalloproteinases, MMPs) that digest the ECM, thus opening a passage across the substrate. Cytoskeleton is critical for the correct occurrence of cell motility and migration.

Cell Biology

Cytoskeleton Cytoskeleton is a network of cytoplasmic proteins, which define the cell “bones.” Many different protein filaments are important for cytoskeleton functions. In particular, microtubules, built from different types of tubulin, originate from specific intracellular structures called microtubules organizing centers (MTOC). Dynamic changes in the polymerization and depolymerization of tubulin maintain microtubule integrity and resulting functions. Furthermore, actin microfilaments form a network of cytoskeleton-associated proteins and connect the focal adhesion with the intracellular cytoskeleton. The dynamic remodeling of microtubules and microfilaments has an impact on cell motility, migration and cell–cell adhesion, ▶ endocytosis, intracellular trafficking, organelle function, cell survival, gene expression, and cell division. Signaling Regulation At the focal adhesion sites, cells accumulate receptors (e.g., growth factor receptors), adaptors (e.g., vinculin), and signaling molecules, as well as structural and motor proteins (e.g., actin, myosin). Migration-specific stimuli (e.g., integrins engagement of ECM, growth factor stimulation, and mechanical stimuli) activate specific biochemical pathways. ▶ Focal Adhesion Kinase (FAK), integrin-linked kinase (ILK), PAK, and ▶ Src play key roles in modulating cell migration and invasion. The FAK/Src complex regulates the assembly and disassembly of focal contacts, F-actin cytoskeleton remodeling, and the formation of lamellipodia and filopodia through the activation of specific downstream cytoskeleton-associated signaling pathways. Further, ILK is also implicated in cell motility and migration by linking integrins with cytoskeleton dynamics through the ▶ PI3K signaling pathway. Also, PAK1 dynamically regulates cytoskeletal changes by coordinating upstream signaling with multiple effectors. By acting on actin reorganization, PAK1 drives directional cell motility and migration. Tumor Biology Cancer is a progressive disease that arises from the clonal expansion of a single transformed cell into

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a mass of uncontrolled proliferating cells. Tumorigenesis is a multistep process and involves progressive conversion of a normal cell into a malignant cell, which subsequently invades the surrounding tissues. The process of tumorigenesis consists of major steps (initiation, promotion, and progression), each involving specific molecular mechanisms, often interlaced with each other, that drive tumor development. Initiation and Promotion In general, initiation of tumorigenesis is referred to as the first oncogenic stimulus. However, such as initial event is not sufficient for tumor induction. In most cases, a second oncogenic stimulus must occur in a restricted time frame, thus promoting an irreversible effect. Chemical (e.g., aromatic compounds (▶ Polycyclic aromatic hydrocarbons)), physical (e.g., ▶ UV radiation), as well as biological (e.g., viruses as Human Papillomavirus) stress have impact on the cells and can induce DNA mutations (e.g., point mutations). In addition, gene deletion or duplication also alters gene function and contributes to the process of tumorigenesis. These genomic changes result in the production of proteins with altered functions or in the overexpression or downregulation of specific proteins, which affects the associated cellular functions. Protooncogenes or oncogenes are genes that encode for proteins involved in the induction of cell proliferation (e.g., cyclin D1, CDK, EGFR, Src, Ras, etc.) and whose overexpression or hyperactivation leads to an uncontrolled cell proliferation. On the other hand, tumor suppressor genes are genes encoding for proteins that negatively regulate cell proliferation (e.g., p53, PARP, CKI, etc.). Inactivating mutations or downregulation of tumor suppressor genes are also critical for enhanced cell proliferation. In addition to DNA damage, oncogenes and tumor suppressor genes, abnormal changes in the epigenetic cellular information (e.g., DNA ▶ methylation) can also participate in clonal evolution of human cancers. Progression The modified balance between the growthinhibitory programs and proliferative networks allows the cell to escape the physiological growth

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restrains. These selective growth advantages produce a population of more aggressive or transformed cells that resist clearance by the immune system (i.e., immune defense escape), and in turn, contributes to the accumulation of additional mutations and eventually, in tumor growth. In this context, an in situ tumor develops, that is the uncontrolled mass of transformed cells stays within the limit of the tissue in which the first cell resided. During this phase, tumor volume increases in parallel with an increased dedifferentiation of the cells that also secrete angiogenic factors (▶ Angiogenesis) to promote blood vessels formation in the tumor. Metastasis Metastasis is the process by which highly vascularized tumor cells acquire the ability to invade the blood-stream and seed in distant organs. Deregulation of cytoskeleton-associated proteins and secretion of protein factors play a critical role in the functionality of the metastatic cells. Stem Cell Biology In 1998, the group of Prof. James Thomson reported the isolation of a human embryonic stem cell line from the blastocyst stage of a human embryo. This cell line showed stability in a specifically developed culture medium and, upon transplantation in the nude mice, had the ability to form tumor-like structures made up of all the major human tissue types. This pioneer study opened the field of stem cell biology. Since then, enormous research efforts have been focused on the understanding of stem cell biology as well as their potential medical and therapeutic implications. Nonetheless, although the last 10 years witnessed an enormous progress, the field of stem cell research is in its infancy. The first controversy is the definition of stem cell itself. For simplicity, a stem cell is a clonal selfrenewing entity that is multipotent and can generate several different cell types. This definition introduces three major characteristic of the stem cells: self-renewal, clonality, and potency. Self-Renewal and Clonality Self-renewal is the process by which a stem cell undergoes an asymmetric mitotic division that

Cell Biology

produces, rather than two identical daughter cells, one cell that is completely identical to the parental stem cell and another cell that is already committed to a more restricted developmental path and more specialized abilities. Thus, stem cells have both the ability to self-maintain their clonal cell population and to produce a population of clones with more differentiated characteristics. In this way, stem cells form a hierarchy of potency. Potency Stem cells have the ability to give rise to a population of daughter stem cells with a reduced differentiation. The totipotent cells are the first embryonic cells that can become any kind of cell type (e.g., zygote). These cells become pluripotent cells, which can differentiate into most but not all cell types (e.g., embryonic stem cells). Next, cells that are committed to produce only a certain lineage of cell types (e.g., ▶ adult stem cells) are the multipotent cells. Some multipotent cells can only generate one specific kind of terminally differentiated cell type and thus, such cells, are called unipotent cells. Environmental Regulation The molecular mechanism by which regulatory processes occur in stem cells are not clear but are believed to be tightly regulated to avoid imbalance in stem cell population or mutation that can lead to tumorigenesis. One possibility is that the asymmetric division produces two daughter cells and, because of intrinsic factors, such cells follow different fates in spite of residing in the same microenvironment. Alternatively, the two daughter cells become functionally different because they are exposed to different extrinsic factors. Most likely, both intrinsic and extrinsic factors are integrated in the milieu of the surrounding microenvironment, also known as the stem cell niche. Signals from the niche determine the type of gene regulation that allows the asymmetric division to take place. In this model, one daughter cell stays in the niche and the other one moves out. Indeed, the importance of the microenvironment in stem

Cell Cycle Checkpoint

cell biology is highlighted by the ability of a particular stem cell to transdifferentiate or to dedifferentiate when put in a different niche. Although the concept of plasticity is debated in the literature, it is part of the “stemness” of a cell, which is the hallmark for a cell to be defined as a stem cell. Social Implications The ability to scientifically manipulate the human embryo or human adult stem cells has opened new perspectives for treatment of several human diseases. However, it has also initiated intense philosophical and political debates on the ethical issues associated with the use of such potential tools in medical practice.

Cross-References ▶ Adhesion ▶ Adult Stem Cells ▶ Angiogenesis ▶ Apoptosis ▶ Autophagy ▶ Bcl2 ▶ Caspase-8 ▶ Cyclin D ▶ Cyclin-Dependent Kinases ▶ Cytochrome P450 ▶ Endocytosis ▶ Estrogen Receptor ▶ Fibroblast Growth Factors ▶ Focal Adhesion Kinase ▶ Inflammation ▶ Invasion ▶ JNK Subfamily ▶ MAP Kinase ▶ Metastasis ▶ Methylation ▶ Migration ▶ Motility ▶ PI3K Signaling ▶ Polycyclic Aromatic Hydrocarbons ▶ Signal Transduction ▶ Src ▶ UV Radiation

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References Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4:143–153 Gearhart J, Hogan B, Melton D et al (2006) Essential of stem cell biology. Academic, London Lowe SW, Cepero E, Evan G (2004) Intrinsic tumour suppression. Nature 432:307–315 Pestell RG, Albanese C, Reutens AT et al (1999) The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr Rev 20:501–534 Potten C, Wilson J (2004) Apoptosis – the life and death of cells. Cambridge University Press, New York

See Also (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi: 10.1007/978-3-642-16483-5_994 (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1362. doi: 10.1007/978-3-642-16483-5_2067 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi: 10.1007/978-3-642-16483-5_3720

Cell Cycle Checkpoint Wenjian Ma National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA

Definition Cell cycle checkpoints are the control mechanisms that stop cell progression during particular stage of the cell cycle to check and ensure the accurate completion of earlier cellular processes and faithful transmission of genetic information before cell division.

Characteristics Cell growth and division proceeds through an ordered set of events called cell cycle, which is divided into four distinct phases namely G1 (the first gap phase), S (DNA synthesis), G2 (the

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second gap phase), and M (mitosis). G1 and G2 are two gap phases that accumulate nutrients, perform biosynthesis, and monitor cell state to get ready for DNA synthesis and mitosis, respectively. DNA replication occurs in S phase and the duplicated chromosomes are separated into two identical sets during mitosis (M phase). Followed by cytokinesis, the mother cell is divided into two daughter cells that are genetically identical to each other. The cell cycle is highly regulated and each phase is monitored by surveillance mechanisms to maintain cellular integrity and faithful transmission of genetic information from mother cell to daughter cell. If a crucial process has not been completed or if a cell has sustained damage, progression into the next cell phase would be prevented. These mechanisms that capable of delaying the cell cycle at specific time points are now referred to as checkpoints, which were first identified in the late 1980s. Various stresses can activate the checkpoint and cause cell cycle arrest, such as nutrient deprivation, mitogenic stimuli, and cytotoxins. However, the most important function of checkpoints is to monitor DNA damages and coordinate repair. Cells are under constant attack by DNA-damaging agents arising from endogenous or exogenous sources such as UV and the reactive oxygen species that inevitably generates during metabolism. These attacks can interfere with DNA replication, transcription, and other cellular functions and finally lead to genome instability. As repairing damaged DNA takes time, it is essential to activate specific checkpoint machinery to temporarily stall the cell cycle progression. In case the damages cannot be dealt with, the checkpoint can also activate other mechanisms such as apoptosis to target the cell for destruction. Multiple checkpoints have been identified from lower eukaryotes to human. Despite variations in molecular details, the controlling mechanisms of different organism share some conserved features in that they are tightly regulated through the interaction of specific protein kinases and adaptor proteins. The transition from one phase of the cell cycle to the next is driven by a group of kinases called cyclin-dependent kinases (CDKs),

Cell Cycle Checkpoint

which become active when bound by their cyclin partners. CDKs phospharylate specific downstream substrates to alter their biochemical function and elicit specific cellular responses. The level of cyclins and CDKs fluctuate during the cell cycle that is controlled by complex negativefeedback loops. Through the oscillation of cyclinCDKs, cellular processes within the cell cycle such as DNA replication, chromosome segregation, and cell division are precisely modulated. Simple eukaryotes such as yeast has only one CDK (Cdc28 in Saccharomyces cerevisae and Cdc2 in pombe), whereas higher eukaryotes have multiple CDKs, and through different combination of CDKs and cyclins, to control different aspects of the cell cycle. For example, S-phase is controlled by cyclin A in combination with CDK2, whereas progression into mitosis is regulated by cyclin B-CDK1 in mammalian cells. So far 16 eukaryotic cyclins and up to nine CDKs have been discovered. CDK activity is also negatively controlled by certain families of inhibitory proteins, and the cell-cycle progression is determined by the relative abundance of positive and negative regulators. The core cell cycle control protein/enzyme machineries sense stress/damage and trigger the cell cycle arrest are not conserved between different eukaryotes. Below describes the major checkpoints in mammalian cells as shown in Fig. 1. G1 checkpoint The G1 checkpoint is located at the end of the G1 phase that ensures everything is ready for DNA synthesis. It is the major restriction point to decide whether the cell continue for a further round of cell division. Under unfavorable environmental conditions, it signals the cell to temporally withdraw from the cell cycle and enter into a resting phase called G0. Once passing this checkpoint, the cell would tend to complete the whole cycle. During G1 phase, the cells may also irreversibly withdraw from the cell cycle into terminally differentiated or senescent states. One of the control pathways acting in G1 checkpoint is through the regulation of the tumor suppressor retinoblastoma protein (Rb) and the transcription factor called E2F. The

Cell Cycle Checkpoint

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Cell Cycle Checkpoint, Fig. 1 The cell cycle checkpoints in mammalian cells

hypophosphorylated form of Rb is active and represses cell cycle progression by inhibiting E2F, which is necessary for S phase entry. Phosphorylation of Rb blocks its inhibition on E2F and brings about the G1 phase progression or G1-S transition. In early G1 phase, increased expression of cyclin D in conjunction with CDK4 or CDK6 (depending on the cell types) leads to Rb phosphorylation. In late G1 phase, Rb is phospharylated by cyclin E/CDK2 complex. Phospharylation of Rb and subsequent release of E2F facilitates the transcription of late G1 genes to get ready for DNA synthesis and S-phase entry. Besides this positive regulation, G1 checkpoint is also negatively regulated by a family of proteins called cyclin-dependent kinase inhibitors (CKIs), which have a function in inhibiting the cyclin/ CDK complexes. In mammalian cells, there are two major families of CKIs – INK4 family (selectively for CDK4 and CDK6) and the CIP/KIP family (has a broader range of inhibition). In addition to the above pathway, another control of the G1 checkpoint is through the tumor suppressor p53 and its negative regulator MDM2. p53 Activation can cause G1 growth arrest via the CIP family member p21Cip1. This pathway, which also works in G2 checkpoint, plays an important regulatory role in DNA repair, senescence, and apoptosis.

Intra S-phase checkpoint Strict control of S-phase is important to ensure the genome stability and precise transmission of genetic information. The intra S-phase checkpoints monitor DNA damage, coordinate DNA repair pathways, and cause transient and reversible inhibition of the DNA replication during the whole S phase. They are activated when the replication fork stalls which can help preventing the conversion of primary DNA damages into lethal lesions such as DNA double strand breaks. There are two major checkpoint pathways in human that are initiated by the sensor proteins ATR or ATM, which delays the cell cycle either through the downstream signal cascade of Chk1(Chk2)/cdc25a/CDK2 or ATM/MRN/SMC1. In the first pathway, it is often triggered by the formation of single-stranded DNA (ssDNA) in replication fork as a result of uncoupling between DNA unwinding and DNA synthesis. ssDNA signals the recruitment of ATR to the stalled forks then activates downstream mediator and transduces the signal to Chk1/2. Phospharylated Chk1/2 then activating other downstream proteins/factors, such as cdc25 and CDK2/cyclin A, to control several cellular processes including cell cycle delay, prevention of late replication origins from firing, and the activation of DNA repair pathways. In the second pathway, ATM is recruited to sites of DNA damage by a component

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of the double strand break repair complex MBN. ATM then phosphorylates another component of the MRN complex called NBS1 as well as the cohesin complex SMC1 and lead to s-phase delay with mechanisms that are poorly understood. G2/M checkpoint G2/M checkpoint is located at the end of G2 phase, which controls the entry into mitosis. It checks a number of factors such as the completion of DNA replication and the genomic integrity before cell division starts. Genomic DNA often contains damaged parts prior to mitosis, which makes G2 checkpoint an important control in preventing transmission of damages to daughter cells. It is especially critical in repairing some lethal damages such as DNA double strand break, which can be repaired precisely by homologues recombination using the intact DNA sequences in sister chromatids as template. The G2-M transition is regulated by the cdc2/ Cyclin B complex. Under favorable conditions, it is activated by the mitosis promoting factor (MPF) for further cell progression. It is maintained in an inactive state by the tyrosine kinases Wee1 and Myt1 as the negative control. Once DNA damages are recognized by the sensory protein kinases DNA-PK or ATM, two parallel signal cascades can be activated and induce growth arrest by inactivating cdc2/Cyclin B. The first pathway is through Chk1/2 kinases and its downstream target cdc25, which prevents cdc2 activation and rapidly inhibits G2-M progression. A second pathway, which is slower, is through the tumor suppressor p53. P53 regulates multiple downstream players such as p21cip1, 14-3-3, and Gadd45, which inactivate cdc2-cyclin B by different mechanisms, to arrest the cell cycle progression. Mitotic checkpoint The mitotic checkpoint, also known as the spindle assembly checkpoint, occurs at the metaphase/ anaphase transition to ensure that all the chromosomes are aligned at the mitotic plate and a bipolar spindle is formed. The central element in this checkpoint is the anaphase promoting complex (APC) which is

Cell Cycle Checkpoint

highly conserved across different eukaryotes. In its activated form, APC can target many cyclins for degradation, which in turn triggers the signal cascade leading to the cuts of the cohesin complex that holds sister chromatids together. APC is negatively controlled by MAD1/2, BUB1/3, BUBR2, and the centromere protein E (CENP-E). Under favorable cellular conditions when chromosomes are correctly aligned, the checkpoint signal that inhibits APC is silenced and renders the latter to target cyclin B for destruction and inactivates CDK1, thereby promoting exit from mitosis and initiating anaphase. Followed by cytokinesis, the cell splits into two cells and the daughter cells enter into G1 to start a new cell cycle. Checkpoints and cancer Checkpoint failure often causes accumulation of genome mutations and rearrangements, which is a major factor in the development of many diseases including cancer. Cell cycle arrest with its vital role in maintaining genome stability is the most important barrier to prevent uncontrolled proliferation, the hallmarks of cancer. Many tumor suppressors are in fact components of the cell cycle checkpoints, such as p53, p16, ATM, and BRCA1/2. Mutation or loss of these tumor suppressors is common in cancer cell, which provides growth advantages over adjacent normal cells that are regulated by growth signals. Cell cycle checkpoints are also one of the most important targets in cancer drug development, which can enhance the efficacy of DNA damage related therapies. Most tumor cells have defects in their G1 checkpoint pathway, and therefore rely more on the efficient S and G2 phase checkpoints for repairing DNA damages and cell survival. Modulating the S and G2/M checkpoints has emerged as an attractive therapeutic strategy for anticancer therapy. Various inhibitors selectively targeting the key players in S or G2/M but not G1 checkpoints, such as Chk1, have been developed and showed promising effects in enhancing the conventional chemotherapy and radiotherapy. Checkpoint inhibition has become an area of intense interest in cancer biology and is continue growing.

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References

Cellular Antigens Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9(3):153–166 Reinhardt HC, Yaffe MB (2009) Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol 21(2):245–255 Sclafani RA, Holzen TM (2007) Cell cycle regulation of DNA replication. Annu Rev Genet 41:237–280

▶ CD Antigens

C Cellular Immunotherapy ▶ Adoptive Immunotherapy

Cell Locomotion ▶ Migration

Cellular Self-cannibalism ▶ Autophagy

Cell Motility ▶ Migration

Cellular Self-digestion ▶ Autophagy

Cell Movement ▶ Motility

Central Neurocytoma ▶ Neurocytoma

b-Cell Tumor of the Islets ▶ Insulinoma

Central Neurofibromatosis ▶ Neurofibromatosis 2

Cell-Free Circulating Nucleic Acids (cfDNA) ▶ Circulating Nucleic Acids

Centroblastic ▶ Diffuse Large B-Cell Lymphoma

Cell-Free Nucleic Acids in Plasma and Serum (CNAPS), Circulating Tumor Centrocytic (Mantle Cell) Lymphoma DNA (ctDNA) ▶ Circulating Nucleic Acids

▶ Mantle Cell Lymphoma

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Centrosome Kenji Fukasawa Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

Synonyms Major microtubule organizing center; MTOC; SPB; Spindle pole body

Definition The centrosome is a nonmembranous organelle (1–2 mm in diameter) normally localized at the periphery of nucleus, and its primary function is to nucleate and anchor microtubules.

Characteristics Structure and Function The centrosome in mammalian cells consists of a pair of centrioles and the surrounding protein aggregates consisting of a number of different proteins (known as pericentriolar material; PCM). The centrioles in the pair structurally differ from each other; one with a set of appendages at the distal ends (mother centriole) and another without appendages (daughter centriole). These appendages are believed to be important for nucleating and anchoring microtubules. The daughter centriole acquires the appendages in late G2-phase of the cell cycle. As the primary function of the centrosome is to nucleate and anchor microtubules, centrosomes organize the cytoplasmic microtubule network during interphase, which is involved in vesicle transport, proper distribution of small organelles, and establishment of cell shape and polarity. In mitosis, centrosomes become the core structures of spindle poles and direct the formation of mitotic spindles (Fig. 1).

Centrosome

Centrosome Duplication Upon cytokinesis, each daughter cell receives only one centrosome. Thus, the centrosome, like DNA, must duplicate once prior to the next mitosis. In other words, cells have either one unduplicated or two duplicated centrosomes at any given time point during the cell cycle. Since DNA and centrosome are the only two organelles that undergo semiconservative duplication once in a single cell cycle, cells are equipped with a mechanism that coordinates these two events, likely to ensure these two organelles to duplicate once, and only once. In late G1/early S-phase, the centrosome initiates duplication by physical separation of the paired centrioles, which is followed by the formation of a procentriole in the proximity of each preexisting centriole. During S and G2, the procentrioles elongate and two centrosomes continue to mature by recruiting PCM. By late G2, two mature centrosomes are generated. The coupling of the initiation of DNA and centrosome duplication is in part achieved by late G1-specific activation of cyclin-dependent kinase 2 (CDK2)/cyclin E. CDK2/cyclin E triggers initiation of both DNA synthesis and centrosome duplication. The activation of CDK2/ cyclin E is controlled by the late G1-specific expression of cyclin E as well as the basal level expression of p53 and its transactivation target p21Waf1/Cip1 (p21), a potent CDK inhibitor. Several potential targets of CDK2/cyclin E for centrosome duplication have been identified, including nucleophosmin, Mps1 kinase, and CP110. For instance, nucleophosmin localizes between the paired centrioles, likely functioning in the pairing of the centrioles. CDK2/cyclin E-mediated phosphorylation promotes dissociation of nucleophosmin from the centriole pairs, leading to physical separation of the paired centrioles (Fig. 2). Abnormal Amplification of Centrosomes and Chromosome Instability in Cancer The presence of two centrosomes at mitosis ensures the formation of bipolar mitotic spindles. Since chromosomes are pulled toward each spindle pole, the bipolarity of mitotic spindles is essential for the accurate chromosome

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Centrosome, Fig. 1 Structure and function of centrosomes. (a) The centrosome consists of a pair of centrioles and surrounding protein aggregates (PCM). (b, c) Mouse embryonic fibroblasts were immunostained for g-tubulin (one of major centrosomal proteins, green – appearing in

yellow) and a- and b-tubulin (primary constituents of microtubules, red). Cells were also counterstained for DNA with DAPI. Panel b: interphase cell, panel c: mitotic cell

segregation into two daughter cells during cytokinesis. Abrogation of the regulation underlying the numeral homeostasis of centrosomes (i.e., regulation of centrosome duplication) results in abnormal amplification of centrosomes (presence of >2 centrosomes), which in turn increases the frequency of mitotic defects (i.e., formation of >2 spindle poles) and chromosome segregation errors/chromosome instability (see Fukasawa 2005 for the full description of the mechanisms for generation of centrosome amplification). Chromosome instability has been recognized as a hallmark of cancer and contributes to multistep carcinogenesis by facilitating the accumulation of genetic lesions required for the acquisition of various malignant phenotypes. To date, a number of studies have shown that centrosome amplification is a frequent event in almost all types of solid tumors, including breast, bladder, brain, bone, liver, lung, colon, prostate, pancreas, ovary,

testicle, cervix, gallbladder, bile duct, adrenal cortex, and head and neck squamous cell, to name a few. Centrosome amplification has also been observed in certain cases of leukemia and lymphoma. Many studies have also shown the strong association between the occurrence of centrosome amplification and a high degree of aneuploidy. Thus, centrosome amplification can be reasonably considered as a major contributing factor for chromosome instability in cancer (Fig. 3). Loss of Tumor Suppressor Proteins and Centrosome Amplification In view of carcinogenesis, it is important to mention that loss or inactivating mutation of certain tumor suppressor proteins, most notably p53 and BRCA1, results in centrosome amplification. For both p53 and BRCA1, they were initially implicated in the control of centrosome duplication and

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Centrosome, Fig. 2 The centrosome/centriole duplication cycle. Late G1-specific activation of CDK2/cyclin E triggers initiation of both DNA and centrosome duplication. Centrosome duplication begins with the physical separation of the paired centrioles, which is followed by formation of procentrioles. During S- and G2-phases, procentrioles elongate and two centrosomes progressively

recruit PCM. In late G2, the daughter centriole of the parental pair acquires appendages (shown as red wedges), and two identical centrosomes are generated. During mitosis, two duplicated centrosomes form spindle poles and direct the formation of bipolar mitotic spindles. Upon cytokinesis, each daughter cell receives one centrosome

Centrosome, Fig. 3 Representative immunostaining images of centrosome amplification in human cancer. The touch preparations of G3 tumor grade bladder cancer specimens and the adjacent normal bladder epithelium samples were subjected to immunostaining for g-tubulin

(centrosome, green) and counterstained for DNA with DAPI (blue). No centrosome amplification can be seen in normal bladder epithelium (a), while a high frequency of centrosome amplification in the G3 tumors (b)

numeral homeostasis of centrosomes by the observations that centrosome amplification and consequential mitotic aberrations were frequent in the embryonic fibroblasts (as well as various

tissues) of p53-null mice as well as mice harboring BRCA1 mutation, which implies that the destabilization of chromosomes due to centrosome amplification contributes to the cancer susceptibility

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phenotype associated with loss or mutational inactivation of p53 as well as BRCA1. Centrosome Amplification and Cancer Chemotherapy In cells inhibited for DNA synthesis (i.e., by exposure to DNA synthesis inhibitors such as aphidicolin (Aph) or hydroxyurea (HU)), centrosomes undergo multiple rounds of duplication in the absence of DNA synthesis, resulting in abnormal amplification of centrosomes. However, this phenomenon preferentially occurs when p53 is either mutated or lost. In the presence of wild-type p53, centrosome duplication is also blocked by exposure to DNA synthesis inhibitors; p53 is upregulated upon prolonged exposure to Aph or HU, leading to the transactivation of p21, which in turn blocks the initiation of centrosome duplication via continuous inhibition of CDK2/cyclin E. In contrast, in cells lacking p53, p21 fails to be upregulated in response to the cellular stress imposed by DNA synthesis inhibitors, allowing “accidental” activation of CDK2/cyclin E, which triggers the initiation of centrosome duplication. Considering the high frequency of p53 mutation in human cancer, it is important to address the effect of commonly used anticancer drugs targeting S-phase (DNA replication) on centrosomes. When p53-null cells were exposed to subtoxic concentrations of the S-phase targeting chemotherapeutic agents (i.e., 50 -fluorouracil, arabinoside-C), centrosome amplification was efficiently induced. Moreover, after removal of drugs, these cells resumed cell cycling and suffered dramatic destabilization of chromosomes. This finding may be significant in the context of cancer chemotherapy using the S-phase targeting drugs. During chemotherapy, not all cells in tumors receive a maximal dose of drugs – such cells may not be killed, but only arrested for cell cycling. If these cells harbor p53 mutations, centrosome amplification occurs during the drug-induced cell cycle arrest. Upon cessation of chemotherapy, these cells resume cell cycling in the presence of amplified centrosomes and suffer significant mitotic aberrations and chromosome instability, which increases the risk of acquiring further malignant phenotypes. This may in part explain why the

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recurrent tumors after chemotherapy are often found to be more malignant than the original tumors. Many S-phase targeting anticancer drugs have been found to be effective, and there is no doubt that DNA duplication should be one of the major targets for future development of more effective anticancer drugs. However, the possibility that the S-phase targeting drugs may exacerbate a chromosome instability phenotype by inducing centrosome amplification should be taken into consideration. Another important issue to be addressed is the concept of centrosome duplication as a target of cancer chemotherapy. Like DNA replication, centrosome duplication occurs only in proliferating cells. Inhibition of centrosome duplication will not only suppress centrosome amplification and chromosome instability but also block cell division and possibly induce cell death – cells with one centrosome fail to form bipolar mitotic spindles, and are often undergo cell death. Moreover, in contrast to genotoxic drugs which impose an increased rate of secondary mutations through interfering with DNA metabolisms, such side effects will likely be minimal in the protocol designed to block centrosome duplication.

Cross-References ▶ Genomic Imbalance ▶ Microtubule-Associated Proteins

References Bennett RA, Izumi H, Fukasawa K (2004) Induction of centrosome amplification and chromosome instability in p53-null cells by transient exposure to sub-toxic levels of S-phase targeting anti-cancer drugs. Oncogene 23:6823–6829 Deng CX (2002) Roles of BRCA1 in centrosome duplication. Oncogene 21:6222–6227 Fukasawa K (2005) Centrosome amplification, chromosome instability and cancer development. Cancer Lett 230:6–19 Hinchcliffe EH, Sluder G (2002) Two for two: Cdk2 and its role in centrosome doubling. Oncogene 21:6154–6160 Tarapore P, Fukasawa K (2002) Loss of p53 and centrosome hyperamplification. Oncogene 21:6234–6240

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Ceramide Katrin Anne Becker and Erich Gulbins Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany

Definition Ceramide belongs to the group of sphingolipids and is constituted by the amide ester of the sphingoid base D-erythro-sphingosine and a fatty acid of C16 through C32 chain length. At present, the differential biological function of different ceramide species is unknown and, thus, the term ceramide is used collectively to represent all long-chain ceramide molecules.

Characteristics Formation of Ceramide Ceramide molecules are very hydrophobic and exclusively present in membranes. Sphingomyelin, the choline-ester of ceramide, is hydrolyzed by acid, neutral, and alkaline sphingomyelinases to release ceramide. Ceramide is also de novo synthesized via a pathway involving the serine-palmitoyl-CoA transferase and a variety of partly cell type-specific ceramide synthases. Under some circumstances, ceramide can be also formed from sphingosine by a reverse activity of the acid ceramidase. Ceramide-Induced Changes of Biological Membranes The formation of ceramide within biological membranes results in a dramatic change of the biophysical properties of the lipid bilayer. Ceramide molecules have the tendency to selfassociate and to form small ceramide-enriched membrane microdomains. These membrane microdomains spontaneously fuse to large ceramide-enriched membrane macrodomains that constitute a very hydrophobic and stable membrane domain. Furthermore, ceramide molecules seem to compete with and displace

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cholesterol from membrane domains. Ceramideenriched membrane platforms serve to reorganize and cluster/aggregate receptor molecules in the membrane resulting in a very high density of receptors within a small area of the cell membrane. At least for some receptors, the transmembranous domain of the receptor determines its preferential partitioning in ceramideenriched membrane platforms. Ceramide-enriched membrane macrodomains are also involved in the recruitment or exclusion, respectively, of intracellular signaling molecules that mediate transmission of signals into the cell via a particular receptor. In general, clustering of receptors in ceramide-enriched membrane domains serves to amplify a weak primary signal. For instance, it was shown that ceramide-enriched membrane platforms amplify CD95 signaling ~100-fold. Ceramide in Receptor-Mediated Signaling Death receptors, in particular CD95 or DR5, activate the acid sphingomyelinase and trigger the translocation of the enzyme onto the extracellular leaflet of the cell membrane. Translocation of the acid sphingomyelinase onto the extracellular leaflet of the cell membrane may occur by fusion of intracellular vesicles that are mobilized upon receptor stimulation with the cell membrane. Surface exposure and stimulation of the acid sphingomyelinase results in very rapid release of ceramide in the cell membrane. Ceramide forms membrane platforms and mediates clustering of the death receptors, which is required for the induction of cell death via these receptors (Fig. 1). However, ceramide is not only involved in the mediation of apoptotic stimuli, but also many other stimuli trigger the release of ceramide including CD40, CD20, FcgRII, CD5, LFA-1, CD28, TNFa, Interleukin-1 receptor, PAF-receptor, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, g-irradiation, measles virus, UV-light, doxorubicin, cisplatin, gemcitabine, disruption of integrin-signaling, and some conditions of developmental death. In general, ceramide is often involved in acute and strong stress responses.

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Ceramide, Fig. 1 Receptors cluster in ceramide-enriched membrane domain to transmit signals into cells. The interaction of a ligand with its receptor results in translocation of the acid sphingomyelinase onto the extracellular leaflet and a concomitant release of ceramide. Ceramide spontaneously forms ceramide-enriched microdomains that fuse to large ceramide-enriched macrodomains. These domains trap activated receptor molecules finally

resulting in clustering of many receptor molecules within a small area of the cell membrane. The high density of receptor molecules and associated intracellular molecules amplifies the primarily weak signal, permits the generation of a strong signal and, thus, efficient transmission of the signal into the cell (Modified from A. Carpinteiro et al. Cancer Letters)

Signaling Molecules Regulated by Ceramide Ceramide interacts with and activates phospholipase A2, kinase suppressor of Ras (KSR; identical to ceramide-activated protein kinase), ceramideactivated protein serine-threonine phosphatases, protein kinase C isoforms, and c-Raf-1. Furthermore, ceramide inhibits the potassium channel Kv1.3 and calcium release activated calcium (CRAC) channels. Lysosomal ceramide specifically binds to and activates cathepsin D resulting in the translocation of cathepsin D into the cytoplasm and induction of cell death via the proapoptotic proteins Bid, Bax, and Bak.

been shown to be involved in the apoptosis response of mitochondria.

Ceramide in Mitochondria and Cell Death Besides a function of ceramide in the plasma membrane and lysosomes for the induction of cell death, ceramide is also generated in mitochondria via the de novo synthesis pathway, a reverse activity of the ceramidase and/or activity of the acid sphingomyelinase. Although at present the function of ceramide in the mediation of mitochondrial proapoptotic events is poorly defined, it was suggested that C16-ceramide molecules form large channels in mitochondrial membranes that may permit the exit of cytochrome c from mitochondria to execute death. Further, ceramide has

Ceramide in g-Irradiation- and UV-A Light-Induced Apoptosis The acid sphingomyelinase and ceramide are critically involved in the response of cells to g-irradiation. Animals or cells lacking the acid sphingomyelinase are resistant to g-irradiationinduced cell death. In particular, endothelial cells in acid sphingomyelinase-deficient mice are resistant to g-irradiation. Ceramide also plays a critical role for UV-light induced apoptosis. UV-A and UV-C light activate the acid sphingomyelinase, trigger the release of ceramide, and the formation of large ceramide-

Genetic Evidence for a Function of Ceramide in Apoptosis The role of the acid sphingomyelinase and ceramide for CD95 and DR5-triggered apoptosis was evidenced by studies on acid sphingomyelinasedeficient cells or mice, respectively, that revealed a resistance of these cells to CD95- and DR5-triggered apoptosis, but also g-irradiationand UV-light- or P. aeruginosa-triggered cell death.

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enriched membrane domains in the cell membrane to initiate death. Ceramide and Chemotherapy In addition to a central role of ceramide in g-irradiation-induced cell death, ceramide is also critically involved in the induction of cell death by at least some chemotherapeutic drugs. Thus, doxorubicin-, cisplatin-, taxol-, and gemcitabineinduced cell death of malignant and nonmalignant cells requires expression of the acid sphingomyelinase, release of ceramide and/or the formation of ceramide-enriched membrane platforms to trigger death. Short Chain Ceramide Short chain ceramide molecules composed of a fatty acid chain with C2 through C12 length are water soluble and, thus, very much differ from endogenous long ceramide molecules (C16-C32). However, they are very efficient reagents to kill tumor cells in vitro. Cationic pyridiniumceramides seem to accumulate in mitochondria of tumor cells and may, thus, serve as a new class of antitumor reagents, although at present no convincing concepts are available to selectively target tumor cells in vivo and to avoid effects of short chain ceramide on normal cells.

Cross-References ▶ Adriamycin ▶ Cisplatin ▶ Death Receptors ▶ Docetaxel ▶ Gemcitabine

References Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811 Gulbins E, Kolesnick RN (2003) Raft ceramide in molecular medicine. Oncogene 22:7070–7077 Jaffrezou JP, Laurent G (2004) Ceramide: a new target in anticancer research? Bull Cancer 91:E133–E161

Cervical Cancers Kolesnick RN, Goni FM, Alonso A (2000) Compartmentalization of ceramide signaling: physical foundations and biological effects. J Cell Physiol 184:285–300 Ogretmen B, Hannun YA (2004) Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 4:604–616

Cervical Cancers Jiro Fujimoto Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan

Definition The regions of the uterus are the corpus and the cervix. Cancer originating from the cervix is defined as cancer of the cervix. When cancers are simultaneously detected in the cervix and corpus, squamous cell carcinoma (SCC) is designated as a cancer of the cervix, and adenocarcinoma is designated as a cancer of the corpus. When cancer occupies both the cervix and vagina without the junctional area (the fornix), the cancer extending to the exocervix is recognized as a cancer of the cervix. Thus, cervical cancer is defined apart from cancer of the uterine corpus (cancer of the uterine endometrium) and cancer of the vagina.

Characteristics The main gynecological cancers originate from the cervix, endometrium, and ovary. Among them, cervical cancer is the most common malignancy in women. Main risk factors are the following: • Young age at first intercourse, especially shortly after the menarche • High number of sexual partners • High number of sexual partners of the partner • High number of children • Excessive douching

Cervical Cancers

Smoking appears to increase the incidence of SCC, but not of adenocarcinoma or adenosquamous carcinoma. Immunosuppression by smoke-derived nicotine and its metabolite cotinine in the cervical mucus may enhance the effects of sexually transmitted disease (STD) including human papillomavirus (HPV) infection. Most epidemiological risk factors for cervical cancer are associated with STDs. HPV induces an STD, human venereal condyloma, which is associated with cervical, vaginal, and vulvar dysplasia, and invasive carcinomas. HPV particles and DNA, especially HPV-16, HPV-18, and HPV-33, are detected in cervical and vulvar dysplasia and in invasive carcinomas. Additionally, it has been demonstrated that HPV transforms human cell lines. HPV infection of the cervix is a main etiology of cervical cancer. Symptoms Main symptoms of cervical cancer are the following: • Vaginal bleeding, which may be recognized as postmenopausal bleeding, irregular menses, or postcoital bleeding • Abnormal vaginal (watery, purulent, or mucoid) discharge In advanced cases, corresponding local symptoms occur. A Pap smear even in unsymptomatic cases is useful for the early detection of cervical dysplasia and cancers. Among women over the age of 18 who have had sexual intercourse, highrisk women should be screened at least yearly. Pathology Histopathological types in cervical cancers are mainly SCC and adenocarcinoma, which account for about 90% of all cervical cancers (adenosquamous carcinoma, glassy cell carcinoma, adenoid cystic carcinoma, adenoid basal carcinoma, carcinoid, small cell carcinoma, and undifferentiated carcinoma also occur). SCCs are keratinizing or nonkeratinizing in most cases and may be verrucous, condylomatous, papillary, or lymphoepithelioma-like carcinomas in a few cases. Adenocarcinomas are classified into

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mucinous, endometrioid, clear cell, serous, and mesonephric adenocarcinomas; mucinous adenocarcinomas are subclassified with endocervical type into adenoma malignum and villoglandular papillary adenocarcinoma and intestinal type adenocarcinoma. Staging Clinical staging represents the degree of advancement of the tumor and is defined by the FIGO classification established in 1994 and by the TNM classification of malignant tumors set by the UICC in 1997 as follows (classified by FIGO [TNM]): • Stage 0 (Tis): carcinoma in situ (preinvasive carcinoma). • Stage I (T1): cervical carcinoma confined to the uterus. • Stage II (T2): tumor invades beyond the uterus but not to the pelvic wall or to the lower third of the vagina. • Stage III (T3): tumor extends to the pelvic wall and/or involves the lower third of the vagina and/or causes hydronephrosis or nonfunctioning kidney. • Stage IVA (T4): tumor invades the mucosa of the bladder or rectum and/or extends beyond the true pelvis. • Stage IVA (Ml): distant metastasis. Stage IA (TIa) has been further classified by microinvasive depth and width into stage IA1 (Tlal) (depth of stromal invasion 3 mm, horizontal spread 7 mm) and stage IA2 (Tla2) (depth of stromal invasion >3 mm, 5 mm; horizontal spread 7 mm). Stage IB (Tlb) has been further classified by tumor size into stage IB1 (Tlbl) (greatest dimension 4 cm) and stage IB2 (Tlb2) (greatest dimension >4 cm). In cases staged IA2 (Tla2) or less advanced, colposcopically directed biopsy in the transformation zone of the cervix, endocervical curettage, or cervical conization is required. Prognosis Unfavorable prognostic factors include younger age, advanced clinical stage, certain

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histopathological types, vessel permeation, large tumor volume, parametrium involvement, and lymph node metastasis. Nodal metastasis is an especially critical prognostic factor after curative resection. Vascular endothelial growth factor (VEGF)-C and osteopontin contribute to the aggressive lymphangitic metastasis in uterine cervical cancers. Platelet-derived endothelial cell growth factor (PD-ECGF) contributes to the advancement of metastatic lesions as an angiogenic factor. PD-ECGF, VEGF-C, and osteopontin levels in metastatic lesions are prognostic indicators. Furthermore, serum PD-ECGF level reflects the status of advancement of cervical cancers and is recognized as a novel tumor marker for both SCC and adenocarcinoma of the cervix, while the tumor marker SCC is well known only as an indicator for SCC of the cervix. VEGF-C and osteopontin contribute to the aggressive lymphangitic metastasis in uterine cervical cancers. Therapy The treatment for cervical cancer consists mainly of surgery and radiation. Chemotherapy is performed in combination with surgery and/or radiation for advanced cases, and immunotherapy is an adjuvant treatment for surgery, radiation, and chemotherapy. The standard treatment for carcinoma in situ is cervical conization or total hysterectomy. The standard treatment for microinvasive carcinoma stage IA (Tla) is modified radical hysterectomy regardless of regional lymphadenectomy. The standard surgical treatment for invasive carcinoma is radical hysterectomy with regional lymphadenectomy. Although oophorectomy can be avoided in some cases during the reproductive period, ovarian metastasis must be considered especially in adenocarcinoma of the cervix. When oophorectomy is avoided, the ovary is better shifted out of radiation area. For patients who undergo oophorectomy, hormone replacement therapy can be useful. In more advanced cases, extended radical hysterectomy or pelvic exenteration is appropriate. After surgery external irradiation is followed in some cases. The standard radiotherapy without surgery for invasive carcinoma is intracavitary and/or

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external irradiation. Neoadjuvant therapy (chemotherapy) has been tried in order to make surgery more successful, and concurrent radiochemotherapy has been tested for the purpose of enhancing the effect of radiation.

References Fujimoto J, Sakaguchi H, Hirose R et al (1999) Clinical implication of expression of platelet-derived endothelial cell growth factor (PD-ECGF) in metastatic lesions of uterine cervical cancers. Cancer Res 59:3041–3044 Fujimoto J, Sakaguchi H, Aoki I et al (2000) The value of platelet-derived endothelial cell growth factor as a novel predictor of advancement of uterine cervical cancers. Cancer Res 60:3662–3665 Fujimoto J, Toyoki H, Sato E et al (2004) Clinical implication of expression of vascular endothelial growth factor-C in metastatic lymph nodes of uterine cervical cancers. Br J Cancer 91:466–469

Cetuximab Definition Trade name Erbitux ® is a chimeric IgG1k monoclonal antibody specifically binding to epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) (EGFR, ErbB-1, HER1 in humans) is the cellsurface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Ligands which induce activation of EGFR are epidermal growth factor and transforming growth factor-a, for example. Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity resulting in activation of several signal transduction cascades which lead to DNA synthesis and cell proliferation. EGFR mutations can lead to EGFR overexpression or overactivity and consequently result in uncontrolled cell division. Mutations of EGFR have been identified in several types of cancer, such as lung cancer and colorectal cancer.

Chelators as Anti-Cancer Drugs

Cetuximab is approved for the treatment of irinotecan-refractory metastatic colorectal cancer (CRC) in combination with irinotecan and for the treatment of locoregional advanced head and neck cancer as monotherapy or in combination with radiation. Dermatological toxicity is a limiting factor to the use of cetuximab. Hypersensitivity to cetuximab (rash, urticaria, fever, dyspnea, and hypotension) is frequent in certain regions and has been related to the presence of IgE antibodies specific for an oligosaccharide, galactose-a-1,3-galactose, which is present on the Fab portion of the cetuximab heavy chain. Cetuximab is under investigation in combination with chemotherapy (carboplatin or irinotecan) in pretreated triple-negative breast cancer (TNBC) with advanced disease. TNBC is estrogen and progesterone receptor negative, as well as HER2 negative, and therefore is not amenable to treatment with hormonal therapy or with trastuzumab. Furthermore, several phase I/II studies with cetuximab in combination with cytotoxic agents or with other targeted therapies, such as trastuzumab, are currently ongoing.

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C-HA-RAS1 ▶ HRAS

Charged Particle Therapy ▶ Proton Beam Therapy

Checkpoint with Forkhead and RING Finger Domain Protein ▶ CHFR

Chelation Therapy ▶ Chelators as Anti-Cancer Drugs

Cross-References

Chelators as Anti-Cancer Drugs

▶ Epidermal Growth Factor Inhibitors

David B. Lovejoy, Yu Yu and Des R. Richardson Department of Pathology, University of Sydney, Sydney, NSW, Australia

See Also (2012) Monoclonal antibody therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2367–2368. doi: 10.1007/9783-642-16483-5_3823

Synonyms Chelation therapy

Definition

CG Antigens ▶ Cancer Germline Antigens

CGP57148 ▶ Imatinib ▶ STI-571

Iron is an element fundamental for life. Many vital cellular processes such as energy metabolism and DNA synthesis consist of reactions that require catalysis by iron-containing proteins. These proteins include cytochromes, and ribonucleotide reductase (RR). The latter is more significant in the context of cellular proliferation due to its role in catalyzing the rate-limiting step of DNA synthesis. Ultimately, the importance of iron is

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highlighted by the fact that iron-deprivation leads to G1/S cell cycle arrest and ▶ apoptosis. Cancer cells, in particular, have a higher iron requirement because of their rapid rate of proliferation. In order to satisfy their iron requirement, some cancer cells have altered iron metabolism. In addition, iron chelators also demonstrate the ability to inhibit growth of aggressive tumors such as ▶ neuroblastoma. For these reasons, irondeprivation through iron chelation is seen as an exploitable therapeutic strategy for the treatment of cancer.

Characteristics Iron Metabolism in Cancer Cells In order to attain more iron, cancer cells have higher numbers of the transferrin receptor-1 molecule (TfR1) on their cell surface. The TfR1 binds the serum iron transport protein, transferrin (Tf). Hence, cancer cells are able to bind more Tf and, thus, take up iron at a greater rate than their normal counterparts. This is reflected by the ability of tumors to be radiolocalized using a radioisotope of gallium, 67Ga, which binds to the iron-binding site on Tf for delivery via TfR1. 67Ga can bind to iron-binding sites of Tf due to the similar atomic properties between gallium(III) and iron(III). Additionally, gene therapy by the administration of antisense TfR1 targeted to the sequences of TfR1 mRNA also showed selective anti-cancer activity, further demonstrating the importance of TfR1 in mediating cancer cell growth. Apart from TfR1 up-regulation, the expression of the iron-storage protein ferritin is also often altered in neoplastic cells, especially neuroblastoma (NB) and breast carcinoma. In childhood NB, serum ferritin levels are elevated at stages III and IV of the disease. In a longitudinal study, it was found that the elevated level was associated with a markedly poorer prognosis of the disease. In addition, serum ferritin levels also exceeded the normal limit in ▶ hepatocellular carcinoma and were found to be directly related to axillary lymph node status, presence of metastatic disease (▶ metastasis), and clinical stages of breast cancer.

Chelators as Anti-Cancer Drugs

Desferrioxamine, an Iron Chelator with Some Anti-Cancer Activity Desferrioxamine (DFO) is a natural ligand secreted by the bacterium Streptomyces pilosus to selectively sequester iron for biological use (Fig. 1). DFO is used clinically for the treatment of iron overload disorders such as the transfusionrelated iron overload in b-thalassemia. DFO is active against aggressive tumors including NB and leukemia in cell culture and clinical trials. The cytotoxicity of DFO in vitro was prevented by co-incubation of the cells with iron or iron saturated DFO, indicating that its antiproliferative activity was due to the depletion of cellular iron. Furthermore, DFO induces a block in cell cycle progression. Therefore, it was proposed that the mechanism of action of DFO involved the depletion of cellular iron, leading to the inhibition of ribonucleotide reductase for DNA synthesis and cell cycle arrest. In human NB cells, 5 days of exposure to DFO resulted in approximately 90% cell death. In contrast, the effect of DFO was minimal on non-NB cells, suggesting that it had selective anti-NB activity. A clinical trial showed that seven of nine NB patients had up to 50% reduction in bone marrow infiltration after a course of DFO administered for 5 days. Other clinical trials using DFO as a single agent and in combination with other chemotherapeutic drugs confirmed the anti-cancer potential of this chelator. However, in some animal studies and clinical trials, DFO was found to exhibit limited or no activity. DFO also suffers a number of limitations as a result of its highly hydrophilic nature. It has poor gastrointestinal absorption and a short plasma half-life of about 12 min due to rapid metabolism. As a result, DFO is not orally active and needs to be administered via subcutaneous infusion for prolonged periods ranging from 8 to 12 h for five to seven times per week. The prolonged infusion results in pain and swelling, which consequently leads to poor patient compliance. DFO is also expensive to produce. Despite these limitations and mixed results in clinical trials, DFO nonetheless provides “proof of principle” that iron chelation therapy may be specific and useful for cancer treatment.

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Chelators as Anti-Cancer Drugs, Fig. 1 Chemical structures of the iron chelators desferrioxamine (DFO), N, N0 ,N00 -tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyridine or tachpyr), 3-aminopyridine-2® carboxaldehyde-thiosemicarbazone (3-AP or Triapine ),

2-hydroxy-1-napthaldehyde isonicotinoyl hydrazone (311), and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) showing coordination to iron (Fe) through pyridyl nitrogen, aldimine nitrogen, and thionyl sulfur donor atoms

Other Chelators with Anti-Cancer Potential The limitations of DFO as an anti-cancer agent have encouraged the search for other active ironchelating drugs against cancer. Other experimental iron chelators include Triapine® (3-AP; Fig. 1), an iron-binding thiosemicarbazone-based drug currently in clinical trials for cancer therapy. Triapine® is a chelator that binds iron via sulfur and two nitrogen donor atoms and is suggested to be one of the most potent inhibitors of RR yet identified. In clinical trials, high doses of Triapine® (160 mg/m2/day) resulted in dose-limiting toxicities, including reduction in white blood cells, jaundice, nausea, and vomiting. Lower doses of Triapine® administered as a 96-h iv infusion at 120 mg/m2/day every 2 weeks was found to be well tolerated. In clinical trials with patients with advanced cancer, Triapine® was combined with the cytotoxic cancer drug gemcitabine, which also targets DNA synthesis. Of the 22 patients examined after treatment with gemcitabine and Triapine®, three were observed to have an objective response, and one patient had evidence of tumor reduction. In this trial, Triapine® was suggested to cause oxidation of hemoglobin to met-hemoglobin. This may have led to or contributed to the hypoxia, acute hypotension, and

electrocardiogram changes in patients receiving this chelator. An asymptomatic myocardial infarction was also observed in one individual administered Triapine® and this may also be related to its oxidative effects. Triapine® continues to be examined in clinical trials, particularly in combination with standard chemotherapy drugs. However, these deleterious effects must be considered when designing future studies with compounds of this class. Tachpyridine (or tachpyr; Fig. 1) is a novel chelator based upon the framework of the triamine cis,cis-1,3,5-triaminocyclohexane. Tachpyridine is cytotoxic to cultured bladder cancer cells with an activity approximately fifteen times greater than that of DFO. Although tachpyridine has the potential to chelate a number of metals, including calcium(II), magnesium(II), manganese(II), copper(II), and zinc(II), toxicity studies on tachypyridine complexes suggest that iron and zinc depletion mediates its cytotoxic effects. Similar to Triapine ®, Tachpyridine induces apoptotic cell death independent of functional p53 (see section “Iron Chelation and Cell Cycle Control Molecules,” below) (▶ p53 family). In addition, tachpyridine-iron complexes produce toxic free radicals (▶ reactive oxygen species),

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which was also thought to contribute to its antitumor activity. Tachpyridine arrests cells at the G2 phase, whereas the majority of iron chelators arrest cells at the G1/S phase due to the inhibition of ribonucleotide reductase. The G2 phase stage of the cell cycle is particularly sensitive to the effects of radiation. Ionizing radiation increases the sensitivity of tumor cells to the action of tachpyridine. Currently, tachpyridine is in preclinical development with the National Cancer Institute, USA. PIH Chelators The most comprehensively assessed alternate chelators for cancer treatment are the pyridoxal isonicotinoyl hydrazone (PIH) analogues. This class of chelators binds iron through the carbonyl oxygen, imine nitrogen, and phenolic oxygen (Fig. 1). Originally conceived for the treatment of iron overload disorders, several chelators of the PIH class were found to inhibit the growth of cancer cells. In fact, the chelator 311 (Fig. 1) was found to be highly active against a range of cancer cells. These compounds also showed marked ability to remove Fe from cells and prevent cellular Fe uptake from transferrin. The marked anti-cancer activity of chelator 311 was attributed to its relatively high lipophilicity, which facilitates entry into the cell. Indeed, a general trend observed with the PIH analogues was that anti-cancer activity increased as the chelator became more lipophilic. Mechanistically, PIH analogues have multiple modes of anti-cancer activity, aside from chelation of iron and inhibition of ribonucleotide reductase. Some members of the PIH class of chelators (e.g., see section “The DpT Chelators: Dp44mT,” below) increase the generation of toxic free radicals (reactive oxygen species) in cancer cells and affect the expression of cell-cycle control molecules (see section “Iron Chelation and Cell Cycle Control Molecules,” below). Additional studies with 311 have also shown that it can markedly induce the expression of the metastasis suppressor protein, Drg-1 in tumor cells. The Drg-1 protein is known to play a critical role in suppressing tumor growth and metastasis. Hence, induction of Drg-1 by potent iron chelators such

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as 311 may significantly contribute to the anticancer activity of these analogues. The DpT Chelators: Dp44mT The DpT class of chelators are structurally related to PIH analogues, but feature a sulfur donor atom instead of the hydrazone oxygen donor atom (Fig. 1). The chelator Dp44mT has been shown to be the most effective of the DpT series of ligands in terms of anti-cancer activity. It acts with selectivity against tumor cells and has much less effect on the growth of normal cells. Dp44mT also showed high iron chelation efficacy and prevented cellular uptake of iron from ironlabeled Tf. Another mechanism of its action involves the generation of toxic free radicals (reactive oxygen species) when Dp44mT interacts with cellular iron pools. Initially, in vivo studies of Dp44mT in mice bearing chemotherapy-resistant M109 lung carcinoma showed a reduction in the size of the tumor by 53% after 5-days of treatment. A later investigation also found marked inhibition of the growth of human lung, neuroepithelioma, and melanoma xenografts growing in mice. In fact, a 7-week administration of Dp44mT in mice bearing human melanoma xenografts resulted in the decrease of tumor growth to 8% of that in untreated control mice. At the dose given, no hematological abnormalities were detected, although at a higher dose, myocardial fibrosis was identified. This side effect at a high dose may be due to the marked redox activity of the Dp44mT-iron complex. However, at a lower dose Dp44mT was well tolerated with no hematological abnormalities and less cardiotoxicity. Other studies with Dp44mT showed that it also markedly increased the expression of the metastasis suppressor protein, Drg-1 in tumor cells. Induction of Drg-1 could potentially be a very significant component of the anti-cancer mechanism of Dp44mT. Further development of DpT series chelators is currently underway. Iron Chelation and Cell Cycle Control Molecules Iron-deprivation generally leads to G1/S phase cell cycle arrest as a result of inhibition of

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ribonucleotide reductase. This has prompted many studies assessing the effect of iron chelation by DFO and chelator 311 on the expression of many cell cycle control molecules, namely, cyclins, ▶ cyclin dependent kinases (cdks), cdk inhibitors, and p53 (p53 gene family). Consistently, these studies found that iron chelation markedly decreased the expression of ▶ cyclin D (D1, D2, and D3) and to a lesser extent cyclin A and B. The expression of cdk2 and cdk1, but not cdk4, were also decreased upon iron chelation. These effects were dependent on irondeprivation, as iron-chelator complexes were unable to induce such effects. Cyclins D, E, and A and cdks 2, 4, and 6 are involved in progression through the G1 phase, although cyclin E, A, and cdk2 are also involved in S phase progression. The formation of the cyclin A-cdk2 complex is essential for G1/S progression. Cyclin B and cdk1, on the other hand, are important for mitosis. During the G1 phase, cyclin D and E bind to cdk4 and cdk2, respectively, to phosphorylate (phosphorylation) the retinoblastoma protein (pRb) (▶ retinoblastoma protein, biological and clinical functions). This results in the release of molecules such as the E2F transcription factor from pRb that promotes the expression of genes for S phase. The decrease in the expression of these cyclins upon iron chelation causes hypophosphorylation of pRb, which in turn leads to the G1/S phase arrest. In addition to cyclins and cdks, iron chelation also affects the expression of cell cycle modulatory molecules. In particular, iron chelators caused a marked increase in the expression of the cyclin-dependent kinase inhibitor p21WAF1/ CIP1 (▶ p21(WAF1/CIP1/SDI1)) at the mRNA level. P21WAF1/CIP1 mediates G1/S phase arrest by directly binding the cyclin-cdk complexes. It was speculated that the increased level of p21WAF1/CIP1 upon iron chelation was consistent with its potential role in the G1/S phase arrest. However, an increase of p21WAF1/CIP1 expression only occurred at the mRNA level, with either no change or a decrease in p21WAF1/CIP1 protein expression being observed. This was unexpected and it was subsequently demonstrated that p21WAF1/CIP1 protein level could be controlled by

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proteasomal (▶ proteasome) degradation after iron chelation. In contrast, investigations examining p53 showed that its protein expression and DNA-binding activity were increased after chelation. P53 is a tumor suppressor and acts as a transcription factor that is involved in the transcription of a variety of genes involved in cell cycle arrest, differentiation, apoptosis, and DNA repair. An increase in p53 after iron chelation may be the result of a decrease in deoxyribonucleotide levels due to the inhibition of RR activity or changes in intracellular redox status. Despite the fact that p21WAF1/CIP1 is a downstream effector of p53, elevated expression of p21WAF1/CIP1 upon iron chelation occurs through a p53-independent pathway. The ability of chelators to potentially inhibit tumor cell growth by a p53-independent pathway is significant, since p53 is the most frequently mutated gene in cancer. This also explains why cells with wild type or mutant p53 are similarly sensitive to the growth inhibitory effects of iron chelators. However, the function of increased p53 expression after chelation remains a subject for further investigation. Conclusions The demonstration that some iron chelators may be clinically useful for cancer treatment followed on from initial observations that rapid cancer cell proliferation requires iron. Currently, the iron chelator, Triapine ®, is being examined in a variety of clinical trials, with focus on a potential role in combination chemotherapy. The search for more effective anti-cancer Fe chelators than DFO has also led to the development of other potent Fe chelators, including Dp44mT and tachpyridine, and significant progress has been made toward understanding their molecular targets. However, further in vivo experiments and pre-clinical studies will be necessary to build upon the promise of these agents.

Cross-References ▶ Apoptosis ▶ Cyclin D

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▶ Cyclin-Dependent Kinases ▶ Hepatocellular Carcinoma ▶ Metastasis ▶ Neuroblastoma ▶ P53 Family ▶ p21 ▶ Proteasome ▶ Reactive Oxygen Species ▶ Retinoblastoma Protein, Biological and Clinical Functions

References Buss JL, Greene BT, Turner J et al (2004) Iron chelators in cancer chemotherapy. Curr Top Med Chem 4:1623–1635 Kalinowski D, Richardson DR (2005) Evolution of iron chelators for the treatment of iron overload disease and cancer. Pharmacol Rev 57(4):1–37 Le NTV, Richardson DR (2004) Iron chelators with high anti-proliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a novel link between iron metabolism and proliferation. Blood 104:2967–2975 Whitnall M, Howard J, Ponka P et al (2006) A class of iron chelators with a wide spectrum of potent anti-tumor activity that overcome resistance to chemotherapeutics. Proc Natl Acad Sci U S A 103:14901–14906 Yu Y, Wong J, Lovejoy DB et al (2006) Chelators at the cancer coalface: desferrioxamine to triapine and beyond. Clin Cancer Res 12:6876–6883

See Also (2012) Cell cycle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi: 10.1007/978-3-642-16483-5_994 (2012) Cytochrome c . In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1043. doi: 10.1007/978-3-642-16483-5_1458 (2012) Drg-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1160. doi: 10.1007/978-3-642-16483-5_1730 (2012) E2F transcription factor . In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1183. doi: 10.1007/978-3-642-16483-5_1770 (2012) Lipophilicity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2058. doi: 10.1007/978-3-642-16483-5_3384 (2012) Ribonucleotide reductase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3308. doi: 10.1007/978-3-642-16483-5_5102 (2012) Chelator. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 755. doi: 10.1007/978-3-642-16483-5_1052 (2012) Phosphorylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2870. doi: 10.1007/978-3-642-16483-5_4544

Chemical Biology Screen

Chemical Biology Screen ▶ Small Molecule Screens

Chemical Carcinogenesis Joseph R. Landolph, Jr. Department of Molecular Microbiology and Immunology, and Department of Pathology; Laboratory of Chemical Carcinogenesis and Molecular Oncology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Health Sciences Campus, University of Southern California, Los Angeles, CA, USA

Definition Chemical carcinogenesis (▶ carcinogenesis) is the process of the genesis of a tumor (carcinoma) and the series of sequential steps that occur when lower animals or humans are treated with chemical carcinogens that lead to tumor development. After all these steps are accomplished, the physiological mechanisms regulating the control of growth in the normal cells are degraded, and the normal cells are degraded and converted into tumor cells. The tumor cells then grow in an unregulated fashion and evade the host immune system, leading to development of visible tumors.

Characteristics Normal Cell Types in Animals and the Tumors They Give Rise To During embryogenesis in mammals (warmblooded animals), there are three primary germ layers of the early embryo which develop into all the basic cell types, tissues, and organs in the body. These are the ectoderm, the endoderm, and the mesoderm. The ectoderm and endoderm are

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epithelial layers. Most of the epithelial organs in the body are derived from the endodermal and the ectodermal germ layers. The epidermis of the skin, the corneal epithelium, and mammary glands develop from the ectoderm. The endoderm layer develops into the liver, pancreas, stomach, and intestines. The mesoderm develops into the kidney and linings of male and female reproductive tracts. Three types of cells are important in chemical carcinogenesis. These cell types are (i) epithelial cells, which form the coverings and internal parts of organs; (ii) fibroblasts, which are connective tissue cells derived from primitive mesenchymal cells; and (iii) cells of the hematolymphopoietic series, which are derived from the blood-forming elements. These cell types all have special and specific characteristics. In humans, 92% of the tumors that arise are derived from epithelial cells (epithelial cell tumors). These tumors are called carcinomas. The remaining 8% of the tumors are derived from a combination of tumors derived from fibroblasts, called sarcomas, and tumors derived from white blood cells, called leukemias and lymphomas. Carcinogens There are a group of molecules and radiations referred to as “carcinogens.” A carcinogen is any molecule, or group of molecules, such as viruses (▶ Virology), or radiation (▶ Radiation carcinogenesis; ▶ radiation oncology), that can cause tumors in lower animals and humans, when they are exposed to this agent. This happens when carcinogens cause normal cells to transform or convert into transformed cells and tumor cells during experiments in vitro, called chemical transformation experiments. Chemicals referred to as chemical carcinogens (chemical carcinogenesis) can cause tumors in lower animals and in humans exposed to them. Examples of chemical carcinogens are vinyl chloride, aflatoxin B1 (a metabolite and biocide of the fungus, Aspergillus flavus) (▶ Aflatoxins), benzo(a)pyrene (a polycyclic aromatic hydrocarbon formed when organic matter is pyrolyzed in the absence of oxygen) (▶ Polycyclic aromatic hydrocarbons), and beta-naphthylamine

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(an aromatic amine used to manufacture dyestuffs that causes bladder cancer in animals and humans) (▶ Aromatic amine). Nitrosamines are another class of chemical carcinogens. An example is dimethylnitrosamine (DMN). Many nitrosamines are synthetic compounds. Some are believed to form in the stomach of humans when amines (derived from fish in the diet) contact nitrous acid (formed from the nitrate from fertilizer that is used to grow foodstuffs) in the acidic conditions (acid pH) of the stomach. Chemicals in all these classes of carcinogens can cause tumors in humans and in lower mammals. There are also a number of radiations that cause tumors in humans and lower animals. These include ionizing radiations, such as alpha particles (charged helium nuclei), beta particles (naked electrons), and gamma particles. There are also tumor viruses, consisting of RNA (RNA tumor viruses) and DNA (DNA tumor viruses). When animals are treated with these viruses, tumors are formed. Examples of RNA tumor viruses are the Rous sarcoma virus, the Abelson leukemia virus, and the Kirsten Ras virus. Examples of DNA tumor viruses are the polyoma virus, the SV40 (simian virus 40) (▶ SV40) virus, the ▶ EpsteinBarr virus, and the human papilloma viruses 16 and 18. Mechanisms of Chemical Carcinogenesis There are two broad mechanisms of chemical carcinogenesis. In the first type, which we refer to here as “complete ▶ carcinogenesis,” a mammal is treated with a large dose of a chemical carcinogen, such as 7,12-dimethylbenz(a) anthracene, and the animals treated eventually develop tumors. Carcinogenesis with complete carcinogens is usually dose dependent, such that the higher doses of carcinogens that the animals are treated with, the high the yield of tumors per animal and in the percentage of animals with tumors. The second mechanism of chemical carcinogenesis, discovered by Dr. Isaac Berenblum of the Weizmann Institute in Israel, is referred to as “two-step carcinogenesis,” or “initiation and promotion.” In initiation and promotion experiments, Berenblum treated mice on the skin of their

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shaved backs with chemical carcinogens at low doses and also with tumor promoters. Berenblum was testing the hypothesis that carcinogenesis was due to irritation and inflammation. Hence, he used croton oil, a product of the plant, Euphorbia lathyris, which the plant uses as a biocide against insects. Croton oil is a very irritating substance, which is important in the plant’s use of it as a biocide against insects. When mice were treated with low doses of 7,12-dimethylbenz(a) anthracene (DMBA, a carcinogenic PAH), one time, they exhibited no tumors. A second group of animals was treated with the tumor promoter, croton oil, once per week, and the animals also exhibited no tumors. When the mice were treated with a low dose of DMBA, and then once weekly with croton oil, they developed many tumors. If the latter treatment was reversed, i.e., the animals were treated first with croton oil once per week and then later treated with a low dose of DMBA, the animals showed no tumors. If the animals were treated with a low dose of DMBA, then no treatment was performed for a significant amount of time and then the animals were treated with croton oil once per week, the animals also developed a high yield of tumors. In this system, treatment of the animals with the low dose of DMBA is referred to as the “initiation step,” and later treatment with croton oil is called the “promotion” step. Initiation is believed to be a genotoxic event, likely a mutation, and is an irreversible step. Initiated cells can be promoted to tumors cells if they are treated with croton oil long enough. The promotion step is believed to be due to the binding of tetradecanoyl-phorbol acetate (TPA, the most active constituent of the mixture of phorbol esters in croton oil), to protein kinase C, triggering signal transduction and cell division in cells bearing mutations in proto-oncogenes. If promotion is interrupted, then tumorigenesis is reversible, i.e., the cellular death rate will equal the cellular growth rate, and the tumor will regress. If promotion is continued long enough, the tumor becomes fixed and will not regress. Eric Hecker of the German Cancer Research Center (Deutsch Krebs Forschung Zentrum) in Heidelberg, Germany, fractionated croton oil used by Berenblum, by high pressure liquid

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chromatography, and found that TPA was the most active tumor promoter in it. From experiments with high doses of chemical carcinogens, and experiments with initiation and promotion, we now have evidence that chemical carcinogens such as DMBA cause mutations in protooncogenes, such as RAS genes, converting them into activated ▶ oncogenes. In complete carcinogenesis experiments, further mutations in other protooncogenes can also occur, leading to activation of additional oncogenes. In addition, activated metabolites of the carcinogens (formed in the animals/mammals by cytochrome P450 or other enzymes of metabolic activation) also cause mutational inactivation of ▶ tumor suppressor genes or breakage of chromosomes bearing them, leading to loss of these tumor suppressor genes. Together, activation of oncogenes and inactivation of tumor suppressor genes leads to the genesis of tumors in mammals. Insights into Mechanisms of Chemical Carcinogenesis from Studies of Chemically Induced Neoplastic Transformation Studies of the abilities of chemical carcinogens to convert normal cells into tumor cells in cell culture dishes have given us substantial insight into the molecular mechanisms of chemical carcinogenesis. In cell culture, normal fibroblasts and normal epithelial cells grow if they are fed properly, until they eventually fill the culture dish, and touch each other. Growth then ceases. This process is called contact inhibition of cell division. Cells can then be removed from the cell culture dish with a protease called trypsin, diluted, and replated into new cell culture dishes. This process can be repeated many times, until the population of total cells has undergone sixty population doublings. At this point, the cells senesce (▶ Senescence and immortalization) or die. This is due to progressive shortening of telomeres (▶ Telomerase), structures at the end of chromosomes, with each successive DNA replication and cell division. Telomere shortening acts as a cellular and molecular “clock,” to mark the lifetime of the cell. This process aids in the control of the normal physiology of the organism, by removing old cells which accumulated many mutations, which could eventually lead to cancer.

Chemical Carcinogenesis

▶ Chemically induced cell transformation is the process by which normal cells are treated with chemical carcinogens in vitro in a cell culture dish or flask, and then their growth control mechanisms degrade, converting or transforming them into transformed cells. There are two mechanisms by which cells can be converted by chemical carcinogens into transformed cells. Firstly, cells can be treated with genotoxic (DNA damaging) (▶ Genetic toxicology) chemical carcinogens. Many of these genotoxic carcinogens are mutagens (▶ Mutation rate). These carcinogens either already are direct mutagens (rare), or more commonly they are pre-carcinogens, and can be converted into mutagenic proximate carcinogens by cytochrome P450 enzymes or other enzyme systems that activate the pre-carcinogens into mutagens. The carcinogens benzo(a)pyrene, aflatoxin B1, and nitrosamines are all examples of pre-carcinogens that are metabolically activated into mutagens by various types of cytochrome P450 enzymes. Most pre-carcinogens are hydrophobic (fat loving) compounds that would bioaccumulate in the body and cause alterations in the properties of enzymes and membranes in cells. Mammals must therefore derive strategies to eliminate hydrophobic pre-carcinogens. The cytochrome P450 enzyme systems, and other enzyme systems, have evolved in order to metabolize these pre-carcinogens, to make them water-soluble, so they can be excreted in the urine and removed from the body. Since these compounds are inherently chemically inert, a necessary first chemical reaction step has evolved, in which cytochrome P450 enzymes attack pre-carcinogens like benzo(a)pyrene (BaP) with molecular oxygen and reducing equivalents (NADPH and NADH) to generate epoxides and diol epoxides from it. These metabolites are mutagens, and this step results in “metabolic activation.” In a second step, which is closely coupled to the first step, these active metabolites are reacted with and conjugated to, molecules of water by the enzyme, epoxide hydrolase, converting them to trans-dihydrodiols and tetraols, which are highly water-soluble, so they are excreted in the urine. The small amount of epoxides and diol epoxides derived from BaP

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then bind covalently to DNA bases, resulting in mutations in proto-oncogenes, activating them into oncogenes, and mutations in tumor suppressor genes, inactivating them. In a second mechanism of ▶ carcinogenesis, chemicals called “non-genotoxic carcinogens” transform normal cells into tumor cells in a different way, by non-mutagenic mechanisms. One example is the chemical, 5-azacytidine, a chemical analog of a normal base. 5-azacytidine binds to DNA methyltransferases (▶ Methylation), inhibiting them. This results in a loss of methylation of the cytidine in DNA. If this occurs in quiescent proto-oncogenes, then these can become transcriptionally activated, leading to cell transformation. Other examples of non-genotoxic carcinogens include hormones, such as testosterone and estrogen. Higher steadystate levels of testosterone and estrogen are believed to lead to aberrantly high numbers of cell divisions in the prostate and breast tissue. The resultant spontaneous mutations that occur are believed to lead to prostate cancer and breast cancer, respectively. The process by which a normal cell is converted into tumor cells, or chemically induced neoplastic transformation (neoplastic cell transformation), occurs in four steps. In the first step, when cells are treated with mutagenic chemical carcinogens, there occur mutations in protooncogenes, activating them to oncogenes, and mutations in tumor suppressor genes inactivating them. The cells then develop the ability to grow in multilayers and form foci. This is particularly true for fibroblastic cells, less so for epithelial cells. This first step in cell transformation is called morphological cell transformation or focus formation. Further genetic changes occur in the transformed cells. The second step that occurs is that the cells become immortal and do not die or senesce. Some activated oncogenes (v-myc) can cause cells to become immortal. This step would be called transformation to cellular immortality. In the third step, cells develop the ability to grow in soft agar, in three-dimensional suspension. This step is called anchorage-independent cell transformation or transformation to anchorage independence. A final step that develops after further genetic

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change is that cells develop the ability to form tumors when injected into athymic (nude) mice. This step is called neoplastic transformation, or the ability of cells to be transformed so that they form neoplasms or new growths, which we call tumors. Often, a number of activated oncogenes, two or more, may cooperate together to perturb normal cellular physiology to cause neoplastic transformation of normal rodent or human cells in culture. It is now believed by scientists that activation of proto-oncogenes into oncogenes, and inactivation of tumor suppressor genes, such that approximately eight total genes are genetically altered, leads to the aberrant expression of approximately 150 genes or more in the tumor cells. This then leads to neoplastic transformation of cells in culture and hence to chemical cacinogenesis in the animal. We believe that chemically induced neoplastic transformation is a good model for how cells in the animal become converted (transformed) into tumor cells when the animal is treated with chemical carcinogens. Significance of Chemical Carcinogenesis The significance of the process of chemical carcinogenesis is twofold. Firstly, the assay for chemical carcinogenesis in lower animals, usually mice and rats, can be used to test chemicals to determine whether they are carcinogens by virtue of their ability to induce tumors in mice and rats. Those chemicals that are able to cause a reproducible, dose-dependent induction of tumors in mice and/or rats are presumed to be human carcinogens. This presumption is due first to the relationship that rodents and humans are both warm-blooded animals and mammals. As such, their biochemistry and physiology are similar. In addition, many chemical carcinogens were first found to be carcinogenic in rodent carcinogenesis bioassays and later found to be carcinogens in humans. Almost all carcinogens that have been shown to be carcinogenic in humans are also carcinogenic in rodents (aflatoxin B1, vinyl chloride, asbestos, cigarette smoke, asbestos, polycyclic aromatic hydrocarbons). Secondly, the process of chemical carcinogenesis as studied in rodents has led to unique insights into the mechanisms of carcinogenesis. Investigators frequently use whole animal carcinogenesis

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bioassays to study how proto-oncogenes are activated into oncogenes, how tumor suppressor genes are inactivated by chemical carcinogens, and how oncogene activation and tumor suppressor gene inactivation lead to induction of tumors in mammals. Studying the mechanisms of carcinogenesis in rodents has also led to the identification of agents that interfere with this process and may eventually be used to prevent the induction of cancer in humans.

Cross-References ▶ Adenocarcinoma ▶ Aflatoxins ▶ Alkylating Agents ▶ Amplification ▶ Anchorage-Independent ▶ Aneuploidy ▶ Aromatic Amine ▶ Benzene and Leukemia ▶ Benzpyrene ▶ Bladder Cancer ▶ Cancer ▶ Cancer Causes and Control ▶ Cancer Epidemiology ▶ Carcinogen Metabolism ▶ Carcinogenesis ▶ Chemically Induced Cell Transformation ▶ Chromium Carcinogenesis ▶ Chromosomal Instability ▶ Class II Tumor Suppressor Genes ▶ Detoxification ▶ DNA Damage ▶ DNA Oxidation Damage ▶ Embryonic Stem Cells ▶ Endocrine Oncology ▶ Endocrine-Related Cancers ▶ Epidemiology of Cancer ▶ Epigenetic ▶ Epigenomics ▶ Epstein-Barr Virus ▶ Fibrosarcoma ▶ Genetic Toxicology ▶ Genomic Instability ▶ Helicobacter Pylori in the Pathogenesis of Gastric Cancer

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▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Hepatitis B Virus ▶ Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention ▶ Hexavalent Chromium ▶ Hormonal Carcinogenesis ▶ Hypomethylation of DNA ▶ Inflammation ▶ Lung Cancer ▶ Lung Cancer Epidemiology ▶ Mesenchymal Stem Cells ▶ Methylation ▶ Mutation Rate ▶ Oncogene ▶ Oxidative Stress ▶ Polycyclic Aromatic Hydrocarbons ▶ Radiation Carcinogenesis ▶ Radiation Oncology ▶ Reactive Oxygen Species ▶ Renal Cancer Pathogenesis ▶ Repair of DNA ▶ Senescence and Immortalization ▶ SV40 ▶ Telomerase ▶ Toxicological Carcinogenesis ▶ Tumor Suppressor Genes ▶ Virology

References Landolph JR Jr, Xue W, Warshawsky D (2006) Whole animal carcinogenicity bioassays, Chapter 2. In: Warshawsky D, Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, pp 25–44 Verma R, Ramnath J, Clemens F et al (2005) Molecular biology of nickel carcinogenesis: identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Mol Cell Biochem 255:203–216 Warshawsky D (2006) Carcinogens and mutagens, Chapter 1. In: Warshawsky D, Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, pp 1–24 Warshawsky D, Landolph JR Jr (2006) Overview of human cancer induction and human exposure to carcinogens, Chapter 13. In: Warshawsky D, Landolph JR

921 Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, pp 289–302 Weinberg RW (2007) Multi-step tumorigenesis, Chapter 11. In: Ram A (ed) The biology of cancer. Garland Science/ Taylor and Francis Group, LLC, New York, pp 399–462

See Also (2012) Carcinogen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 644. doi:10.1007/978-3-642-16483-5_839 (2012) Cytochrome P450 enzymes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1043. doi:10.1007/978-3-642-16483-5_1465 (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 12911292. doi:10.1007/978-3-642-16483-5_1958 (2012) Fibroblasts. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1398. doi:10.1007/978-3-642-16483-5_2176 (2012) Genotoxic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1540. doi:10.1007/978-3-642-16483-5_2393 (2012) Mutagen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2409. doi:10.1007/978-3-642-16483-5_3907 (2012) Mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Neoplastic cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2474. doi:10.1007/978-3-642-16483-5_4013 (2012) Proto-oncogenes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3107-3108. doi:10.1007/978-3-642-16483-5_6656 (2012) Tumor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3792. doi:10.1007/978-3-642-16483-5_6014 (2012) Tumor promoter. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6047 (2012) Two-step carcinogenesis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3821. doi:10.1007/978-3-642-16483-5_6071

Chemical Genetic Screen ▶ Small Molecule Screens

Chemical Mutagenesis ▶ Genetic Toxicology

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Chemically Induced Cell Transformation Joseph R. Landolph, Jr. Department of Molecular Microbiology and Immunology, and Department of Pathology; Laboratory of Chemical Carcinogenesis and Molecular Oncology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Health Sciences Campus, University of Southern California, Los Angeles, CA, USA

Definition Chemically induced cell transformation is the series of sequential steps that occur when mammalian cells are treated with ▶ Chemical Carcinogenesis and converted into tumor cells. The intermediate cell phenotypes (cell properties) are acquired one at a time, including first cellular immortality, then morphological transformation (change in cell shape, leading to crisscrossing of cells in abnormal patterns), then anchorage independence (growth of cells as colonies or balls of cells in three-dimensional suspension of agar, without attachment to the plastic dishes cells are usually grown on), and finally neoplastic transformation (neoplastic cell transformation), or the ability of cells to form tumors when injected into nude (athymic) mice.

Characteristics Normal Growth of Normal Cells In the mammalian organism (warm-blooded animal), there are many types of cells. In general, these cell types are divided into (i) epithelial cells, which form the coverings of organs; (ii) fibroblasts, which are connective tissue cells; and (iii) cells of the hemato-lymphopoietic series, which are derived from the blood-forming elements. These cell types all have special and specific characteristics.

Chemically Induced Cell Transformation

These three general cell types can be grown outside the body in an artificial situation, in cell culture medium in plastic cell culture dishes. This constitutes a model system in which the physiology of cells can be studied outside of the complicated conditions of the body. When grown in cell culture, epithelial cells and fibroblastic cells attach to the cell culture dish, by virtue of the surface charge of the cell relative to that of the plastic of the cell culture dish. These normal fibroblastic and epithelial cells must anchor to the bottom inside of the cell culture dish in order to be able to replicate their DNA and divide. This is called anchorage dependence of cell growth. These cells continue to grow if fed properly with cell culture medium, containing 5–10% fetal calf serum and cell culture medium. Cell culture medium consists of sugars, amino acids, salts, and buffers, along with an indicator to detect the acidity of the culture medium (pH indicator), all dissolved in water. In cell culture, the normal fibroblasts and normal epithelial cells continue to grow if they are fed properly, until they eventually fill the culture dish, and touch each other. Growth then ceases. This process is called contact inhibition of cell division. These cells can then be removed from the cell culture dish with a protease called trypsin, diluted and replated into new cell culture dishes. This process can be repeated many times, until the population of total cells has undergone approximately 60 population doublings. This is called the “Hayflick limit,” after Dr. Leonard Hayflick, who discovered it. At this point, the cells undergo cellular senescence (▶ Senescence and immortalization) or die. This is due to progressive shortening of telomeres (▶ Telomerase), structures at the end of chromosomes that are progressively shortened with each successive DNA replication and cell division. Hence, telomere shortening acts as a cellular and molecular “clock,” to mark the lifetime of the cell. This process is believed to aid in the control of the normal physiology of the organism, and to rid it of old cells which have many mutations, which could eventually lead to cancer. If these normal cells are injected into mice lacking an immune system (athymic or “nude” mice), they will not grow and will not form tumors.

Chemically Induced Cell Transformation

In contrast, cells of the hemato-lymphopoietic series grow in three-dimensional suspension (the blood) in vivo. Hence, when grown in vitro (outside the body), these cells must also be grown in three-dimensional suspension. A common practice is to grow the cells in varying concentrations of agar. When injected into athymic or “nude” mice, these normal cells, whether cells of the hematopoietic (red blood cell) or lymphoid (white blood cell) lineages, will not form tumors. Carcinogens There are a group of chemical molecules, radiations, and viruses referred to as “carcinogens.” A carcinogen is any chemical or group of molecules, such as viruses (▶ Virology) or radiation (▶ Radiation carcinogenesis; ▶ radiation oncology) that can cause tumors in lower animals when they are treated with this agent. These agents can also cause normal cells to transform (convert) into transformed cells and tumor cells. There are a group of chemicals referred to as chemical carcinogens (▶ Chemical carcinogenesis). These are specific chemicals that can cause tumors in animals treated with them. Examples of these are vinyl chloride, aflatoxin B1 (a metabolite and biocide of the fungus, Aspergillus flavus) (▶ Aflatoxins), benzo(a)pyrene (a polycyclic aromatic hydrocarbon formed when organic matter is burned in the absence of oxygen) (▶ Polycyclic aromatic hydrocarbons), and beta-naphthylamine (an aromatic amine used to manufacture dyestuffs that causes bladder cancer in animals and humans) (▶ Aromatic amine). Another class of chemical carcinogens is called nitrosamines. An example is dimethylnitrosamine (DMN). Many nitrosamines are synthetic compounds. Some are believed to form in the stomach of humans when amines (derived from fish in the diet) contact nitrous acid (formed from the nitrate from fertilizer that is used to grow foodstuffs) in the acidic conditions (acid pH) of the stomach. Chemicals in all these classes of carcinogens can cause tumors in humans and in lower mammals. There are also a number of radiations (radiation carcinogenesis) that can cause tumors in humans and lower animals. These include ionizing radiations, such as alpha particles (charged helium

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nuclei), beta particles (naked electrons), and gamma particles. In addition, there are also tumor viruses, consisting of RNA (RNA tumor viruses) and DNA (DNA tumor viruses). When animals are treated with these viruses, tumors are formed. Examples of RNA tumor viruses are the Rous sarcoma virus, the Abelson leukemia virus, and the Kirsten Ras virus. Examples of DNA tumor viruses are the polyoma virus, the SV40 (simian virus 40) (▶ SV40) virus, the ▶ Epstein-Barr virus, and the human papilloma viruses 16 and 18. Chemically Induced Cell Transformation: Description and Mechanisms Chemically induced cell transformation is the process by which normal cells are treated with chemical carcinogens in vitro in a cell culture dish or flask, and they then convert or transform into transformed cells. There are two mechanisms by which cells can be converted by chemical carcinogens into transformed cells. Firstly, cells can be treated with genotoxic (DNA damaging) (▶ Genetic toxicology) chemical carcinogens. Many of these genotoxic carcinogens are mutagens (▶ Mutation rate). These carcinogens either already are direct mutagens (rare), or more commonly they are pre-carcinogens, and can be converted into mutagenic proximate carcinogens by cytochrome P450 enzymes or other enzyme systems that activate the pre-carcinogens into mutagens. The pre-carinogens benzo(a)pyrene, aflatoxin B1, and nitrosamines are all examples of pre-carcinogens that are metabolically activated into mutagens by various types of cytochrome P450 enzymes. The perspective for this process is that most pre-carcinogens are hydrophobic (fat loving) compounds that would bioaccumulate in the body and cause alterations in the properties of enzymes and membranes in cells. Hence, the organism must derive a strategy to eliminate these hydrophobic pre-carcinogens. Therefore, the cytochrome P450 enzyme systems, and other enzyme systems, have evolved in order to metabolize these pre-carcinogens, to make them watersoluble, so they can be excreted in the urine and removed from the body. Since these compounds are inherently chemically inert, a necessary first

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chemical reaction step has evolved, in which cytochrome P450 enzymes first attack pre-carcinogens like benzo(a)pyrene (BaP) with molecular oxygen and reducing equivalents (NADPH and NADH) to generate epoxides and diol epoxides from it. These metabolites are mutagens, and this step results in “metabolic activation.” In a second step, which is closely coupled to the first step, these active metabolites are reacted with and conjugated to, molecules of water by the enzyme, epoxide hydrolase, converting them to trans-dihydrodiols and tetraols, which are highly water-soluble, so they are excreted in the urine. The small amount of epoxides and diol epoxides derived from BaP then go on to bind covalently to DNA bases, resulting in mutations in proto-▶ oncogenes, activating them into ▶ oncogenes, and mutations in ▶ tumor suppressor genes, inactivating them. In a second mechanism of ▶ carcinogenesis, chemicals called “non-genotoxic carcinogens” transform normal cells into tumor cells in a different way, by non-mutagenic mechanisms. One example is the chemical, 5-azacytidine, a chemical analog of a normal base. 5-azacytidine binds to DNA methyltransferases (▶ Methylation), inhibiting them. This results in a loss of methylation of the cytidine in DNA. If this occurs in quiescent proto-oncogenes, then these can become transcriptionally activated, leading to cell transformation. Other examples of non-genotoxic carcinogens include hormones, such as testosterone and estrogen. Higher steadystate levels of testosterone and estrogen are believed to lead to aberrantly high numbers of cell divisions in the prostate and breast tissue. The resultant spontaneous mutations that occur are believed to lead to prostate cancer and breast cancer, respectively. The process of chemically induced neoplastic transformation, or the process of generating a tumor cell, falls into at least four steps. In the first step, when cells are treated with mutagenic chemical carcinogens, there occur mutations in proto-oncogenes, activating them to oncogenes, and mutations in tumor suppressor genes, inactivating them. The cells then develop the ability to grow in multilayers and form foci. This is particularly true for fibroblastic cells, less so for

Chemically Induced Cell Transformation

epithelial cells. This first step in cell transformation is called morphological cell transformation or focus formation. Further genetic changes occur in the transformed cells. The second step that occurs is that the cells become immortal and do not die or senesce. Some activated oncogenes (v-myc) can cause cells to become immortal. This step would be called transformation to cellular immortality. A third step that occurs is that the cells develop the ability to grow in soft agar, in three-dimensional suspension. This step is called anchorageindependent cell transformation or transformation to anchorage independence. A final step that develops after further genetic change is that the cells develop the ability to form tumors when injected into athymic (nude) mice. This step is called neoplastic transformation, or the ability of the cell to be transformed so that it forms neoplasms or new growths, which we call tumors. Often, a number of activated oncogenes, two or more, may cooperate together to perturb normal cellular physiology to cause neoplastic transformation of normal rodent or human cells in culture. Significance of Chemically Induced Neoplastic Transformation The significance of the process of chemically induced neoplastic transformation is two-fold. Firstly, the assay for chemically induced morphological cell transformation can be used an assay to detect chemical carcinogens. Those chemicals that have the ability to induce foci of morphologically transformed cells are highly likely to be able to induce tumors in animals. Hence, this assay can detect chemical carcinogens by virtue of their ability to induce foci of morphologically transformed cells. Secondly, the study of chemically induced morphological, anchorage-independent, and neoplastic transformation in vitro is frequently used as a model system to study the process of chemical carcinogenesis. Investigators frequently use these assays to study how proto-oncogenes are activated into oncogenes, and how tumor suppressor genes are inactivated by chemical carcinogens, and how oncogene activation and tumor suppressor gene inactivation leads to induction

Chemically Induced Cell Transformation

of morphological transformation, cellular immortality, anchorage-independent transformation, and neoplastic transformation.

Cross-References ▶ 5-aza-20 Deoxycytidine ▶ Aflatoxins ▶ Anchorage-Independent ▶ Aromatic Amine ▶ Benzpyrene ▶ Cancer ▶ Carcinogen Metabolism ▶ Carcinogenesis ▶ Cervical Cancers ▶ Chemical Carcinogenesis ▶ Class II Tumor Suppressor Genes ▶ DNA Damage ▶ Epigenetic ▶ Epithelium ▶ Epstein-Barr Virus ▶ Estrogenic Hormones ▶ Genetic Toxicology ▶ KRAS ▶ Methylation ▶ Mutation Rate ▶ Oncogene ▶ Polycyclic Aromatic Hydrocarbons ▶ Radiation Carcinogenesis ▶ Radiation Oncology ▶ Senescence and Immortalization ▶ SV40 ▶ Telomerase ▶ Tumor Suppressor Genes ▶ Virology

References Kumar V, Abbas AK, Fausto N (2005) Neoplasia, Chapter 7. In: Robbins and Cotran’s pathologic basis of disease, 7th edn. Elsevier Saunders, Philadelphia, pp 269–342 Landolph JR Jr (2006) Chemically induced morphological and neoplastic transformation in C3H/10T1/2 mouse embryo cells, Chapter 9. In: Warshawsky D, Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, pp 199–220

925 Pitot HC, Dragan YP (2001) Chemical carcinogenesis, Chapter 8. In: Klaassen CD (ed) Casarett and Doull’s toxicology, the basic science of poisons, 6th edn. McGraw-Hill, New York, pp 239–320 Verma R, Ramnath J, Clemens F et al (2005) Molecular biology of nickel carcinogenesis: identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Mol Cell Biochem 255:203–216 Weinberg RW (2007) Multi-step tumorigenesis, Chapter 11. In: The biology of cancer. Garland Science/Taylor and Francis Group, LLC, New York, pp 399–462

See Also (2012) Anchorage-independent cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 173. doi:10.1007/9783-642-16483-5_263 (2012) Carcinogen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 644. doi:10.1007/978-3-642-16483-5_839 (2012) Cellular senescence. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 743. doi:10.1007/978-3-642-16483-5_1019 (2012) Cytochrome P450 enzymes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1043. doi:10.1007/978-3-642-164835_1465 (2012) Contact inhibition of cell division. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 974. doi:10.1007/978-3-642-164835_1324 (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1291– 1292. doi:10.1007/978-3-642-16483-5_1958 (2012) Fibroblasts. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1398. doi:10.1007/978-3-642-16483-5_2176 (2012) Genotoxic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1540. doi:10.1007/978-3-642-16483-5_2393 (2012) Morphological cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2373. doi:10.1007/978-3-642-164835_3836 (2012) Mutagen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2409. doi:10.1007/978-3-642-16483-5_3907 (2012) Mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Neoplastic cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2474. doi:10.1007/978-3-642-164835_4013 (2012) Transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3757–3758. doi:10.1007/978-3-642-16483-5_5913

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Chemoattractant Cytokine ▶ Chemokines

Chemoattraction Jose Luis Rodríguez-Fernández Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Madrid, Spain

Synonyms Directed migration; Directed motility

Definition Chemoattraction is the process whereby a cell detects a chemical gradient of a ligand called chemoattractant and, as a consequence, gets oriented and subsequently moves in the direction from a low to a high concentration of the chemoattractant. Chemoattraction is controlled by specific chemoattractant receptors that are able to detect selectively these ligands. Chemoattraction is called chemotaxis or haptotaxis when the chemical gradient of the chemoattractant is presented to the cell either in a soluble or bound to a substrate form, respectively. As it is not clear which one of these two types of motile processes takes place in vivo, it is more appropriate to refer to these directional motile processes with the more general term of chemoattraction.

Characteristics Chemoattractants use specific chemoattractant receptors to guide different migratory cell types toward specific sites in the organism. These receptors, upon binding to the chemoattractant, transform the information of this ligand in intracellular signals that result in the movement of the migratory

Chemoattractant Cytokine

cell toward the positions where chemoattractant is present at high concentration. Therefore, the analysis, in a specific context, in one hand, of the type of chemoattractant receptors expressed by a certain migratory cell and, on the other hand, the position in the organism of the chemoattractants recognized by these receptors, allow to make predictions on the potential tissues where this cell can be attracted. Upon arrival to the position where the chemoattractant is at a high concentration, adhesive receptors may contribute to slow down (function largely performed by selectin adhesive receptors for cells in blood vessels) and eventually attach (cells use integrin receptors for this function in most cell types) the cells to these sites. Chemoattractants can be conveniently classified according to the type of receptor that they bind. In this regard, the first and the largest group include chemoattractants that bind members of the G protein-coupled receptor (GPCR) superfamily. In this first group is included the family of ▶ chemokines. A second group is formed by chemoattractants that bind tyrosine kinase receptors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF)). A third group includes ligands that bind receptors different of the two aforementioned families (e.g., laminin and fibronectin, which bind integrin receptors). This article deals mainly with the chemokines because they have been the chemoattractant family most studied in relation to ▶ cancer and ▶ metastasis. Chemokines Chemokines (chemotactic chemokines) are a family of peptides (60–100 amino acids (aa)) that includes some 50 members (Fig. 1). Based on the number and spacing of the conserved cysteine (C) residue in the N-terminus of the protein, chemokines are subdivided into four families (C, CC, CXC, CX3C), where X is any intervening amino acid between the cysteines. Chemokine receptors transmit intracellular signals that can control either chemoattraction or other functions (Fig. 1). The chemokine receptors (some 20 members) are included in the G protein-coupled receptor (GPCR) superfamily. They are classified based on the class of chemokines that they bind, i.e.,

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Common name IL-8 GCP-2 NAP-2 ENA-78 GROa GROb GROg IP-10 Mig I-TAC SDF-1a/b BCA-1 BRAK MCP-1 MCP-4 MCP-3 MCP-2 MIP-1b MIP-1a S MIP-1a P RANTES MPIF-1 HCC-1 HCC-2 HCC-4 Eotaxin-2 Eotaxin-3 Eotaxin TARC MDC MIP-3a ELC SLC I-309 TECK CTACK PARC Lymphotactin SCM-1b Fractalkine

Chemokine receptor

New name CXCL8 CXCL6 CXCL7 CXCL5 CXCL1 CXCL2 CXCL3 CXCL10 CXCL9 CXCL11 CXCL12 CXCL13 CXCL16 CXCL14 CCL2 CCL13 CCL7 CCL8 CCL4 CCL3 CCL3LI CCL5 CCL23 CCL14 CCL15 CCL16 CCL24 CCL26 CCL11 CCL17 CCL22 CCL20 CCL19 CCL21 CCL1 CCL25 CCL27 CCL18 XCL1 XCL2 CX3CL1

CXCR1 CXCR2

CXCR3 CXCR7 CXCR4 CXCR5 CXCR6 Unknow CCR2

CCR5

CCR1

CCR3

CCR4 CCR6 CCR7 CCR8 CCR9 CCR10 Unknown XCR1 CX3CR1

Chemoattraction, Fig. 1 Classical and new names of chemokines are included. Red identifies “inducible” or “inflammatory” chemokines, green “homeostatic” agonists, and yellow ligands belonging to both realms. BCA B cell-activating chemokine, BRAK breast and kidney chemokine, CTACK cutaneous T-cell attracting-chemokine, ELC Epstein-Barr virus-induced receptor ligand chemokine, ENA-78 epithelial cell-derived neutrophil-activating factor (78 amino acids), GCP granulocyte chemoattractant

receptors that bind to C, CC, CXC, and CX3C chemokines are called, respectively, CR, CCR, CXCR, and CX3CR receptors. Based largely on studies performed in the immune system, chemokines have been classified in three functional groups: homeostatic, inducible, and dual function (Fig. 1). The first group, which includes chemokines constitutively produced by “resting cells” in specific organs or in tissues inside these organs, controls homeostatic migratory processes that determinate the correct location of different cell types in the organism under normal conditions. The second group is inducible or inflammatory chemokines, which are secreted in different tissues in emergency situations and serve to attract to these places’ specialized cell types that contribute to the resolution of the emergency situation. The third group is formed by dual function chemokines, which can be either homeostatic or inducible depending on the context (Fig. 1). Although chemoattraction is the function most commonly regulated by chemokines, however, studies performed mainly on leukocytes have demonstrated that these peptides, acting through specific chemokine receptors, may control additional cellular functions, including proliferation, ▶ adhesion, ▶ motility, survival, or protease secretion, among other functions. By controlling these activities, chemokines may contribute to modulate the functions of leukocytes and other cell types. Chemokines and Cancer Cancer is a disease where cells have disrupted the mechanisms that regulate their normal growth ä

Chemokines

Chemoattraction, Fig. 1 (continued) protein, GRO growth-related oncogene, HCC human CC chemokine, IP IFN-inducible protein, I-TAC IFN-inducible T-cell a chemoattractant, MCP monocyte chemoattractant protein, MDC macrophage-derived chemokine, Mig monokine induced by gamma interferon, MIP macrophage inflammatory protein, MPIF myeloid progenitor inhibitory factor, NAP neutrophil-activating protein, PARC pulmonary and activation-regulated chemokine, RANTES regulated upon activation normal T cell expressed and secreted, SCM single C motif, SDF stromal cell-derived factor, SLC secondary lymphoid tissue chemokine, TARC thymus and activation-related chemokine, TECK thymus-expressed chemokine

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and, consequently, proliferate without control. This affliction becomes life threatening when cancer cells become metastatic, that is, they acquire the ability to leave their original sites of growth (primary tumor) and invade other tissues or organs where the uncontrolled growing cells can form new colonies (▶ metastasis) that can interfere with vital functions. The process leading to metastasis formation has been divided into several steps. In the first step, the cancer cells detach from the substrate and from the neighboring cells and escape from the primary tumors. The second step involves the penetration of the cancer cells into the blood or lymphatic vessels and their ▶ migration through these vessels. In the case of cells that migrate through the afferent lymphatics, they migrate first to the lymph nodes from where they can exit through the efferent lymphatics, eventually ending up in the blood vessels. In the third stage, cancer cells extravasate from blood vessels and home into new sites in the organism where new metastatic colonies can be formed. During these migratory processes, the cells undergo changes in their adhesive properties that are regulated by modulation of the activities and/or levels of integrin receptors. Moreover, cancer cells and/or associated stromal cells secrete proteases which, by degrading extracellular matrix (ECM) proteins of connective tissues, facilitate the moving of the cells and the ▶ invasion of other tissues. Finally, at the metastatic sites, the cancer cells attach and grow as secondary colonies. In addition, they may secrete chemokines and other soluble factors that induce new vascular vessel formation (▶ angiogenesis) and contribute to maintain the growth of the metastatic cells. Although millions of cells may be shed into the blood from primary tumors, however, only a reduced percentage of these cells are able to form metastases, suggesting that metastatic cells develop mechanisms that increase their survival in the face of a hostile environment. Chemoattraction: A Key Process to Attract Cancer Cells to New Biological Niches Since the work of Stephen Paget in the second half of the nineteenth century, it is known that metastatic cells do not move randomly, displaying in

Chemoattraction

contrast a marked tropism toward specific organs (Table 1). A variety of experimental data indicates that chemokines may play an important role in determining this bias of the metastatic cells. Analysis of the phenotype of multiple metastatic cell types shows that these cells express specific sets of chemokine receptors (Table 1). Furthermore, a clear correlation has been observed between the expression of a specific chemokine receptor by a metastatic cell and the presence of its respective ligands in the metastatic sites, suggesting the involvement of these receptors in the homing processes (Table 1). Finally, a direct role for chemokines and their receptors in the control of the tropism of metastatic cells is corroborated in studies that show that interference with the binding to the chemokine receptors impairs the ability to metastasize to specific organs. For instance, antibody neutralization of ▶ CXCR4 in breast cancer cells reduced the ability of these cells to form metastases in the lung, both upon intravenous injection and after orthotopic implantation of the cells. Conversely, overexpression of CCR7 in B16 melanoma resulted in a dramatic enhancement in the ability of these cells to form metastases in the draining lymph nodes upon intravenous injection of the cells in mice. From these studies it has also emerged that CCR7 and CXCR4 are the chemokine receptors most commonly expressed by metastatic cells. This finding contributes to explain the ability of multiple metastatic cell types that express these receptors to colonize the lymph node and other organs where CXCL12 (ligand for CXCR4 and CXCR7) and CCL19 and CCL21 (both ligands of CCR7) are expressed (Table 1). Premetastatic niche is the name given to the specific regions, whose formation is induced by soluble factors released by primary tumor cells, which eventually become colonized by distant metastatic cells from the primary tumors. It has been shown that chemokine expression may confer premetastatic niches the ability to attract metastatic cells from the distant primary tumor. In this regard, it has been shown that chemokines S100A8 and S100A9, expressed by myeloid and endothelial in premetastatic niches in the lung, are responsible of attracting incoming

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Chemoattraction, Table 1 Chemokine receptors involved in cancer metastases Chemokine/s receptor/s/ligand/ s CXCR3/CXCL9, CXCL10, CXCL11 CXCR4/ CXCL12

Site/s of metastases Lung, bone, lymph node Lung, bone, lymph node

C Breast, ovarian, prostate, glioma, pancreas, melanoma, esophageal, lung (small cell lung cancer), head and neck, bladder, colorectal, renal, stomach, astrocytoma, cervical cancer, squamous cell cancer, osteosarcoma, multiple myeloma, intraocular lymphoma, follicular center lymphoma, rhabdomyosarcoma, neuroblastoma, B-lineage acute lymphocytic leukemia, B-chronic lymphocytic leukemia, non-Hodgkin lymphoma, acute myeloid leukemia, thyroid cancer, acute lymphoblastic leukemia, chronic myelogenous leukemia Head and neck, chronic myelogenous leukemia

Chemoattraction, angiogenesis, survival, growth

Breast, cervical carcinoma, glioma, lymphoma, lung carcinoma

Adhesion, survival, growth

Skin

Cutaneous T-cell lymphoma

Chemoattraction

Lymph node

Breast, melanoma, lung (non-small cell lung cancer), head and neck, colorectal, stomach, chronic lymphocytic leukemia Melanoma, prostate

Chemoattraction

CXCR5/ CXCL13 CXCR7/ CXCL11, CXCL12 CCR4/CCL17, CCL22 CCR7/CCL19, CCL21

Lymph node Lymph node

CCR9/CCL25

Small intestine Skin

CCR10/CCL27

Cancer cell types Acute lymphoblastic leukemia, chronic myelogenous leukemia, colon, melanoma

Function/s regulated by chemokine receptor Chemoattraction

Melanoma, cutaneous T-cell lymphoma

Lewis lung carcinoma metastatic cells to these niches because neutralization of the chemokines with antibodies reduced the metastases in these areas. In sum, chemokine/chemokine receptor pairs are important factors that control the colonization of cancer cells to specific sites in the organism. Other Biological Effects of Chemokines on Cancer Cells Apart from Chemoattraction Chemokines may affect cancer not only by regulating chemoattraction but also by regulating other functions that control cancer progression. Chemokines Can Contribute to Regulate the Growth of Cancer Cells Uncontrolled growth is a hallmark of cancer cells. Considering that chemokines may control cell growth in different cell types, the effect of

Chemoattraction

Chemoattraction Chemoattraction, growth, survival

chemokines on the proliferation of cancer cells is not unexpected. The growth of tumor cells may be affected by chemokines that can be either released in an ▶ autocrine signaling fashion by the cancer cells or secreted by the stromal tissues associated to the cancer cells. As an example of the first case, it is known that CXCL1, CXCL2, CXCL3, and CXCL8, secreted as autocrine growth factors by melanoma, pancreatic, and liver cancer cells, regulate the proliferation of all these cell types. As an example of the second case, it has been reported that CXCL12, which is secreted in the lungs and lymph nodes, leads to the increase in the growth of glioma, ovarian, small cell lung, basal cell carcinoma, and renal cancer, all cancer cell types that colonize the aforementioned organs. The effects of chemokines on growth can be complex because, for instance, interference with CCR5 seems to increase the proliferation of xenografts

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of human breast cancer, suggesting that CCR5 inhibits the growth of this cancer cells. Chemokines Can Contribute to Regulate the Survival of Cancer Cells A reduced susceptibility to ▶ apoptosis, leading to a concomitant extended survival, is also an important factor to explain the uncontrolled growth and the ability of cancer cells to form metastases. Chemokines have been involved in regulating survival in leukocytes and other cells; therefore these ligands may potentially contribute to regulate the carcinogenic phenotype by modulating this function. Stimulation of melanoma B16 cells expressing CCR10 with its ligand CCL27 enhances the resistance of these cells to the apoptosis induced by stimulation of the death receptor CD95. These in vitro results are consistent with in vivo experiments that show that the neutralization of CCL27 ligand with antibodies results in the blocking of tumor cell formation. Also, stimulation of glioma cells with CXCL12 protects these cells from the apoptosis induced by serum deprivation. It has been shown that CXCR7, a novel second receptor for CXCL12, is expressed in a variety of cancer cells. It has been indicated that CXCR7 may regulate survival, growth, and adhesion. Thus, it is possible that CXCR7 may also contribute to control all these functions in cancer cells. Chemokines Can Contribute to Regulate the Adhesion to New Sites in Cancer Cells Migratory cancer cells experience changes in adhesion, including processes of attachment and detachment, as they move through the organism. Enhanced adhesion is particularly crucial at the final stages of cancer progression where these cells require attaching to the new metastatic sites. Stimulation of cancer cells with chemokines may change the adhesion of these cells either by increasing the activity of integrins or by inducing changes in the expression levels on the membrane of these receptors. As an example of the first case, it has been observed that stimulation of B16 melanoma cells with CXCL12 leads to an increase in the affinity of the b1 integrin by the ligand VCAM-1 both in in vitro and in in vivo

Chemoattraction

experiments. As an example of the second case, stimulation of prostate tumor cells with CXCL12 induces enhanced expression of the integrins a3 and b5. Chemokines Can Contribute to Control Protease Secretion in Cancer Cells Metalloproteins are largely responsible for ECM remodeling and play key roles in solid tumor cell invasion. In this regard, it has been shown that chemokines enhance in protease secretion in some cancer cell types. For instance, stimulation of myeloma cells with CXCL12 induces metalloproteinase secretion. Chemokines Can Contribute to Control Angiogenesis in Cancer Cells At metastatic sites cancer cells induce formation of new vessels (angiogenesis), which allow the nourishment of the metastatic colonies. Angiogenesis is a finely orchestrated process where endothelial cells proliferate, secrete proteases, change their adhesive properties, migrate, and, finally, differentiate into new vessels. Chemokines can act as positive or negative regulators of the angiogenesis in the tumor microenvironment. In this regard, the members of the CXC chemokine family play an important role during this process. The CXC family has been divided into two groups. The first group includes members that present the triplet glutamic acid-leucinearginine (ELR) before the first Cys (ELR+ CXC chemokines), and the second group includes the members that lack this three amino acids (ELR CXC chemokines). Although there are exceptions, by and large, ELR+ CXC chemokines (including CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) play pro-angiogenic roles, promoting vessel formation through the stimulation of the CXCR2 receptor. For instance, in human ovarian carcinoma, CXCL8 induces both angiogenesis and tumorigenesis. Furthermore, treatment of mice that bear CXCL8-producing non-small cell ▶ lung cancer cells with anti-CXCL8 antibodies blunted the growth of these tumors in the mice. Exceptions to the rule ELR+ CXC=angiogenic chemokines are the ELR+ CXC members CXCL1 and

Chemoattraction

CXCL2, which are angiostatic, i.e., they inhibit angiogenesis. ELR CXC chemokines, including CXCL9, CXCL10, and CXCL11, are generally angiostatic. For instance, CXCL9 and CXCL10 inhibit Burkitt lymphoma tumor formation probably by blocking blood vessel formation. An exception to the rule ELR CXC=angiostatic chemokine is CXCL12 that is angiogenic, as suggested by CXCL12 and CXCR4 KO mice that display cardiovascular development defects. It is believed that the angiogenic effects of CXCL12 are mediated by the vascular endothelial growth factor (VEGF) that is secreted by endothelial cells upon stimulation with CXCL12. The latter chemokine can be secreted in the tumor microenvironment by both the cancer cells and associated stromal cells. Finally, apart from CXC chemokines, other chemokines families may also regulate angiogenesis. In this regard, the CC chemokine CCL21 is angiostatic. In contrast, three CC family members (CCL1, CCL2, CCL11) and one CX3C family member (CX3CL1) can induce angiogenesis. All these chemokines, secreted inside the tumor, may potentially regulate the growth of the metastatic cells. Therapeutical Aspects The multiple points at which chemokines may regulate cancer progression make them attractive targets to develop anticancer drugs. Several strategies have been adopted to harness the power of chemokines against cancer, including the use of antibodies against the overexpressed chemokine receptors in the target cancer cells to induce apoptosis of these cells. One common strategy has been the development of inhibitors to block the binding of the chemokines to the receptors and consequently the function of these receptors. The fact that chemokine receptors are on the membrane and that much information is available on the sequences, both on the ligands and on the receptors, necessary for receptor-ligand binding have enabled the development of numerous peptide or small molecule inhibitors that interfere with chemokine function. Some of these inhibitors have been developed against CCR1, CCR5, CXCR7, and CXCR4. Most of these inhibitors

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relay on their ability to inhibit survival or angiogenesis in the target cells. As CXCR4 is one of the most broadly expressed chemokine receptor in cancer cells, at least six peptides or small molecule inhibitors of the function of CXCR4 have been developed and used in preclinical cancer models. CXCR4 is particularly interesting due to its pro-angiogenic functions. A variety of data indicate that the growth and persistence of tumors and their metastases depend on an active angiogenesis at the tumor sites. In this regard, interference with this process is a powerful strategy to inhibit tumor growth. Interference with CXCR4 has been used in several cancer models, including many of the cancers indicated in Table 1. Although peptide inhibitors of chemokine receptors may not have by itself tumoricidal affects, however, along with other strategies may be a powerful therapy against tumors. Summary and Final Conclusions Upon becoming carcinogenic and metastatic, a variety of cancer cells upregulate the expression of chemokine receptors. In this regard, the microenvironment conditions inside the tumors are also known to induce chemokine receptor expression in some cases. For instance, the low oxygen concentration (▶ hypoxia) inside a tumor induces CXCR4 expression which concomitantly leads to a more aggressive metastatic phenotype in cancer cells. Chemokine receptors endow cancer cells with “postal codes” that determine their migration to tissues where the ligands of these receptors are expressed and therefore are important for the metastatic ability of these cells. In addition, these receptors may confer or modulate cancer cells functions that, by regulating different steps in cancer progression, may contribute to the carcinogenic and metastatic phenotype of these cells. The case of the Kaposi sarcoma herpesvirus (KSHV), which induces cancer lesions similar to that of the Kaposi sarcoma, is a dramatic example that shows the important role that chemokines and their receptors may play in cancer. Interestingly, this virus encodes a constitutively active receptor that displays a high degree of sequence similarity to chemokine receptors CXCR1 and CXCR2 and which can even be further activated by the

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CXCR2 ligands CXCL1 and/or CXCL8. KSHV is also pro-angiogenic and induces survival effects in the cancer cells where it is expressed. Further supporting a causative role of CXCR2 in cancer, a constitutive form of CXCR2, can induce cell transformation in susceptible cell types.

Cross-References ▶ Adhesion ▶ Angiogenesis ▶ Apoptosis ▶ Autocrine Signaling ▶ Cancer ▶ Chemokine Receptor CXCR4 ▶ Chemokines ▶ G Proteins ▶ Hypoxia ▶ Invasion ▶ Lung Cancer ▶ Metastasis ▶ Migration ▶ Motility

Chemokine Receptor CXCR4 Heidelberg, p 1587. doi: 10.1007/978-3-642-164835_2294 (2012) Haptotaxis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1631. doi: 10.1007/978-3-642-16483-5_2565 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi: 10.1007/978-3-642-16483-5_3084 (2012) Orthotopic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2661. doi: 10.1007/978-3-642-16483-5_4264 (2012) Xenograft. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3967. doi: 10.1007/978-3-642-16483-5_6278

Chemokine Receptor CXCR4 Jonathan Blay Department of Pharmacology, Dalhousie University, Halifax, NS, Canada

Synonyms CD184; Fusin; Receptor for CXCL12; Receptor for stromal cell-derived factor-1 alpha; SDF-1a

References Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540–550 Ben-Baruch A (2006) The multifaceted roles of chemokines in malignancy. Cancer Metastasis Rev 25:357–371 Kakinuma T, Hwang ST (2006) Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol 79:639–651 Sánchez-Sánchez N, Riol-Blanco L, Rodríguez-Fernández JL (2006) The multiples personalities of the chemokine receptor CCR7 in dendritic cells. J Immunol 176:5153–5159 Zlotnik A (2006) Chemokines and cancer. Int J Cancer 119:2026–2029

See Also (2012) Chemotaxis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 793. doi: 10.1007/978-3-642-16483-5_1081 (2012) Glioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1557. doi: 10.1007/978-3-642-16483-5_2423 (2012) G-protein Couple Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin

Definition CXCR4 is a cell surface protein that acts as a receptor for the molecule CXCL12 (stromal cellderived factor-1 alpha, SDF-1a). CXCL12 is one of a class of signaling molecules called chemokines that regulate the movement and other activities of cells throughout the body. Although CXCL12 and CXCR4 play major roles in regulating stem cells and cells of the immune system, CXCR4 is also found on many cancer cells and plays a part in metastasis, spread of the cancer cells being influenced by tissue levels of CXCL12.

Characteristics Chemokines are a class of peptide mediators that play important roles in controlling cellular

Chemokine Receptor CXCR4

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homing and migration both in embryonic development and in the regulation of cell populations in the adult. There are at least 40 different chemokines that fall into four classes depending upon their peptide structure. The different classes are “C,” “CC,” “CXC,” and “CX3C” chemokines, for which characteristic sequence motifs involve residues of the amino acid cysteine (C) either in sequence or separated by one or three other amino acids (X or X3). The chemokines themselves are peptides that can exist freely in solution in biological fluids and act by binding to corresponding ▶ receptors. In the language of molecular interactions, a chemokine is therefore known as a ligand. Chemokines are denoted by the letter L within their name. CXCL12 is thus a ligand and a chemokine of the CXC class of chemokine mediators. The chemokine receptors are named according to the chemokine class of their binding partner (or ligand), with the letter “R” to designate their receptor status. CXCR4 is therefore a receptor. As for chemokines, the numbers serve to distinguish individual members of the overall family. The partnership between chemokine receptors and the chemokines is not monogamous, and some chemokine receptors may bind as many as ten Chemokine Receptor CXCR4, Fig. 1 The cellular signaling pathways of CXCR4. When the chemokine ligand CXCL12 binds to its receptor CXCR4, one or more of several pathways can be activated through initial links involving G proteins that associate with the receptor. These pathways, which are shown only in outline, involve a further network of interactions that eventually lead to a cellular response that may ensure cell growth, migration or survival

different chemokines. However, most receptors have between one and three distinct partners. With very few exceptions, these partnerships are within a particular chemokine class (e.g., CXCL chemokines bind selectively to certain CXCR receptors). At this point, the only chemokine factor known to bind to CXCR4 is CXCL12, although CXCL12 itself is able to bind to an alternate receptor (CXCR7, previously known as RDC-1) as well as to CXCR4. Chemokine receptors such as CXCR4 are seven-transmembrane, G protein-coupled receptors. The protein chain of CXCR4 therefore winds back and forth across the outer membrane of the cell so that it crosses the membrane a total of seven times. One end of the protein chain (the amino terminus) protrudes from the outside of the cell. This region of the protein, together with certain parts of the three extracellular loops, forms the binding domain for CXCL12. The part of the receptor that protrudes from the inner face of the membrane (composed of the carboxy-terminus and three intracellular loops) contains the characteristics that allow it to provoke a cascade of events within the cell (Fig. 1). These steps are initiated firstly by a linkage to one or more of a CXCL12

CXCR4 4 Exterior Cell membrane Interior G proteins

Phospholipase-Cγ

Inositol trisphosphate

Release of Ca2+

Diacylglycerol

Protein kinase C

Ras

Phosphatidylinositol 3-kinase

Raf

Phosphatidylinositol (3,4,5)-trisphosphate

MAPK

Protein kinase B/Akt

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small family of proteins that interact directly with the receptor, called ▶ G proteins (in this case primarily Gai and Gaq). G protein involvement leads to the activation of three major signaling pathways: (i) the phospholipase C-diacylglycerol/IP3 pathway, (ii) the Ras-RafMAP kinase pathway, and (iii) the PI3-kinase pathway. CXCR4 is a crucially important member of the chemokine receptor family. If CXCR4 or CXCL12 is absent during embryonic development, the organism is unable to survive. The key dependence on CXCL12 and CXCR4 reflects the importance of this signal/receptor pair in marshaling the correct formation of cells as tissues are formed from their more rudimentary cellular precursors in the embryo. The CXCL12-CXCR4 axis, as it is often called, is a central part of the normal development of the central nervous system (the brain itself) and the exquisitely organized tissue that replenishes the different cells of the blood through adult life (the hematopoietic system). In addition, CXCR4 and CXCL12 seem to play a particular role in the development of the gut, and their participation is important for the proper development of the blood vessel system that is required for efficient intestinal function in the adult. In adult organisms, CXCR4 and CXCL12 partly reprise their developmental role during tissue damage by participating in repair processes. Once the organism is fully formed, the most evident role for CXCR4 and CXCL12 in a normal individual is that of continued regulation of the hematopoietic system. This takes place mainly in the bone marrow, which acts as a reservoir for the ancestral cells (stem cells and other progenitor cells) that are needed for the continued production of various white cells (leukocytes) and other progeny that are required to ensure a proper defense against infection or injury or to deal with replacement and remodeling of damaged tissues. These stem cells – which need to be maintained safely by the body until required to respond – are located within the protected environment of the bone marrow and are supported and nourished by a specialized grouping of cells that together are referred to as the “microenvironmental niche.”

Chemokine Receptor CXCR4

These supporting cells or “stromal cells” secrete a number of factors that serve to nourish the stem cells and to keep them within a safe environment in their primitive and “resting” state. Notable among these factors is CXCL12 (the “stromal cell-derived factor”), which can bind to CXCR4 on the stem cells. The binding of CXCL12 to its receptor has several effects on cell behavior, but the principal outcome is to attract cells toward the source of CXCL12. In the case of stem cells in the bone marrow, this results in retention within the microenvironmental niche or directs migrant stem cells back to this location. This ability of the CXCL12:CXCR4 axis to direct cell movement is what underlies its key role in orchestrating tissue development and repair. The phenomenon can be demonstrated in experiments using isolated cells, such that cells that have the CXCR4 receptor can be induced to migrate through pores in an artificial filter in response to an upward concentration gradient of CXCL12 in the fluid. This is a cellular response known as chemotaxis, and CXCL12 is referred to as a chemoattractant. Unfortunately, this normal and very important process by which CXCL12 and CXCR4 assist directed cell movement has been subverted by cancer cells to assist the spread of a cancer or metastasis. Normal tissues that are not subject to inflammation or repair processes typically have very low levels of CXCR4. However, when cancers are formed the affected cells frequently experience a dramatic increase (“upregulation”) of CXCR4. This has been shown for the common adult cancers (carcinomas of the breast, colon, lung, prostate, cervix, etc.), which arise in the membranous linings (epithelia) of certain organs; but CXCR4 levels are also elevated in cancers arising in the bone (e.g., osteosarcoma), muscle (e.g., rhabdomyosarcoma), nervous tissue (e.g., glioblastoma), or white cells (various leukemias). This is such a consistent finding that in many cancers the level, or “expression,” of CXCR4 can be used as cancer biomarker. The levels of CXCR4 that are present on the cells give an indication of how the cancer is likely to behave in the future and what therapeutic steps might need to be considered. Levels are assessed using a technique

Chemokine Receptor CXCR4

called immunohistochemistry. In this approach very thin slices or “sections” – no more than 0.005 mm thick – are taken from the suspect tissue onto glass slides. Special protein reagents called antibodies are used that recognize any molecules of CXCR4 in the tissue, and additional steps in the process generate color wherever the antibody has bound. The resulting picture under a microscope tells the pathologist not only about the architecture of the tissue and the characteristics of the cells but whether or not they have high levels of CXCR4. High levels (expression) of CXCR4 are associated with cancer aggressiveness, a likelihood that the cancer will spread or metastasize and means that the outlook for the patient is likely to be poorer. The link between cancer aggressiveness/ metastasis exists because the CXCL12:CXCR4 axis has a similar role of “directing traffic” in cancer as it does in normal circumstances. In this situation it is the cancer cells that possess the receptor – CXCR4 – and have levels at the cell surface that are much greater than are found on their normal counterparts. The exact reasons for these elevated levels of the chemokine receptor are not fully understood. Undoubtedly the genetic changes that are characteristic of cancer cells lead to alterations in transcription of the CXCR4 gene Primary tumor e.g. colon or breast cancer

Increased levels of receptor (CXCR4) on tumor cell surface

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that may provide certain subpopulations with greater amounts of the CXCR4 protein, and these cells have a selective advantage. However, there are also indications that factors within the environment of the tumor can make the situation worse by stimulating the cell to make even more CXCR4. The hypoxic nature of tumor tissue causes an increase in CXCR4 gene transcription through a pathway involving ▶ hypoxiainducible factor 1 alpha (HIF-1a). Various smallmolecular-weight and polypeptide mediators have also been shown to enhance the cellular expression of this chemokine receptor. The cancer cells are therefore equipped to be attracted toward sources of CXCL12 and to be captured within environments that are high in concentrations of CXCL12. Thus, it is no coincidence that the tissues that are high in CXCL12 are also those in which cancers form secondary tumors or metastases. Such tissues include the lymph nodes – central filters in the system that drain fluid from all tissues – as well as the liver, lung, and bone marrow. CXCL12 is believed to be one of the major factors driving metastasis (Fig. 2). As a colorectal cancer develops in the large intestine, for example, and small groups of tumor cells are shed into the blood circulation and the lymphatic drainage, circulating cells will find Metastatic site e.g. lung, liver, bone

CXCL 12 in tissue of e.g. lung, liver, bone:

1. Stimulates entry of tumor cells into tissue 2. Stimulates cell division/ growth of the metastasis

Chemokine Receptor CXCR4, Fig. 2 How CXCR4 and CXCL12 work together to facilitate metastasis. Tumor cells have increased levels of the receptor at their cell surface. When the tumor grows sufficiently for the cancer cells to find their way into the bloodstream, some cells lodge in tissues (e.g., lungs, liver, and bone marrow) that

have high concentrations of CXCL12, the molecule for which CXCR4 is the receptor. CXCL12 both encourages the entry of cells into the tissue and promotes growth of the cell population, facilitating metastatic spread. Tissues that have low levels of CXCL12 are much less likely to accept metastases

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an attractive home as they encounter lymph nodes in the mesenteric fat around the intestinal wall, when they are delivered to the liver through the portal circulation or as they lodge in the capillary beds of the lung after traversing the systemic circulation. Conversely, they have a much reduced probability of taking up residence in sites such as the heart or skeletal (voluntary) muscle, which are low in CXCL12. In addition to being attracted and retained in tissues that have high concentrations of CXCL12, the CXCR4-bearing cancer cells may respond in other ways. Although this may not be the case for all cancers, in some types (e.g., carcinomas of the colon and prostate), there is evidence that once the cells have settled in to their new location, the presence of CXCL12 acting through CXCR4 also enhances their ability to grow and colonize the tissue. In this way, CXCL12 can also be regarded as a growth factor, alongside other polypeptide growth stimulators that participate in tumor expansion. One additional factor that makes CXCR4 of interest for many different clinicians and researchers is that it is one of the two major coreceptors by which the AIDS virus infects human cells. One of the proteins that is present within the outer surface of the HIV-1 virus, called gp120, binds to CXCR4, although at a slightly different site to CXCL12. When the virus binds to its major target (the CD4 protein) on susceptible cells, it requires a coreceptor in order to complete its cellular attack. This allows it to complete the molecular changes that allow it to infect the cell. Depending on the exact cell and viral type, the coreceptor may be CXCR4 or another chemokine receptor, CCR5. While the link with AIDS has limited direct relevance to most cancers, the two fields of research have synergized to extend our present understanding of CXCR4.

Chemokines

See Also (2012) Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 208. doi: 10.1007/978-3-642-16483-5_312 (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 408–409. doi: 10.1007/978-3-642-16483-5_6601 (2012) Chemotaxis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 793. doi: 10.1007/978-3-642-16483-5_1081 (2012) G-protein Couple Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1587. doi: 10.1007/978-3-642-164835_2294 (2012) Hematopoietic System. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1645. doi: 10.1007/978-3-642-16483-5_2621 (2012) Ligands. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2040. doi: 10.1007/978-3-642-16483-5_3352 (2012) Microenvironmental Niche. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi: 10.1007/978-3-642-164835_3721 (2012) Stromal Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3544. doi: 10.1007/978-3-642-16483-5_5535 (2012) Transcription. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3752. doi: 10.1007/978-3-642-16483-5_5899

Chemokines Lei Fang and Sam T. Hwang Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Synonyms Chemoattractant cytokine; Chemotactic cytokine

Definition Cross-References ▶ G Proteins ▶ Hypoxia-Inducible Factor-1 ▶ Receptors

Chemokines are a large group of small proteins that play multiple biological roles, including stimulating directional migration (chemotaxis) of leukocytes and tumor cells via their membranebound receptors.

Chemokines

The name comes from “chemotactic cytokines,” these small cytokines induce migration of diverse immune cells. The family of the chemokines is quite numerous, as are the chemokine receptors, and often there is “promiscuity,” in that a single chemokine can activate multiple receptors and multiple chemokines can activate a single receptor. These molecules direct trafficking of leucocytes. Two chemokine receptors are also the principal coreceptors for HIV involved in viral entry: CCR5, expressed on monocytes and macrophages as well as other cells, and the more widely expressed CXCR4. The tropism of specific chemokine receptors is associated with HIV clinical effects, with CCR5 linked to infection and CXCR4 tropism linked to progression to AIDS.

Characteristics Chemokines are divided into four subgroups (C, CC, CXC, and CX3C) based on the spacing of the key cysteine residues near the N terminus of these proteins. The CC and CXC families represent the majority of known chemokines. Chemokines signal through seven-transmembrane-domain receptors, which are coupled to heterotrimeric Gi-proteins. Activation of phospholipase C (PLC) and phosphatidylinositol-3-kinase g (PI3Kg) by bg subunits of ▶ G-proteins is well established. So far, approximately 50 chemokines and 18 chemokine receptors have been identified. Some chemokine receptors bind to multiple chemokines and vice versa, suggesting possible redundancies in chemokine functions. Chemokine receptors permit diverse cells to sense small changes in the gradient of soluble and extracellular matrix-bound chemokines, thus facilitating the directional migration of these cells toward higher relative concentrations of chemokines. While soluble chemoattractants can induce directional migration, chemokines (due to their net positive charges) will often be bound to and presented by negatively charged macromolecules such as endothelial cell-derived proteoglycans in vivo. Chemokine gradients bound to solid surfaces are

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capable of mediating haptotaxis of leukocytes and other cells. Chemokine receptor activation can also trigger conformational changes in membrane integrins, permitting strong cell–cell adhesion in the presence of appropriate integrin receptors. This signaling pathway is particularly relevant in triggering cellular integrins found on leukocytes and cancer cells to bind to their respective receptors (e.g., ICAM-1) on vascular endothelial cells, facilitating stable binding and spreading of cells to endothelium. The stable binding of metastatic tumor cells to vascular endothelial cells at distant sites of metastasis is likely to be a crucial early step in the process of ▶ metastasis. Circumstantial evidence supports the idea that tumor cells use chemokines to promote their own survival and metastasis through multiple mechanisms. For example, certain chemokines secreted by tumor cells contribute to tumor growth and ▶ angiogenesis. Members of chemokines that contain an ELR motif (Glu–Leu–Arg) act as angiogenic factors, which are chemotatic for endothelial cells in vitro and can stimulate in vivo. In contrast, members without an ELR motif inhibit angiogenesis. Chemokine-mediated tumor cell activation through cellular kinases such as PI3K, ▶ Akt signal transduction pathway in oncogenesis), and other downstream mediators (Fig. 1) influences tumor cell resistance to apoptotic death. For example, activation of the chemokine receptor CCR10 prevents Fas-mediated tumor cell death induced by cytolytic antigenspecific T cells. Selected chemokine receptors are upregulated in a large numbers of common human cancers, including breast, lung, prostate, colon, and melanoma. Chemokine receptors expressed on tumor cells coupled with chemokines preferentially expressed in a variety of organs are believed to play critical roles in cancer metastasis to vital organs as well as draining lymph nodes. CXCR4 is by far the most common chemokine receptor expressed on most cancers. In addition, CXCL12, the ligand for CXCR4, is highly expressed in lung, liver, bone marrow, and lymph nodes, which represent the common sites of metastasis of many cancers. Chemokine receptor expression

C

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Chemokines Chemokine

N terminus

Chemokine receptor

Plasma membrane

C terminus

Gα1

Gβγ

DAG IP3 Ca2+

PLC

PKC

Src PI3K P85/p110

PI3Kγ

PKB

on cancer cells may influence the conversion of small, clinically insignificant foci of cancer cells at metastatic sites to rapidly growing, clinically serious secondary tumors. Cancers that upregulate CCR7 expression also facilitate their entry into lymphatic vessels, which strongly express the CCR7 ligand (CCL21), and subsequent retention within CCL21-rich secondary lymphoid organs. Upregulation of chemokine receptors such as CCR7 may be a major reason for efficient lymph node metastasis observed in many epithelial cancers. Chemokines released by tumor cells have been shown to attract ▶ regulatory T cells, thus suppressing host responses to invasive tumors. Moreover, chemokine and their receptors are involved in ▶ dendritic cell maturation, B and T cell development, and T1 and T2 polarization of the T-cell response. These actions suggest the possibility that chemokines may play a role in altering the magnitude and polarity of host immune responses to cancer cells. Although individual chemokine and chemokine receptor appear to affect many aspects of cancer cell survival, migration, angiogenesis, and the host response to cancer cells, it is still unclear which of these functions predominate in the multistep establishment of primary tumors and secondary metastases.

PLC DAG IP3 PKC PI3Kγ Src PKB

phospholipase C Diacylglycerol inositol-1,4,5-triphosphate protein kinase C phosphatidylinositol-3 kinase γ Src-like kinase protein kinase B

Cross-References ▶ Akt Signal Transduction Pathway ▶ Angiogenesis ▶ Chemokine Receptor CXCR4 ▶ Dendritic Cells ▶ G Proteins ▶ Metastasis ▶ Regulatory T Cells ▶ Src

References Kakinuma T, Hwang ST (2006) Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol 79:639–651 Müller A, Homey B, Soto H et al (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50–56 Murphy PM (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacol Rev 54:227–229 Rossi D, Zlotnik A (2000) The biology of chemokines and their receptors. Annu Rev Immunol 18:217–242 Thelen M (2001) Dancing to the tune of chemokines. Nat Immunol 2:129–134

See Also (2012) Chemotaxis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 793. doi: 10.1007/978-3-642-16483-5_1081

Chemoprevention (2012) FAS. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1379. doi: 10.1007/978-3-642-16483-5_2121 (2012) Haptotaxis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1631. doi: 10.1007/978-3-642-16483-5_2565 (2012) Integrin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi: 10.1007/978-3-642-16483-5_3084 (2012) TH1 Cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3600. doi: 10.1007/978-3-642-16483-5_5647

Chemokinesis ▶ Motility

Chemoprevention Definition Chemoprevention involves the use, in healthy people, of natural or laboratory-made substances to prevent cancer or reduce cancer risk both in high-risk individuals and in the general population. The aim is to reduce the cancer burden in humans. Most work is being done to reduce the risk for ▶ oral cancer, prostate cancer (see “▶ Prostate Cancer Clinical Oncology”), ▶ cervical cancer, ▶ lung cancer, ▶ colorectal cancer, and ▶ breast cancer. The first chemopreventive agent to reach the clinic – and possibly the best known – was ▶ tamoxifen, which has been shown to cut breast cancer incidence in high-risk women by 50%. It was followed by finasteride, found to reduce prostate cancer (see “▶ Prostate Cancer Clinical Oncology”) incidence by 25% in men at high risk for the disease. However, the large-scale trials that confirmed these benefits brought to light a troublesome issue: the drugs caused serious side effects in some patients. This is an issue of particular concern when considering long-term administration of a drug to healthy people who may or may not develop cancer. Obviously, this is raising a number of ethical issues. An effective chemopreventive agent should not significantly alter

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quality of life and should be ideally inexpensive, safe, well tolerated, and effective in preventing more than one cancer. Experience with ▶ celecoxib (Celebrex) and other COX-2 inhibitors illustrates the importance of an assessment of the risk/benefit ratio for patients. COX-2 inhibitors have shown impressive efficacy in the prevention of colon cancer and several other forms of cancer, but they also increase the risk of serious cardiovascular side effects. Attention has focused on ▶ nutraceuticals and phytochemicals (see “▶ Phytochemicals in Cancer Prevention”) as chemopreventive agents. ▶ Curcumin (found in the curry spice turmeric) has shown dramatic anticancer results in preclinical studies owing to its significant anti-▶ inflammation properties. Curcumin has been used for thousands of years in the diets of people in the Middle and Far East and therefore is believed to have a low probability of serious side effects. Under investigation for their potential in breast cancer chemoprevention are aromatase inhibitors (see “▶ Aromatase and Its Inhibitors”), a class of estrogen blockers, which are approved to treat metastatic breast cancer in postmenopausal women. While the idea of cancer chemoprevention is extremely attractive, much research remains to be done to make this a generally applicable option for reducing the human cancer burden. An important element will be to identify informative biomarkers to assess individual cancer risk and to possibly provide information of patient’s tolerance toward individual chemopreventive agents.

Cross-References ▶ Aromatase and Its Inhibitors ▶ Breast Cancer ▶ Celecoxib ▶ Cervical Cancers ▶ Chemoprotectants ▶ Colorectal Cancer ▶ Curcumin ▶ Detoxification ▶ Inflammation

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▶ Lung Cancer ▶ Nutraceuticals ▶ Oral Cancer ▶ Photochemoprevention ▶ Phytochemicals in Cancer Prevention ▶ Prostate Cancer Clinical Oncology ▶ Tamoxifen

Chemoprotectants

serum enzyme levels, or induce significant injury to the tissues/organs. These chemoprotectants include anticancer, antitumor, anti angiogenic, and antioxidant compounds and are used as an adjuvant in cancer ▶ chemotherapy.

Characteristics See Also (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 408–409. doi:10.1007/978-3-642-16483-5_6601 (2012) Cyclooxygenase-2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1035. doi:10.1007/978-3-64216483-5_1435 (2012) Estrogens. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1333. doi:10.1007/978-3-642-16483-5_2019 (2012) Finasteride. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1407. doi:10.1007/978-3-642-16483-5_2191

Chemoprotectants Debasis Bagchi Department of Pharmacy Sciences, Creighton University Medical Center, Omaha, NE, USA

Synonyms Chemoprevention; Chemoprotection

Definition Chemoprotectants are natural or synthetic chemical compounds which exhibit the ability to ameliorate, mimic, or inhibit the toxic or adverse effects of structurally different chemotherapeutic agents, radiation therapy, cytotoxic drugs, or naturally occurring toxins, without compromising the anticancer or antitumor potential of the chemotherapeutic drugs. Chemoprotectants should not affect the therapeutic efficacy of the chemotherapeutic agents, radiation, or drugs, disrupt the

According to the World Health Organization (WHO), cancer accounts for 7.6 million (or 13%) of all deaths in 2005, and the incidence of cancer is expected to rise with an estimated 9 and 11.4 million deaths from cancer in 2015 and 2030, respectively. Cancer chemotherapy and radiation therapy are the most promising choice available for the cancer patients. The global outlook of cancer therapy has made dramatic improvement since the discovery of various synthetic and natural chemoprotectants which slow down the progress of this deadly disease and enhance the life span of the cancer patients. Chemoprotectants may exert toxic effects. Thus, it is very important to determine the right dosage and exposure scenario for each chemoprotectant prior to the exposure to demonstrate adequate safety. Synthetic Chemoprotectants Amifostine. A white powder, water-soluble organic thiophosphate compound, chemically known as 2-[(3-aminopropyl)amino]-ethanethiol dihydrogen phosphate (ester) or 2-(3-aminopropylamino)ethylsulfanyl phosphonic acid or aminopropylaminoethyl thiophosphate (Fig. 1a), and used as a cytoprotective adjuvant in cancer chemotherapy to reduce the incidence of ▶ neutropenia-related fever and infection caused by DNA-binding chemotherapeutic agents including cyclophosphamide and cisplatin. Amifostine (empirical formula C5H15N2O3PS; molecular weight 214.22; trade name Ethyol, synonyms: ethiofos, ethanethiol, gammaphos, WR2721, NSC-296961) is used to decrease the cumulative nephrotoxicity caused by cisplatin in patients with ovarian or lung cancer, as well as to reduce the incidence of moderate to severe xerostomia (dry mouth) in patients undergoing radiotherapy

Chemoprotectants

941 O NH O

HO

S

N H

P HO

a Amifostine

b

(R)-2-acetamido-3-mercaptopropanoic acid

SH H N

O

HO NH2

N H

O

HN

NH2

O

O

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O H N

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O HS OH

O

2-amino-5-{[2[(carboxymethyl)amino]1-(mercaptomethyl)-2-oxoethyl]amino}5-oxopentanoic acid

Na+

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O Dexrazoxane 4-[1-(3, 5-dioxopiperazin-1-yl) propan-2-yl] piperazine-2, 6-dione

d Mesna

Sodium-2-sulfanylethanesulfaonate

HS

e N-Acetylcysteine

(R)-2-acetamido-3mercaptopropanoic acid

Chemoprotectants, Fig. 1 Structures and IUPAC nomenclature of (a) amifostine, (b) dexazoxane, (c) glutathione, (d) mesna, and (e) N-acetylcysteine

for head and neck cancer. Amifostine is dephosphorylated by alkaline phosphatase in tissues to a pharmacologically active free thiol metabolite, which readily scavenge noxious reactive oxygen species (ROS) generated by exposure to either cisplatin or radiation, as well as detoxify reactive metabolites of platinum and other alkylating agents. Pharmacokinetic studies show that amifostine is rapidly cleared from the plasma with a distribution half-life of 3 g/day) are required for an adrenolytic effect. Although responses to mitotane alone may occur in 20–30% of cases, most responses are transient, and the prospect for long-term survival is uncertain. The antitumor effect of mitotane is influenced by its pharmacokinetics and by the duration of its therapeutic exposure. Serum concentration plateaus after 8–12 weeks of treatment and antitumor responses occur only when a serum concentration of at least 14 mg/mL is maintained for a prolonged period. The severe gastrointestinal (nausea, vomiting, diarrhea, and abdominal pain) and neurologic (somnolence, lethargy, ataxia,

Childhood Adrenocortical Carcinoma

depression, and vertigo) toxic effects of mitotane reduce patient adherence. Because mitotane is adrenolytic, all patients receiving this agent should be considered to have severe adrenal insufficiency and treated accordingly. ▶ Cisplatin-based regimens, usually including etoposide and doxorubicin, are used in combination with mitotane, although less than 40% of patients respond. The use of radiotherapy in pediatric ACT has not been consistently investigated, although ACT is generally considered to be radioresistant. Furthermore, because many children with ACT carry germline TP53 mutations that predispose them to cancer, radiation may increase the incidence of secondary tumors. For most patients with metastatic or recurrent disease that is unresponsive to mitotane and chemotherapy, repeated surgical resection is the only alternative. However, given the infiltrative nature of the disease, complete resection is difficult. Image-guided tumor ablation with radiofrequency currently offers a valid alternative for these patients. Prognosis Complete tumor resection is the single most important prognostic indicator. Patients who have distant or local with gross or microscopic residual disease after surgery have a dismal prognosis. Long-term survival (5 years or more after the diagnosis) is about 75% for children after complete tumor resection. Among those who undergo complete tumor resection, tumor size has prognostic value. The estimated event-free survival is 40% for those with tumors weighing more than 200 g and 80% for those with smaller tumors. Children whose tumors produce excess glucocorticoid appear to have a worse prognosis than children who have pure virilizing manifestations. Classification schemes or disease staging systems (Table 2) are still evolving. Prognosis will likely be further refined by adding other predictive factors, including those from gene expression studies. Concluding Remarks Adrenocortical tumors remain difficult to treat, and little progress has been made in developing effective chemotherapeutic regimens. The rarity of ACT hinders the opportunity to conduct

Childhood Cancer Childhood Adrenocortical Carcinoma, Table 2 Staging criteria for childhood adrenocortical tumor Stage I

II

III

IV

Description Tumor totally excised, tumor size A; W212X, incomplete protein). Known substrates for CYP2C19 are the proton pump inhibitors (omeprazole, pantoprazole), in which the treatment of individuals for Helicobacter pylori was found to be less effective in patients with two active alleles compared to patients having one or two deficient alleles. In cancer treatment, a correlation between CYP2C19 genotype and the activation of cyclophosphamide was demonstrated, thereby affecting survival, implicating potential use in cyclophosphamide therapy. Because of the involvement of CYP2C19 in the metabolism of thalidomide, pharmacogenetic analyses for this enzyme in the treatment of chronic myeloid leukemia or prostate cancer might be an interesting option.

CYP2D6

CYP2D6 activity displays a trimodal distribution in Caucasians, differentiating between poor metabolizers (PMs), extensive (normal) metabolizers (EMs), and ultrarapid metabolizers (UMs) (Fig. 4). Also, an intermediate metabolizer group can be distinguished, although phenotypically this group displays substantial overlap with the extensive metabolizers and is usually characterized on the genetic level. For CYP2D6, 5–10% of the Caucasian population is practically deficient, which was discovered using the probe drugs sparteine and debrisoquine. This deficiency is due to inheritance of two defective CYP2D6 alleles, the most common polymorphism being a G to A conversion on position 1846 of the CYP2D6 gene, leading to a RNA splicing defect, characteristic for the CYP2D6*4 variant allele. The second most prominent CYP2D6 allele is the *5 allele, in which the whole CYP2D6 gene is deleted. Over 50 variant alleles have been described until now for CYP2D6, which can be

Cytochrome P450

divided in those encoding no activity (null alleles), decreased activity (decreased function alleles), or normal activity (functional alleles). The frequency of variants in the population depends very much on ethnicity. The CYP2D6*17, for instance, is mainly found in Africans, while in Asians the decreased activity allele CYP2D6*10 is found much more frequent than in Caucasians. Patients with a CYP2D6 deficiency who are treated with tamoxifen for breast cancer showed decreased effectiveness of therapy, due to decreased activation. In addition to genetic deficient alleles, CYP2D6 can also be present as gene duplications in certain individuals, bringing the total number of CYP2D6 alleles to 3, or even higher. In Sweden, a family was identified that had 13 copies of the CYP2D6 gene. This gene duplication is thought to be a compensating mechanism invented by evolution to circumvent the poor inducibility of CYP2D6. The frequency of this gene duplication displays an interesting north–south gradient, with duplications being present in 1–2% of individuals in Sweden, 3.6% in Germany, 7–10% in Spain, 10% in Italy, 20% in Saudi Arabia, and 29% in Ethiopia. The frequency of poor metabolizers shows the reverse, with 1–2% of poor metabolizers in Ethiopia to 5–10% in Northern Europe (Fig. 5). This gene duplication is accompanied by an increased activity and leads to an ultrarapid metabolizer phenotype. Individuals having these gene duplications may experience severe toxic effects with codeine, which is converted in the liver to morphine by CYP2D6; fatalities have been documented where the breast milk of a mother having codeine contained extreme high levels of morphine, leading to fatal morphine exposure in the child. This high morphine concentration in the milk was a result of the mother being a CYP2D6 ultrarapid metabolizer. In the treatment of cancer, CYP2D6 also plays a minor role in the metabolism of the new anticancer agent imatinib, but thus far the clinical implications of being a poor metabolizer for this kind of therapy are not known. CYP3A4

This cytochrome is regarded as the most important enzyme and is involved in the metabolism of over

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50% of commonly described drugs. It is highly susceptible to induction and inhibition, with a resulting large interindividual variability in activity. Although extensive research has been done identifying genetic polymorphisms in this enzyme, most SNPs and the 20 variant alleles described until now proved to have low frequencies ( G), which has an allele frequency of 2–9% in Caucasians, 35–67% in blacks, but was rarely found in Asians. In vitro experiments demonstrated a slightly higher transcription, and thus CYP3A4 activity, but the clinical consequences of this are not clear yet. Interestingly, this CYP3A4 polymorphism was shown to be linked genetically to a polymorphism in the CYP3A5 gene. It was suggested that effects, attributes to the CYP3A4 polymorphism, were, in fact, due to the CYP3A5 polymorphism. However, several studies have shown correlations with the CYP3A4*1B allele without any correlation with CYP3A5 variant alleles. In study on cyclophosphamide, a decreased metabolism and a decreased median survival in breast cancer patients were demonstrated having the CYP3A4*1B allele. Another variant, the CYP3A4*16B (554C > G; Thr185Ser) allele, was indirectly shown by the use of tagging SNPs to be correlated with a 20% decrease in paclitaxel metabolism in Japanese patients, a population in which the allele frequency of this variant was found to be 1.7%. CYP3A5

For a long time, the interindividual expression of CYP3A5 was poorly understood. However, its expression in only 20% of Caucasians appeared to be caused by a highly frequent genetic polymorphism, 6986A > G, which is characteristic of the CYP3A5*3 allele, and causes aberrant splicing. About 80% of Caucasians, but only 30% of the African American population, are deficient for this enzyme, most of them having the CYP3A5*3/*3 genotype. Because of the substantial substrate overlap with CYP3A4, it is not always clear to what extent CYP3A5 contributes to the metabolism of certain drugs. This polymorphism does have an impact in the metabolism of

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Cytochrome P450

Cytochrome P450, Fig. 5 Geographical variation in frequency of CYP2D6 ultrarapid metabolizers (UMs) and poor metabolizers (PMs)

the immunosuppressive drug tacrolimus, which was slower in CYP3A5*3/*3 individuals. Cancer Incidence A number of the detoxifying CYP450 enzymes have been associated with cancer incidence,

mostly because of their involvement in the conversion of procarcinogens to carcinogenic metabolites (Table 1). The xenobiotic benzo(a)pyrene, for instance, is a common environmental pollutant produced from the burning of coal, from the combustion of tobacco products, from food barbeque

Cytochrome P450

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Cytochrome P450, Table 1 Correlations described for cytochrome P450 enzymes and the incidence of various cancers, either directly or in combination with environmental exposure 1A1 Bladder Breast Colorectal Dermatologic Gastric Gynecological Head and neck Hematologic Hepatocellular Lung Esophageal Prostate Renal

• • • •

1A2

2A6



1B1

2C9



2C19 •

2D6







• •

3A4

3A5

17

19







C

• • •

• •

2E1

• •



• •

• • •

on charcoal briquettes, and from industrial processing. It is a weak carcinogen, but is converted to a potent carcinogen by CYP1A1, CYP1A2, and CYP1B1, making that variable activities in these enzymes correlate with cancer incidence. CYP1A1 alleles have been implicated in lung, gastric, colorectal, hepatocellular, breast, prostate, and dermatologic cancer, while CYP1B1 variant alleles correlated with colorectal, hepatocellular, prostate, head and neck, renal, and gynecological cancers. For CYP3A4, the CYP3A4*1B allele was discovered because it correlated with a more aggressive form of prostate cancer. In nonaggressive bladder cancer, a lower frequency of CYP2C19 poor metabolizers is found, while, in contrast, for squamous cell carcinoma, a higher frequency of CYP2C19 poor metabolizers was apparent. Also correlations with lung cancer and colorectal cancer have been reported, while CYP2C9 variant alleles showed correlation with colorectal cancer. CYP2E1 polymorphisms were associated with an increased risk of lung, esophageal, head and neck, gastric, colorectal, and hepatocellular cancer, while CYP2D6 poor metabolizers appeared to be relatively protected for hepatocellular carcinoma but were at some increased risk for prostate cancer in patients who were using tobacco. Although not all mechanisms behind the correlation between variant CYP alleles and cancer incidence are known, it is obvious from the described observations that CYP450

• • •





• • •





enzymes not only play a role in the treatment of cancer but may also be involved in the development of cancer.

Cross-References ▶ Docetaxel ▶ Paclitaxel ▶ Tamoxifen

References http://www.cypallele.ki.se Ingelman-Sundberg M, Oscarson M, McLellan R (1999) Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharmacol Sci 20:342–349 Nelson DR, Koyman L, Kamataki T et al (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1–42 Solus JF, Arietta BJ, Harris JR et al (2004) Genetic variation in eleven phase I drug metabolism genes in an ethnically diverse population. Pharmacogenomics 5:895–931 van Schaik RHN (2005) Cancer treatment and pharmacogenetics of cytochrome P450 enzymes. Invest New Drugs 23:513–522

See Also (2012) Single nucleotide polymorphism. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3412. doi:10.1007/978-3-64216483-5_5316

1290 (2012) SNP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3460. doi:10.1007/978-3-642-16483-5_5395 (2012) Variant allele. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3885. doi:10.1007/978-3-642-16483-5_6152 (2012) Wild-type. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3947. doi:10.1007/978-3-642-16483-5_6247 (2012) Pharmacogenetics. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2840. doi:10.1007/978-3-642-16483-5_4496

Cytokine Definition Cytokine is any of a variety of secreted polypeptides that control the development, differentiation, and proliferation of hematopoietic cells. The effects of cytokines on lymphocytes are usually mediated through membrane-bound cytokine receptors and are especially critical during immune responses. A group of proteins mainly functioning as soluble signal transmitters in the immune system are primarily released by immune cells, but many other cell types can also release cytokines. Cytokines direct and mediate various functions of the adaptive and innate immunity and modulate functions of immune cells and other responsive cell types. Their local of systemic effects, elicited by binding to specific cell surface receptors, contribute mainly to innate and adaptive immune responses, so that their production is involved in a range of infectious, immunological, and inflammatory diseases. To this class belong interleukins, interferons, TNF-like molecules, etc. These molecular signals are similar to hormones and neurotransmitters and are used to allow one cell to communicate with another.

Cytokine

Cytokine Receptor ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy

Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy Koji Kawakami Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Kyoto, Japan

Synonyms Cancer vaccines; Cytokine receptor; Immunotherapy; Immunotoxins

Definition Cancer ▶ immunotherapy is the treatment of cancer by improving the ability of a tumor-bearing individual to reject the tumor immunologically. In the case of immunotherapy targeting cytokine receptors, delivery of tumor antigen proteins induces immune response against cancer cells bearing such tumor antigen to eliminate them. Cytokine receptor-targeting immunotoxins are proteins containing a bacterial toxin or chemical compound along with an antibody or a ligand that binds specifically to its target receptor. Immunotoxins then internalize through the receptors and achieve cytotoxic effect derived from the toxin moiety.

Cross-References Characteristics ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶ Signal Transducers and Activators of Transcription in Oncogenesis

When cancer cells are generated in a body, a variety of mechanisms cooperate in order to kill the cancer cells. Among these mechanisms, the

Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy

immune system has an important role to eliminate tumor tissues. First, immunocytes like ▶ macrophages and natural killer (NK) cells are activated to attack the newly formed foreign body. If they were unable to completely remove the cancer cells, T-cells and B-cells are the next players to fight against cancer. Many scientists have tried to find how the natural immune system works in order to control and to beat cancer. As cancer cells are known to express specific antigens or cytokine receptors on their cell surface, these molecules may be utilized as a target for tumor immunotherapy and immunotoxin therapy. Cytokine Receptor as the Target for Immunotherapy Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors. For example, administration of interferon can activate the systemic immunity. Therapeutic cancer vaccine is also included in this category, which is designed to activate the host immune system against tumor cells. Cancer vaccines take advantage of the fact that certain molecules on the surface of cancer cells are either unique or more abundant than those found on normal or noncancerous cells. These molecules act as antigens, stimulating the immune system to evoke a specific immune response. There are few licensed therapeutic vaccines to date. However, several cancer vaccines are in large-scale ▶ clinical trials. The Her2/neu (c-erbB2), the target protein of ▶ Herceptin, which is the world’s first therapeutic antibody to treat ▶ breast cancer, has long been studied as a target of cancer immunotherapy. The immunization methodology is variable, including administration of plasmid DNA, recombinant protein, or intracellular domain (ICD) peptide; virus vector delivery; dendritic cell pulse therapy; and combination therapy with adjuvant like granulocyte-macrophage colony stimulating factor (GM-CSF) or with anticancer reagents. Her2/ neu immunotherapy has been tested in Phase II clinical trials to treat breast cancer, and in Phase I for various cancer types including ▶ ovarian cancer, ▶ prostate cancer, and ▶ nonsmall-cell lung carcinoma. In the Phase II trial contributed by Washington University, the ICD of HER-2

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vaccine immunizes breast cancer patients with CD4 + helper T epitopes derived from the HER-2 protein. During ▶ preclinical testing, the rat neu peptide vaccine is effective because it circumvents tolerance to rat neu protein and generates rat neu-specific immunity. In Phase I study, patients underwent intradermal immunization once a month for a total of six immunizations with GM-CSF as an adjuvant. The endpoint of the study was to evaluate the toxicity and both the cellular and humoral HER-2/neu-specific immunity when the vaccinations were completed. The majority of patients (24 of 27 patients, 89%) developed HER-2/neu ICD-specific T-cell immunity. Out of 27 patients, 22 patients (82%) also developed HER-2/neu-specific IgG antibody immunity, and over half of the assessable patients retained HER-2/ neu-specific T-cell immunity 9–12 months after the completion of immunizations. ▶ Vascular endothelial growth factor receptor2 (VEGFR2) is highly expressed in neovascular endothelial cells in a tumor tissue. The epitope peptides of VEGFR2 were identified, and stimulation using these peptides induces CTLs with potent cytotoxicity in the ▶ HLA class I-restricted fashion against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR2. In A2/Kb transgenic mice expressing a1 and a2 domains of human HLA-A*0201, vaccination using these epitope peptides in vivo was associated with significant suppression of the tumor growth and prolongation of the animal survival. A clinical trial has been initiated to verify its effectiveness on breast and ▶ gastric cancer at University of Tokyo, Japan. Another approach attempts to avoid immunologic tolerance to cancer vaccines. Cancer immunotherapy utilizes the host immune system, and tolerance is one of the major causes weakening vaccine efficiency. If immunologic tolerance could be controlled, it is expected to enhance the effect of a cancer vaccine. There are a variety of other strategies which have shown antitumor activity in mouse model of cancer, e.g., dendritic cells pulsed with EphA2 (ephrin-A2, an angiogenic factor) epitope peptide, KLH-bound EGF receptor variant III peptide, VEGFR1 epitope peptide, and plasmid DNA of IL-13 receptor a2.

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Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy

Cytokine Receptor as the Target for Immunotoxin Therapy Immunotoxins are protein toxins connected to a cell binding ligand or antibody. Classically, immunotoxins were created by chemically conjugating an antibody to a whole protein toxin, or, for more selective activity, by using a protein toxin devoid of its natural binding domain. Immunologic proteins that are smaller than monoclonal antibodies (MAbs), like growth factors and ▶ cytokines, have also been chemically conjugated and genetically fused to protein toxins. Targeted cancer therapy such as immunotoxin therapy which targets tumor-specific cell surface receptors is one of the most effective strategies against cancer. The targeted agents require a threshold level of receptor expression on the cancer cells to achieve their antitumor activity. At present, only one agent targeting cytokine receptor, which contains human interleukin (IL)-2 and truncated diphtheria toxin (ONTAK), is approved for use in cutaneous T-cell lymphoma (CTCL). ONTAK, or denileukin diftitox, is a fusion protein designed to direct the cytocidal action of diphtheria toxin to cells which express the IL-2 receptor (IL-2R). Among three forms of IL-2R, the high affinity form consisting of CD25/ CD122/CD132 subunits is usually found only on activated hemocytes as T lymphocytes, B lymphocytes, and macrophages. A Phase III randomized, double-blind clinical trial was conducted in 71 patients with recurrent or persistent Stage Ib to IVa CTCL whose malignant cells express the CD25 component of the IL-2R. Administered with 9 or 18 mg/kg/day of ONTAK as an intravenous infusion daily for 5 days every 3 weeks (median: 6 courses), 7 patients (10%) achieved a complete response and 14 patients (20%) achieved a partial response. In 1999, the US FDA approved ONTAK indicated for the treatment of patients with persistent or recurrent CTCL whose malignant cells express CD25 component of the IL-2R. In the case of the IL-13 receptor (IL-13R) system, these receptors are constitutively overexpressed on a variety of human solid cancer cells including renal cell carcinoma, glioma, AIDS-associated ▶ Kaposi sarcoma, head and

neck cancer, ovarian cancer, and prostate cancer. To target IL-13R, a recombinant fusion IL-13 cytotoxin termed IL13-PE38QQR, or cintredekin besudotox, has been developed that is composed of IL-13 and a mutated form of Pseudomonas exotoxin. In early-phase studies, cintredekin besudotox was administered via intraparenchymal ▶ convection-enhanced delivery (CED) after resection of supratentorial recurrent malignant glioma. CED is a novel approach for the delivery of small and large molecules in solid tissues, utilizing a pressure gradient to distribute macromolecules to clinically significant volumes of tissue by bulk flow. The CED of cintredekin besudotox was fairly well tolerated, with a reasonable benefit/risk profile for treatment of patients with glioma. It has received orphan drug designation in Europe and the USA as well as fast-track drug development program status from the FDA (Fig. 1). The ▶ interleukin-4 receptor (IL-4R), which is related to the IL-13R, is also expressed by a variety of solid tumors and ▶ hematologic malignancies. The IL-4 cytotoxin, IL4(38–37)PE38KDEL, which is composed of circular permuted IL-4 and a mutated form of Pseudomonas exotoxin, is highly active in killing IL-4Rexpressing tumor cells in vitro and in vivo. In a Phase I/II trial of 31 glioma patients, tumor necrosis was observed in 71% of patients, and one patient experienced long-term survival. The Phase II intratumoral study had been completed, but further development has stalled because of severe adverse events as infusion concentration of drug was possibly too high. TransMID™, a modified diphtheria toxin conjugated to transferrin, is currently in Phase III clinical trials indicated for the treatment of glioblastoma multiforme. Transferrin receptors are particularly prevalent on rapidly dividing cells, and the high level of transferrin receptor expression on glioma cells makes it an ideal target for brain cancer. Phase I and Phase II clinical trials for TransMID™ have been successfully completed in patients suffering from inoperable, recurrent high grade gliomas who have failed to all other forms of treatment. In Phase II study, a reduction in tumor size of 50% or more was noted in 35% of evaluable patients, with a corresponding increase

Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy, Fig. 1 Model for cytokine receptor-targeted cancer therapy. In this model, IL-13R expressing cancer cells are treated with IL-13 immunotoxin. When IL-13 moiety of this immunotoxin binds to IL-13 receptor, the receptor–immunotoxin complex immediately internalizes into cytosol. Then domain II of Pseudomonas exotoxin (PE) is degraded in endosome and domain III of PE shows cytotoxic effect through its irreversible inhibition of eF2

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Pseudomonas Exotoxin (PE)

III II

IL-13

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in life expectancy in those patients that did respond. In this study, median survival for patients receiving TransMID™ was ~37 weeks. TransMID™ received fast-track status from the FDA, and orphan drug designation in the USA, Europe, and Japan. TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-a and a genetically engineered form of the Pseudomonas exotoxin, PE38. A Phase I trial was conducted to define the maximum tolerated dose (MTD) and dose limiting toxicity of TP-38 delivered by CED in patients with recurrent malignant brain tumors. The Phase II studies, in which TP-38 was administered to patients with recurrent glioblastoma using CED, have shown initial encouraging results. DT388GMCSF has been developed for the treatment of ▶ acute myeloid leukemia (AML). This molecule is composed of amino acids 1–388 of diphtheria toxin (DT), a histidine–methionine linker, and amino acids 1–124 of human GM-CSF. Phase I clinical trial has been completed in patients with AML, and 4 of 37 patients showed clinical remission of disease.

Another unique DT-based approach is VEGF121-DT385 and VEGF165-DT385 immunotoxins, which are a chemical conjugate containing VEGF165, VEGF121, and truncated DT. ▶ Vascular endothelial growth factor (VEGF) is the most critical inducer of blood vessel formation. In vivo animal studies have shown that these molecules are able to inhibit angiogenesis and tumor growth.

Cross-References ▶ Acute Myeloid Leukemia ▶ Breast Cancer ▶ Clinical Trial ▶ Convection-Enhanced Delivery ▶ Cytokine ▶ Gastric Cancer ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Herceptin ▶ HLA Class I ▶ Immunotherapy ▶ Interleukin-4

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▶ Kaposi Sarcoma ▶ Macrophages ▶ Non-Small-Cell Lung Cancer ▶ Ovarian Cancer ▶ Preclinical Testing ▶ Prostate Cancer ▶ Vascular Endothelial Growth Factor

References Kawakami K, Nakajima O, Morishita R et al (2006) Targeted anticancer immunotoxins and cytotoxic agents with direct killing moieties. Scientific World J 6:781–790 Pastan I, Hassan R, Fitzgerald DJ et al (2006) Immunotoxin therapy of cancer. Nat Rev Cancer 6:559–565 Schuster M, Nechansky A, Kircheis R (2006) Cancer immunotherapy. Biotechnol J 1:138–147

See Also (2012) CTL. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1012. doi:10.1007/978-3-642-16483-5_1406 (2012) Cytokine Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1052. doi:10.1007/978-3-642-16483-5_1475 (2012) Diphtheria Toxin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1124. doi:10.1007/978-3-642-16483-5_1636 (2012) Pseudomonas Exotoxin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3112. doi:10.1007/978-3-642-16483-5_4841 (2012) Renal Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 32253226. doi:10.1007/978-3-642-16483-5_6575

Cytokine Toxin Fusions or Conjugates ▶ Immunotoxins

Cytoplasmic Scaffolding Apoptotic Protease Activating Factor ▶ APAF-1 Signaling

Cytokine Toxin Fusions or Conjugates

Cytoskeleton Francisco Rivero1 and Huajiang Xiong2 1 Centre for Cardiovascular and Metabolic Research, The Hull York Medical School, University of Hull, Hull, UK 2 Department of Zoophysiology, Zoological Institute, Christian-Albrechts-University of Kiel, Kiel, Germany

Definition The cytoskeleton is a complex network of interconnected filaments and tubules that extends mainly throughout the cytosol, reaching from the nuclear envelope to the inner surface of the plasma membrane. It gives shape to the cell, mediates anchoring to the substrate and to other cells, facilitates cell movements and movement of organelles, and is necessary for cell division. Although mainly cytosolic, some cytoskeleton components play roles within the nucleus. Typical eukaryotic cells possess three cytoskeleton systems (Fig. 1) that can be distinguished on the basis of their diameter: microfilaments (MFs) (or actin filaments), intermediate filaments (IFs), and microtubules (MTs).

Characteristics All three cytoskeleton systems are filamentous polymers built from small subunits. Contrary to what its name may suggest, the cytoskeleton is a highly dynamic structure: filaments grow and shrink, resulting in continual remodeling. This constant assembly and disassembly is subject to regulation in response to both intracellular and environmental cues. Moreover, none of the three cytosketon systems exists in isolation: they are connected to each other and to other cellular components (nucleus, organelles, plasma membrane). Numerous classes of associated proteins are responsible for the dynamic properties and the connectivity of the cytoskeleton. They also constitute the targets of signaling networks that control the spatial

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Cytoskeleton, Fig. 1 Schematic depiction of the composition of the three cytoskeleton systems. Microfilaments (actin filaments) are composed of filamentous (F-) actin, linear polymers of globular (G-) actin. Each actin monomer binds one ATP nucleotide (depicted as yellow spheres). ATP hydrolyses when the filament ages (depicted as grey spheres). Actin filaments are polar, with a fast growing (plus or barbed) end and a slow growing (minus or pointed) end. Microtubules are hollow tubules built from stable ab-tubulin heterodimers. Both tubulins bind a GTP nucleotide, but only the one bound to b-tubulin (depicted as red

spheres) can be hydrolyzed and exchanged. GTP hydrolyzes when the microtubule ages (depicted as grey spheres). Microtubules are polar, with a fast growing (plus) end and a slow growing (minus) end. Intermediate filament proteins form parallel dimers that then assemble to staggered antiparallel tetramers, the basic subunits. Eight tetramers associate laterally into unit-length filaments (ULF) that can extend from both ends (intermediate filaments lack polarity). The resulting filaments compress radially (not depicted) into mature 10 nm intermediate filaments

and temporal remodeling of the cytoskeleton. Figure 2 depicts schematically the three cytoskeleton systems and some common specialized assemblies of cytoskeleton-associated components.

exist that are similar in overall structure to actin and play distinct roles. In its monomeric form, actin is referred to as G-actin (G stands for globular). The monomers polymerize to yield F-actin (F stands for filamentous). Most cells contain almost equal proportions of G- and F-actin. Actin monomers polymerize end-to-end, giving rise to a polar filament with a fast growing (plus or “barbed”) and a slow growing (minus or “pointed”) end. Each actin monomer binds one ATP nucleotide and it is in its ATP-bound form that actin polymerizes preferentially. Hydrolysis of ATP destabilizes the filament at the minus end and facilitates depolymerization. MFs contribute to developing and maintaining cell shape and attachment to other cells and to the

Microfilaments (Actin Filaments) Microfilaments are about 8 nm in diameter and constitute the smallest of the three cytoskeleton polymers (Fig. 1). They are linear polymers of actin, a 42 kDa globular protein. Actin is the most abundant protein (1–5% of the total weight of nonmuscle cells and up to 20% in muscle). Actins are encoded by six genes and are distributed in three classes, a-actin predominantly in muscle cells and b and g-actin in nonmuscle cells. Several actin-related proteins (Arps) also

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Cytoskeleton

Cytoskeleton, Fig. 2 Schematic of the three cytoskeleton systems and some specialized assemblies. The hypothetical cell depicted here displays features of an epithelial cell on the right half and of a mesenchymal cell on the left side. Examples of microtubule and microfilamentassociated motor proteins carrying a vesicle are depicted.

Examples of components of LINC complexes (SUN and KASH proteins) that attach the nuclear lamina to the cytoskeleton are also provided. See text for details. ONM, outer nuclear membrane; INM, inner nuclear membrane; ER, endoplasmic reticulum

extracellular matrix and are essential for cell movements (cell migration, muscle contraction), movement of organelles, and completion of cell division. With the help of actin-binding proteins (ABPs), actin filaments are organized in networks, bundles, and complex structures. Dozens of ABPs have been described and have been classified according to functional or structural criteria, although many display more than one activity on actin. Some examples will be considered. Several ABPs control remodeling of MFs by affecting their formation, stabilization or disassembly and constitute critical elements in signaling cascades that precisely modulate where and when actin remodeling takes place. Many ABPs are regulated by Ca2+ ions, binding to phospholipids (in particular phosphoinositides) or phosphorylation. A prominent role in the regulation of

the actin cytoskeleton is played by small GTPases of the Rho family. De novo formation of MFs is an unfavorable process if left to occur spontaneously. This situation is overcome in vivo by nucleation factors that stabilize the formation of the so-called nuclei or seeds (dimeric and trimeric complexes) that then elongate rapidly. There are three classes of nucleation factors, the Arp2/3 complex, formins, and spire proteins. The Arp2/3 complex produces branched filaments that form dendritic networks. This complex requires activation by proteins like ▶ cortactin and members of the WASP/Scar family that are themselves ABPs. Formins and spire, by contrast, produce filaments that are not branched. Monomeric ABPs like thymosin b4 and profilin sequester G-actin and maintain a pool of monomeric actin. Profilin also promotes

Cytoskeleton

actin polymerization by facilitating the exchange of ADP by ATP and binding to nucleation factors, ensuring a supply of fresh monomers to growing filaments. ▶ Gelsolin and cofilin/actin depolymerizing factor control the length of filaments by severing them and blocking the plus end or promoting the dissociation of actin monomers at the minus end. Capping proteins bind to and stabilize the end of the filament. CapG and the heterodimeric protein CapZ bind to the plus end and prevent elongation of the filament. Tropomodulin, on the contrary, caps the minus end and prevents the loss of actin subunits. Proteins like tropomyosin and nebulin bind along the actin filament and stabilize it. Myosins are motor proteins that move along MFs using the energy provided by ATP hydrolysis. All myosins consist of a conserved motor domain that binds one or more light chains with regulatory function, and a variable tail. Many myosins have been described and have been distributed into classes on the basis of sequence similarities of the motor domain. The best know myosin is myosin II, which in muscle cells makes up the thick filaments of the sarcomere and powers muscle contraction. Other myosins are involved in association of the actin cytoskeleton with the plasma membrane (myosin I, myosin VII) and organelles (myosin V). Actin filaments are organized in networks and bundles by actin crosslinking and bundling proteins. In networks, the MFs criss-cross and are loosely packed, whereas in bundles they are densely packed in parallel or antiparallel arrays. Filamin, spectrin, and a-actinin are examples of crosslinking proteins. Fascin, fimbrin (plastin), villin, and espin are common bundling proteins. Actin networks and bundles are found in a variety of protrusive structures. In motile cells, MFs are prevalent at cell edges, particularly in filopodia, pseudopodia, lamellipodia, and ruffles. Filopodia are thin fingerlike protrusions supported by parallel bundles of MFs. Lamellipodia and ruffles are sheetlike protrusions supported by MF networks. Pseudopodia are thick fingerlike protrusions characteristic of cells that exhibit amoeboid movement, like white blood cells. In cells that adhere tightly to the substrate actin, filaments organize in

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bundles called stress fibers that attach to focal adhesions, specialized complexes where the actin cytoskeleton is anchored through talin and other ABPs to integrins, which in turn bind to proteins of the extracellular matrix. MFs organize in antiparallel bundles at the cleavage furrow, a structure that, powered by myosin, constricts the cytoplasm of the dividing cell and contributes to separate the daughter cells. Networks of actin also assemble around endocytic structures at the plasma membrane like phagocytic cups and around endosomes and vesicles of the Golgi apparatus. Cells of the intestinal and renal epithelia display fingerlike protrusions called microvilli that cover their apical pole and contribute to extend the cellular surface for digestion and transport of metabolites. Like filopodia, microvilli are built from parallel arrays of actin filaments, but they are more densely bundled and are attached to the plasma membrane by specialized myosins. Much thicker and longer bundles of MFs are characteristic of the stereocilia of hair cells of the inner ear, involved in sensing tiny movements caused by sound vibrations and changes of posture. Other ABPs like spectrin, dystrophin, talin, proteins of the ERM (ezrin, radixin, moesin) family, and some unconventional myosins are responsible for the association of MFs to the plasma membrane. In association with short actin filaments, spectrin forms a network underneath the plasma membrane of the erythrocyte. In muscle cells, dystrophin is responsible for attaching the sarcomere to a glycoprotein complex which is anchored to the extracellular matrix. Actin also assembles at cell-cell contacts mediated by cadherin, the ▶ adherens junctions or by occludins, the tight junctions. In epithelial cells, these contacts constitute a circumferential band at the boundary between the apical and basolateral poles of the cell. Nesprins are ABPs of the outer nuclear membrane that connect the cytoplasmic cytoskeleton to the nuclear lamina through SUN-domain proteins and contribute to positioning of the nucleus and stability of the nuclear envelope. The sarcomere, the contractile unit of the muscle cell, constitutes one of the most complex

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assemblies of cytoskeletal proteins. In the sarcomere, MFs and associated proteins build up the thin filaments and are arranged in a hexagonal pattern around thick filaments, constituted by bundles of myosin. Numerous structural proteins are responsible for maintaining the precise organization of the sarcomere, including some IF proteins (desmin, syncoilin, and synemin). Sliding of thin filaments past thick filaments is powered by ATP hydrolysis by myosin and results in shortening of the sarcomere and, on a large scale, muscle contraction. Numerous MF-targeting agents have being used to perturb the assembly of actin into MFs (Table 1). Although several of them have demonstrated in vitro and in vivo anticancer efficacy, none of them has been approved for cancer therapy, usually because of their high toxicity, and only one, MKT-077 has been clinically evaluated. This compound crosslinks F-actin and produces aberrant MFs but part of its actions is due to the inhibition of mitochondrial Hsp70 and induction of apoptosis. Microtubules Microtubules are stiff hollow tubes of 25 nm diameter (Fig. 1). They are built from stable dimers of a and b tubulin, two similar globular proteins of approximately 55 kDa. Each tubulin monomer binds a GTP nucleotide but only the one bound to b-tubulin is exchangeable. Tubulin dimers polymerize end-to-end, giving rise to a polar protofilament with a fast growing (plus) and a slow growing (minus) end. On a cross section, a singlet MT is composed of 13 protofilaments around a hollow core, but other arrangements exist in centrioles (triplets) and the axonemes of cilia and flagella (doublets). GTP is needed for MT assembly. A GTP-tubulin cap prevents the peeling away of subunits from the plus end of a growing MT. Hydrolysis of GTP by b-tubulin results in an unstable tip and facilitates rapid depolymerization, an event called MT catastrophe. If the MT regains a GTP-tubulin cap it can resume growth, an event called MT rescue. Globally, this behavior is called dynamic instability. There are two assemblies of MTs, cytoplasmic and axonemal. Cytoplasmic MTs build a very

Cytoskeleton

dynamic network that extends from the ▶ centrosome at the vicinity of the nucleus toward the cell periphery. They are mainly used as tracks for directional movement of organelles and also contribute to maintaining cell shape and polarity. During cell division, cytoplasmic MTs arrange into the mitotic spindle, a cage-like structure that captures the chromosomes and pulls the chromatids apart toward their respective daughter cell. The centrosome constitutes the MT organizing center (MTOC) of cytoplasmic MTs. It is composed of two centrioles surrounded by a cloud of amorphous pericentriolar material. The centrioles are two cylinders placed at right angles to each other. Each centriole is made of a radial array of 9 triplet MTs and contains additional tubulin isoforms (d, e). Interphase cells possess one centrosome that duplicates when the cell undergoes mitosis. Cytoplasmic MTs are not initiated in the centrioles, but in the pericentriolar material, form a protein complex called the g-tubulin ring complex (g-TuRC), that contains one more tubulin isoform, g-tubulin, and 5 or 6 additional proteins. Axonemal MTs are stable and found in specialized fingerlike appendages called cilia and flagella. Cilia are short (2–10 mm) and numerous and are characteristic of epithelial cells of the respiratory tract epithelium, where they produce an oarlike pattern of beating that contributes to carry mucus and foreign matter out of the airway. The flagellum is large (10–200 mm) and usually unique and is responsible for the undulatory beating that propels the sperm cells. Cilia and flagella have the same structure, with a core of MTs arranged in a radial array of nine double MTs around a core of two single microtubules in association with more than 100 accessory proteins. This structure, the axoneme, is an extension of a modified centriole, the basal body. Most mammalian cells possess a unique so-called primary cilium that emerges from one of the centrioles after cell division. The primary cilium is usually nonmotile and functions as a sensory antenna for many signal transduction pathways. The outer segments of rod and cone photoreceptors in the eye are formed from derivatives of primary cilia and possess a basal body and a vestigial axoneme.

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Cytoskeleton, Table 1 Microfilament, microtubule, and intermediate filament-targeting agents. The anticancer effects of most of the agents listed have been investigated Agent family Microfilaments Cytochalasins

in vitro and in vivo. Compounds “under investigation as cancer drug” indicates that they have been or are being tested in clinical studies

Examples

Origin

Mechanism of action

Use

Cytochalasin B, cytochalasin D

Several species of fungi

Cell biology research

Jasplakinolide

Chaetogobosin A, chaetoglobosin K Jasplakinolide

Several species of fungi Marine sponge (Jaspis johnstoni)

Latrunculins

Latrunculin A

MKT-077

MKT-077

Marine sponge (Latrunculia magnifica) Synthetic

Bind to the barbed end and inhibit elongation Similar to cytochalasins Induces polymerization and stabilizes MFs Sequesters actin monomers

Staurosporine

Staurosporine

Under investigation as anticancer drug Biochemistry research

Scytophycins

Tolytoxin

Phalloidin

Phalloidin

Death cap fungus (Amanita phalloides)

Crosslinks MFsInhibits Hsp70 Thinning and loss of MFsProtein kinase inhibitor Inhibit actin polymerization and depolymerize MFs Stabilizes MFs by preventing depolymerization

Vincristine, vinblastine, vindesine, vinorelbin Dolastatin 10, dolastatin 15, monomethyl auristatins Eribulin, halichondrin B

Madagascar periwinkle (Catharanthus roseus) Sea hare (Dolabella auricularia)

Bind tubulin and inhibit assembly into MTs

Approved for cancer therapy

Similar to vinca alkaloids

Approved for cancer therapy

Marine sponge (Halicondria okadai) Meadow saffron (Colchicum autumnale) Cyanobacteria (Nostoc) Synthetic

Similar to vinca alkaloids

Approved for cancer therapy

Similar to vinca alkaloids

Approved as antiinflammatoryCytogenetics

Similar to vinca alkaloids Similar to vinca alkaloids Bind and stabilize MTs

Under investigation as anticancer drug Cell biology research

Chaetoglobosins

Microtubules Vinca alkaloids

Dolastatins

Eribulin

Bacterium (Streptomyces staurosporeus) Cyanobacteria (Scytonemataceae)

Colchicine

Colchicine, colcemid

Cryptophycin

Cryptophycin 1

Nocodazole

Nocodazole

Taxanes

Paclitaxel, docetaxel

Pacific yew tree (Taxus brevifolia)

Epothilones

Ixabepilone, epothilone B (patupilone), sagopilone

Myxobacterium (Sorangium cellulosum)

Similar to taxanes

C Under investigation as anticancer drug Cell biology research

Cell biology research

Research

Cell biology research

Approved for cancer therapyCell biology research Approved for cancer therapy

(continued)

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Cytoskeleton, Table 1 (continued) Agent family Peloruside

Examples Peloruside A

Laulimalide

Laulimalide

Discodermolide

Discodermolide

Intermediate filament Withanolides

Withaferin A

Origin Marine sponge (Mycale hentscheli) Marine sponge (Cacospongia mycofijiensis) Deep-sea sponge (Discodermia dissoluta)

Mechanism of action Similar to taxanes

Use Under investigation as anticancer drug

Similar to taxanes

Under investigation as anticancer drug

Similar to taxanes

Under investigation as anticancer drug

Winter cherry (Withania somnifera)

Bind vimentin at a Cys residue and inhibit assembly

Under investigation as anticancer drug

As with MFs, numerous MT-associated proteins (MAPs) control remodeling of MTs, formation of bundles, formation of networks with other cytoskeletal systems, and association to cellular structures. Like in the case of MFs, de novo formation of MTs is an unfavorable process that in vivo occurs in an organized manner at the MTOC or at basal bodies that function as nucleation centers. Most MAPs bind along the length of the MTs and stabilize them. They are particularly abundant in nervous tissue. Examples include the tau family (tau, MAP2, MAP4), MAP1A and 1B, STOP, tektin, and ▶ doublecortin. Some of these are restricted to specialized cells while others are expressed widely. Phosphorylated, proteolytically modified, and cross-linked tau is the main component of the neurofibrillary tangles that characterize Alzheimer disease. Other MAPs promote the destabilization of MTs. Stathmin/Op18 (oncoprotein 18), a protein expressed at unusually high levels in many malignant cells; sequesters tubulin heterodimers, preventing them from polymerizing. Catastrophin (MCKA, mitotic centromere associated kinesin) is an unusual kinesin that binds the plus ends of MTs and promotes the detachment of subunits. Katanins sever MTs into short fragments that then depolymerize. A group of proteins capture and stabilize the plus ends at the cell periphery, like CLIP-170 and EB1, or at kinetochores, like MAST/Orbit. MT-associated motor proteins use the energy provided by the hydrolysis of ATP to walk along MTs. They belong to two major families, dynein

and kinesin. In general, members of the large family of kinesins move toward the plus end and are involved in the transport of multiprotein complexes and organelles and in various phases of the cell division. Dyneins move toward the minus end and can be grouped into two classes, cytoplasmic (involved in transport of organelles and cell division) and axonemal (responsible for flagellar and cilliary beating). Drugs that affect MT dynamics (Table 1) are used as effective antitumor drugs because blocking reorganization of the mitotic spindle preferentially affects rapidly dividing cells. This blocks the progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis. Vinca alkaloids like Vincristine (Oncovin™) and vinblastine bind to tubulin and cause it to aggregate, thereby preventing the assembly of MTs. They are used in combination with other cytostatic drugs. Vinblastine is used to treat Hodgkin lymphoma, nonsmall cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. It is also used to treat Langerhans cell histiocytosis. Vincristine is mainly used to treat nonHodgkin’s lymphoma, acute lymphoblastic leukemia, and nephroblastoma. Monomethyl auristatin E (Vedotin) is more potent than vinca alkaloids but due to its toxicity it is linked to monoclonal antibodies that direct the drug to cancer cells. It is used to treat relapsed or refractory Hodgkin lymphoma and relapsed or refractory systemic anaplastic large cell lymphoma (Brentuximab

Cytoskeleton

Vedotin). Taxanes stabilize MTs and protect them from disassembly, and as a result interfere with their normal dynamic breakdown during cell division. Paclitaxel (▶ Taxol™) is used to treat patients with lung, ovarian, breast and head and neck cancer, and advanced forms of Kaposi sarcoma. The clinical effectiveness of taxanes is often limited by primary or acquired resistance. This has led to the introduction of novel MT targeting agents like epothilones and eribulin. Epothilones have higher efficacy and lower toxicity than taxanes. Ixabepilone is used in the treatment of aggressive metastatic or locally advanced breast cancer no longer responding to currently available chemotherapies. Eribulin (Halaven™) is used to treat patients with metastatic breast cancer who have received at least two prior chemotherapy regimens for late-stage disease. Due to its relatively low therapeutic index, colchicine is not used in tumor therapy, but continues to be used for its anti-inflammatory properties for the treatment of acute flares of gout and familial Mediterranean fever. Intermediate Filaments Intermediate filaments owe their name to their diameter, 10 nm, intermediate between MFs and MTs (Fig. 1). In contrast to these, IFs vary largely in composition and size among different cell types. However, the subunits that make up IFs are structurally related rod-shaped proteins with a central a-helical coiled-coil domain that forms the core of the filaments and variable globular domains on either side. IF proteins assemble into parallel coiled-coil dimers where the central domains are intertwined. Dimers assemble into antiparallel half-staggered tetramers (protofilaments). Eight tetramers associate laterally into unit-length filaments that anneal longitudinally to form 16 nm filaments which, unlike MFs and MTs, lack polarity. It follows a compaction phase during which filaments are compressed radially into mature 10 nm IFs. Assembly of IFs does not require nucleotide hydrolysis. IFs are flexible, more stable than MFs and MTs and more abundant than these in neurons and epidermal cells. IFs form a meshwork that spans

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the whole cytoplasm, where they play a major structural role allowing cells to withstand stretching and shearing forces, although roles in cell signaling, epithelial polarity, growth, and apoptosis are emerging. IFs also make up the nuclear lamina that reinforces the nuclear envelope. IFs are not involved in cell movements. Although biochemically stable, IF networks undergo rearrangements, particularly during cell spreading, wound healing, cell division, and in response to environmental mechanical stresses. The IF family consists of 73 gene products that have been grouped into six major types or sequence homology classes. Types I–IV are cytoplasmic, type V comprises lamins, present in the nucleus of all cells, and type VI comprises proteins found exclusively in the eye lens. Mutations in almost all genes encoding IF proteins have been found to cause or predispose to human disease, with more than 80 conditions described. Type I and II IFs are acidic and basic keratins (or cytokeratins), respectively, that always associate into heterodimers of one acidic with one basic keratin. The keratin family comprises 54 functional genes, 37 epithelial keratins, and 17 hair keratins. Keratins are characteristic of epithelial cells of the skin and digestive, respiratory and urinary tracts and are an important component of structures that grow from skin, like hair and nails. Each type of epithelium expresses a characteristic combination of type I and type II keratins. For example, in the skin basal keratinocytes express K5/K14, whereas suprabasal keratinocytes express K1/K10. Keratins are widely used tumor markers when diagnosis using conventional microscopic techniques is inconclusive, based on the fact that tumor cells retain the IF proteins characteristic of the tissue of origin. Staining with a pan-keratin specific antibody, for example, distinguishes epithelial from nonepithelial tumors. The precise pattern of keratin expression of metastases allows prediction of the origin of the primary tumor and can also be useful in predicting tumor prognosis. Circulating keratins and fragments thereof released form apoptotic and necrotic tumor and nontumor cells and have been used as markers of disease progression, response to treatment, and

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recurrence. The most commonly used markers are TPA (tissue polypeptide antigen, a mixture of K8, K18, and K19), TPS (tissue polypeptidespecific antigen, derived from K18), and CYFRA 21–1 (cytokeratin fragment 21–1, derived from K19). Type III IFs comprise vimentin, desmin, glial fibrillary acidic protein (GFAP), peripherin, and syncoilin. Desmin is found in muscle and is responsible for stabilizing the sarcomere, where it has a fixed position and anchoring it to the plasma membrane. Syncoilin is also found at stress points in muscle cells. Vimentin is expressed in leukocytes, endothelial cells, and mesenchimal cells. GFAP is found in glial cells and peripherin in the peripheral nervous system. Type IV IFs include neurofilaments (a triplet of low, mid, and high molecular weights), a-internexin, nestin, and synemin. Neurofilaments are found in neurons, where they determine axon diameter. Nestin and a-internexin are also expressed in neurons at early stages of development. Synemin is localized to stress points in muscle cells. Type V IFs are made of lamins. They do not form ropes, but a planar meshwork underneath the inner nuclear membrane, the nuclear lamina. Three genes encode several alternatively spliced lamins that can be grouped into B-type (expressed in all cell types) and A-type (restricted to more differentiated cell types) lamins. Lamins interact with numerous proteins of the inner nuclear membrane, like lamin B receptor, emerin, and LAP (lamin-associated protein). The lamina is connected to the cytoskeleton through LINC (linker of nucleoskeleton and cytoskeleton) complexes composed of SUN proteins of the inner nuclear membrane interacting with nesprins of the outer nuclear membrane. LINC complexes are important for nuclear positioning and migration. Lamins become phosphorylated and disassemble at the onset of mitosis, concomitantly with the breakdown of the nuclear envelope, and reassemble after mitosis. Type VI IFs group two proteins, Bfsp1 (filensin) and Bfsp2 (phakinin), responsible for the exceptional biochemical stability and optical clarity of the eye lens.

Cytoskeleton

Unlike MFs and MTs, which are found in all cells, cytoplasmic IFs display complex patterns of cell and tissue distribution and the expression of some of them is markedly regulated during development and cell differentiation. Diverse IF-associated proteins (IFAPs) crosslink IFs to each other into bundles and networks, or link them to other cytoskeleton networks or specialized structures at the plasma membrane. As opposed to MFs and MTs, no IFAPs have been identified that possess IF-dependent nucleating, capping, or severing activity. IF remodelling is regulated primarily (albeit not exclusively) by phosphorylation. IFs also do not function as tracks for motor proteins, although IF particles can be themselves cargo of MT- and MF-dependent motor proteins. The most widespread IFAPs are the plakins, a family of large multifunctional proteins. Prominent examples of this family are plectin, BPAG1 (bullous pemphigus antigen 1), epiplakin, periplakin, envoplakin, and desmoplakin. Plectin is a very large protein that crosslinks IFs to each other, to the plasma membrane, and to MFs and MTs. BPAG1 cross-links neuronal IFs to MFs. BPAG1 and plectin connect keratin filaments to hemidesmosomes. The other plakins mentioned above connect keratin filaments to desmosomes. ▶ Desmosomes and hemidesmosomes are specialized structures at the plasma membrane that allow the transmission of mechanical forces from one cell to another and to the extracellular matrix, respectively. Examples of proteins that crosslink IFs are filaggrin and KAPs (keratin associated proteins), which interact with keratin in the epidermis and the hair shaft. The wide diversity of IF proteins complicates the search for potential therapeutic agents that specifically target IFs (Table 1). Currently, withaferin A is under investigation as an inhibitor of type III IFs, particularly vimentin. Withaferin A binds covalently to a conserved cysteine residue in vimentin and inhibits the assembly of the IF.

Cross-References ▶ Adherens Junctions ▶ Centrosome

Cytotoxic T Cells

▶ Cortactin ▶ Desmosomes ▶ Doublecortin ▶ Gelsolin ▶ Taxol

References Campellone KG, Welch MD (2010) A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 11:237–251 Godsel LM, Hobbs RP, Green KJ (2007) Intermediate filament assembly: dynamics to disease. Trends Cell Biol 18:28–37 Wade RH (2009) On and around microtubules: an overview. Mol Biotechnol 43:177–191

Cytosolic Transglutaminase ▶ Transglutaminase-2

Cytotactin ▶ Tenascin-C

Cytotoxic T Cells Marc Schmitz Institut für Immunologie, Technische Universität Dresden, Dresden, Germany

Definition CD8+ cytotoxic T cells (CTLs) are major cellular components of the adaptive immune system. They efficiently recognize and destroy virus-infected host cells or tumor cells, which expose antigenic peptides bound to major histocompatibility complex (MHC) class I molecules.

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Characteristics Dendritic Cells Play a Major Role in the Induction of CD8+ T Cell Responses CD8+ CTLs can be efficiently activated by “professional” antigen-presenting cells (APCs) such as dendritic cells (DCs), which display the appropriate MHC class I-peptide complexes on their surface. DCs are the most effective APCs for stimulating naïve T cells that have not recognized and responded to antigens. Besides their extraordinary capability to initiate CD8+ T cell responses, DCs also essentially contribute to the maintenance and regulation of previously activated CD8+ CTLs. Macrophages and B lymphocytes also function as APCs, but mostly for previously stimulated T cells rather than for naïve T cells. DCs originate from bone marrow progenitor cells, circulate through the blood, and migrate into various tissues such as the epidermis of the skin and the epithelia of the gastrointestinal and respiratory tracts. These so-called immature DCs are ideally located at common sides of pathogen entry to perform a sentinel function in the immune defense. They are characterized by the expression of different cell membrane receptors, which mediate an efficient endocytosis of pathogenassociated antigens. Furthermore, they also internalize antigens from the extracellular fluid receptor-independent by pinocytosis. For CD8+ T cell activation, DCs utilize the “classical” MHC class I pathway for processing and presentation of cytosolic proteins. In this context, ubiquitinated cytosolic proteins such as viral or tumor-associated proteins are degraded by proteasomes representing multiprotein enzyme complexes. The generated cytosolic peptides are translocated by a specialized transporter, termed transporter associated with antigen processing, into the endoplasmic reticulum (ER). Inside the ER, the peptides bind to the cleft of newly synthesized and assembled MHC class I molecules consisting of an a-chain and b2-microglobulin. Subsequently, stable MHC class I-peptide complexes move through the Golgi complex and are transported to the cell surface by vesicles. In addition to the “classical” MHC class I pathway, DCs have the extraordinary capacity to ingest virus-infected or tumor cells and

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Cytotoxic T Cells

Cytotoxic T Cells, Fig. 1 DCs display an extraordinary capacity to induce and maintain CD8+ T cell responses. Mature DCs are characterized by the high surface expression of MHC class I-peptide complexes and costimulatory molecules. Therefore, they are well equipped to efficiently

activate and expand CD8+ CTLs. Activated CD8+ CTLs possess a profound capability to recognize and destroy pathogen-infected or tumor cells. Furthermore, they secrete important cytokines such as IFN-gamma, which can stimulate additional immune cell populations

present peptides derived from extracellular viral or tumor antigens in association with MHC class I molecules. This alternative pathway is called cross-presentation. In contrast to their pronounced capacity to capture pathogen-associated antigens, immature DCs are not able to efficiently initiate CD8+ T cell responses, which is based on the low expression of MHC, adhesion and costimulatory molecules on their surface. DC maturation is induced by pathogen-associated molecular patterns such as lipopolysaccharide that are recognized by Toll-like receptors and other pattern recognition receptors. Furthermore, proinflammatory cytokines produced by various immune cells promote the maturation of DCs. Mature DCs migrate from the tissues into draining lymph nodes via afferent lymphatic vessels, a process which is mediated by the interaction of the chemokine receptor CCR7 on DCs and the chemokines CCL 19 and CCL 21 that are produced in the T cell zone of lymph nodes. This leads to a colocalization of antigen-bearing DCs with naïve T cells. Naïve T lymphocytes circulate throughout the body in a nonstimulated state and perform their functional capabilities only after activation. Due to the high surface expression of

MHC class I-peptide complexes as well as adhesion and costimulatory molecules, mature DCs are able to efficiently induce CD8+ T cell responses. Activation of CD8+ T cells Cytokine secretion and clonal expansion of CD8+ T cells as well as the differentiation of naïve T cells into effector and memory T lymphocytes require antigen recognition, costimulation, and cytokines that are provided by “professional” APCs, such as DCs, and by the T cells themselves. Antigen recognition is the important first signal for the activation of CD8+ T lymphocytes, ensuring that the resultant immune response remains specific for the antigen (Fig. 1). In lymphoid organs, naïve CD8+ T cells recognize via their T cell receptors (TCRs) antigen-derived peptides displayed by MHC class I molecules on the surface of DCs. In contrast to naïve CD8+ T cells, differentiated effector T cells can respond to antigens presented by cells other than DCs. In addition to antigen recognition, naïve CD8+ T cells require a second signal provided by costimulatory molecules on DCs (Fig. 1). In the absence of costimulation, T cells that encounter antigens either fail to respond and undergo apoptosis or enter a state of unresponsiveness called anergy.

Cytotoxic T Cells

The best characterized costimulatory pathway in T cell activation involves the T cell surface receptor CD28, which binds the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) expressed on activated DCs. Previously activated effector CD8+ T cells are less dependent on costimulation than naïve T cells. This property enables effector CD8+ T cells to kill other cells that do not express costimulatory molecules. CD28 engagement together with TCR-mediated antigen recognition promotes survival and proliferation of T cells as well as the differentiation of naïve T cells into effector and memory T lymphocytes. Another costimulatory pathway for T cell activation is based on the interaction between inducible costimulator (ICOS) expressed on T cells and its ligand, ICOS-L, which is displayed on the surface of various cell types such as DCs. In the meantime, additional activating and inhibitory molecules have been identified that can regulate T cell responses. In addition to antigen recognition and costimulation, the activation of naïve CD8+ T cells and their differentiation into CD8+ effector and memory T cells may require the participation of CD4+ T helper cells. Thus, CD4+ T helper cells can improve the capacity of DCs to induce CD8+ CTLs by the interaction between CD40 on DCs and CD40 ligand on activated CD4+ T cells. Furthermore, CD4+ T cells provide help for the expansion of CD8+ CTLs by secreting cytokines such as interleukin (IL)-2. Antigen-stimulated CD8+ T cells are characterized by the expression of various surface molecules such as CD69 and CD25. The expression of CD25, a component of the IL-2 receptor, enables T cells to respond to the growth-promoting cytokine IL-2. In addition, activated CD8+ T cells produce IL-2 that promotes the survival, proliferation, and differentiation of T cells. Furthermore, antigen recognition, costimulation, and growth factors such as IL-2 induce T cell proliferation. The result of this proliferation is clonal expansion, which generates a large number of CD8+ T cells from a small pool of naïve antigen-specific CD8+ T lymphocytes. Antigen recognition and other activating stimuli also promote the differentiation of naïve T cells into effector and memory T lymphocytes. CD8+

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memory T cells have an ability to survive for long periods, persist in the circulating lymphocyte pool, mucosal tissues, skin, and lymphoid organs, respond rapidly to subsequent encounter with the same antigen, and generate new CD8+ effector cells that contribute to antigen elimination. They mount larger and more rapid responses than naïve T cells to antigen challenge. Activated CD8+ effector T cells, which are characterized by a strong capacity to lyze target cells and to produce cytokines such as interferon (IFN)-gamma after antigenic stimulation, migrate to peripheral tissues and essentially contribute to the elimination of pathogen-infected or tumor cells. Cytotoxic Potential of Activated CD8+ T Cells CD8+ effector CTLs efficiently destroy targets such as pathogen-infected cells, which express the same MHC class I-antigenic peptide complexes on their surface that triggered the proliferation and differentiation of naive CD8+ T cells (Fig. 1). This specificity of CTL function ensures that normal cells are not killed by CTLs reacting against infected cells. In addition, the target cell killing is highly specific because an immunologic synapse is formed at the site of contact of the CTL and the antigen-expressing target, and the molecules that perform the killing are secreted into the synapse and cannot diffuse to other nearby cells. The process of CD8+ CTL-mediated killing of targets consists of antigen recognition, CTL activation, delivery of the lethal hit that kills the target cells, and release of the CTLs. CD8+ CTLs recognize their target cells by the interaction of the TCR with the appropriate MHC class I-antigenic peptide complex and the accessory molecule leukocyte function antigen-1 with intercellular adhesion molecule-1. CTL recognition leads to activation and target cell killing. One important mechanism of CTL-mediated killing is the delivery of cytotoxic proteins stored within cytoplasmic granules to the target cell, thereby triggering apoptosis. The cytotoxic proteins in the granules include granzymes, which are serine proteases and the membrane-perturbing molecule perforin. Activation results in the release of granule contents from the CTL into the target cell through the area of contact. CTLs also use granule-

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independent mechanisms of killing that is mediated by interactions of membrane molecules on CTLs and target cells. For example, CTLs can express Fas ligand that binds to the death receptor Fas on target cells. This interaction also results in apoptosis of target cells. After delivering the lethal hit, the CTL is released from the target cell and kill other target cells. In addition to their high cytotoxic potential, activated CTLs can produce important proinflammatory cytokines such as IFN-gamma, which can stimulate various immune cell populations. Due to their functional properties, CD8+ CTLs can essentially contribute to the elimination of pathogen-infected cells and tumor cells. In addition, CD8+ CTLs emerged as promising candidates for cancer immunotherapy. In this context, clinical trials revealed that the adoptive transfer of CD8+ T cells can mediate tumor regression in cancer patients.

Cross-References ▶ Adoptive T-Cell Transfer ▶ Chimeric Antigen Receptor on T Cells ▶ Dendritic Cells ▶ HLA Class I ▶ Proteasome

Cytovillin

References Andersen MH, Schrama D, Thor Straten P, Becker JC (2006) Cytotoxic T cells. J Invest Dermatol 126:32–41 Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811 Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, Sherry R, Restifo NP, Hubicki AM, Robinson MR, Raffeld M, Duray P, Seipp CA, Rogers-Freezer L, Morton KE, Mavroukakis SA, White DE, Rosenberg SA (2002) Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298:850–854 Mollderm JJ, Lee PP, Wang C, Felio K, Kantarjian HM, Champlin RE, Davis MM (2000) Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 6:1018–1023 Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 8:299–308 Russell JH, Ley TJ (2002) Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 20:323–370 Williams MA, Bevan MJ (2007) Effector and memory CTL differentiation. Annu Rev Immunol 25:171–192

Cytovillin ▶ ERM Proteins

D

3-(1,3-Dihydro-3-oxo-2H-indol-2ylidene)-1,3 dihydro-2H-indol-2-one ▶ Indirubin and Indirubin Derivatives

Cross-References ▶ Wilms’ Tumor

See Also

DAC ▶ 5-Aza-20 Deoxycytidine

(2012) Actinomycin D. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 19. doi:10.1007/978-3-642-16483-5_46 (2012) Germ cell tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905

Dacogen DAF ▶ 5-Aza-20 Deoxycytidine

▶ Decay-Accelerating Factor

Dactinomycin Damage Response Definition ▶ Stress Response Dactinomycin is a trade name for actinomycin D. It is a chemotherapy drug that is given as a treatment for some types of cancer, most commonly some cancers that occur in children such as ▶ Wilms’ tumor and germ cell tumors, although it may sometimes be used for adults.

# Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3

DANCE, EVEC, UP50, FBLN-5 ▶ Fibulins

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Dap160 (Dynamin-Associated Protein of 160 kDa)

Characteristics

Dap160 (Dynamin-Associated Protein of 160 kDa) ▶ Intersectin

DAP6 ▶ Daxx

DARC ▶ Duffy Antigen Receptor for Chemokines

Daxx Sheng-Cai Lin Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian, China

Synonyms BING2; DAP6; Death-associated protein 6; MGC126245; MGC126246

Definition Daxx was originally identified as a protein factor that binds to a transmembrane receptor called Fas (FAS/APO-1/CD95), one member of the ▶ tumor necrosis factor receptor superfamily (Yang et al. 1997). The extracellular region of Fas is where its ligand, FasL, binds. The intracellular tail shares sequence similarity with another member of the TNF receptor family, TNF receptor I (TNFRI). The shared sequence is termed death domain for its critical role in signaling cell death upon ligand binding.

Since its identification in 1997, Daxx has been intensively investigated for its biological functions. However, up to date the three-dimensional crystal structure of Daxx has not been resolved. Human Daxx protein is a polypeptide of 740 amino acid residues in length. As depicted in the schematic diagram (Fig. 1), Daxx contains several putative domains and many binding sites for interacting with a wide spectrum of proteins including transcription factors, kinases, tumor suppressors, and chromatin remodeling factors. It is worth to note that for the influence from its acidic region, Daxx displays an aberrant molecular weight during electrophoretic migration on gels, being 120 kDa instead of the calculated 81.3 kDa (Yang et al. 1997). Basic Information Human Daxx gene is mapped to 6p21.3 in the major histocompatibility complex (MHC) region and transcribes a single 2.4 Kb RNA. It is ubiquitously expressed in human tissues and is highly conserved in mammals with 69% identity between the human and mouse proteins. Currently at least 35 molecules have been known to interact with Daxx, which were identified by various methods including yeast two-hybrid screening, coimmunoprecipitation, and immunofluorescent costaining in the cell (Salomoni and Khelifi 2006). Most notably, Daxx interacts with the tumor suppressor ▶ p53, Axin (Axis inhibitor), ubiquitin ligase Mdm23 (mouse double minute 2), the Ser/Thr kinase HIPK2 (homeodomain-interacting protein kinase 2), and the product of the promyelocytic leukemia (PML) gene. Subcellular Distribution of Daxx Protein Daxx is predominantly localized in the nucleus. Daxx and PML interact with each other and are colocalized in nuclear punctuate structures known as nuclear bodies (NBs) that have been involved in various processes such as transcriptional regulation, apoptosis, genome stability, and tumor suppression (Salomoni and Khelifi 2006). PML protein is an important tumor suppressor that is essential for the formation of the nuclear bodies,

Daxx

1309 Fas, PML, Ubc9, JNK/ASK1 Pax3, Pax5, CENP-C NLS activation region binding sites 389 394 434 493 501 625 D/E S/P/T- rich 740

HIPK2/Axin binding sites

1

PAH1

PAH2

180 217 CC

Daxx, Fig. 1 Schematic diagram showing various domains of Daxx. Human Daxx is a protein with 740 amino acid residues in length that contains several putative domains: two N-terminal paired amphipathic helices (PAH1 and PAH2), a coiled-coil region, an acidic domain (D/E), and a Ser/Pro/Thr-rich domain (S/P/T).

Glucose deprivation, Fas, TGF b

Mdmx

Daxx

Mdmx

UV

HAUSP

Daxx, Fig. 2 Schematic representation of two possible pathways through which Daxx mediates cell death. Upon stimulation by stress signals such as UV and nutrition deprivation, Daxx becomes associated with ASK1, leading to activation of JNK MAP kinase that is required for cell death induction. In parallel, UV irradiation causes dissociation of p53-destabilizing complex to release p53 and Daxx which then assemble into a p53-activating complex consisting of p53, Axin, Daxx, and HIPK2. Upon complex formation, HIPK2 is activated and activates p53 by phosphorylating it at Ser46. As a result, p53 target genes related to apoptosis are activated

Axin and HIPK2 associate with Daxx through binding sites on the N-terminal portion. A region between amino acid 501 and 625 is required for ASK1 binding and the subsequent activation of JNK. The Ser/Pro/Thr-rich domain contains sites for interaction with a series of proteins, such as Fas, PML, Ubc9, and CENP-C

Ub Ub Ub

Ub

Mdm2 Ub

Mdm2

Daxx

Ub Ub

Ub

p53 p53 p53

HIPK2

Ub Ub Ub

Ask1

Daxx

Degradation

Axin

MKK4/ MKK7

Ub

p53 p53 Daxx HIPK2 Axin

JNK

Degradation

as deletion of the PML gene by genetic knockout can fully abrogate the formation of these structures. In pml/ cells that lack PML-NB structures, the ability of Daxx to induce cell death is abolished, underscoring the importance of PML for cell death induction by Daxx-mediated signaling. Daxx Mediates Stress-Induced Cell Death Daxx has been implicated in the modulation of apoptosis induced by a wide range of stimuli, including ultraviolet radiation, hydrogen peroxide, arsenic trioxide, Fas ligand, transforming

Target genes

P

4

S 6

p53

Cell death

growth factor beta (TGF-b), and interferon-g. As summarized in Fig. 2, two main apoptotic pathways are thought to be mediated by Daxx. • The first reported mechanism for Daxx to induce apoptosis is through association and subsequent activation of apoptotic signalregulating kinase 1(ASK1), an upstream kinase of JNK MAP kinase that leads to activation of c-jun N-terminal kinase (JNK) (Salomoni and Khelifi 2006). Glucose deprivation as a stress can also stimulate the association of Daxx to ASK1 and activate

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Daxx-dependent ASK1 activation. TGF-b is a multifunctional growth factor that has principal roles in growth control. In a yeast two-hybrid screening using the cytoplasmic domain of the type II TGF-b receptor as bait, Daxx was found to interact with this receptor and mediate TGF-b-induced apoptosis via facilitating JNK activation. Interference of Daxx function by truncated Daxx mutants or by knockdown of Daxx expression can inhibit TGF-b-, UV-, or oxidative stress-induced apoptosis. • Another means adopted by Daxx to induce cell death is through Axin/HIPK2/p53 complex (Li et al. 2007). Currently, the best characterized role of Daxx is its ability to modulate the function of the tumor suppressor p53, which is mutated in over 50% of all tumor tissues. p53 exerts its tumor suppressional roles by acting as a transcription factor. Upon DNA damage, p53 is activated, which results in cell cycle arrest or apoptosis by inducing expression of a series of proteins such as p21, GADD45 (growth arrest and DNA damage), and ▶ PUMA (p53-upregulated modulator of apoptosis). HIPK2, an upstream serine/threonine kinase of p53, can specifically phosphorylate p53 at serine 46 upon UV irradiation and plays an important role in UV-induced p53-mediated apoptosis. Axin is a negative regulator of Axis formation in the development of mouse embryos, its deficiency can lead to axis duplication. Accumulating evidence demonstrates that this protein functions as a tumor suppressor. It has been shown that UV irradiation can enhance the association of Daxx and Axin. Further investigation demonstrates that Axin tethers Daxx to p53 and cooperates with Daxx to stimulate HIPK2-mediated Ser46 phosphorylation and transcriptional activity of p53. Axin is required for Daxx-induced p53 activation and apoptosis, as in Axin/ cells the ability of Daxx to induce cell death is dramatically attenuated. Downregulation of HIPK2 by specific siRNA remarkably suppresses Daxx-induced apoptosis, suggesting that HIPK2 plays crucial role in Daxx-induced cell death. All above evidence indicates that

Daxx

formation of a new complex containing Daxx/ Axin/p53/HIPK2 is important for Daxxinduced cell death. Knockdown of Axin and Daxx by ▶ siRNA can strongly attenuate UV-induced cell death, demonstrating that Daxx and Axin complex plays a critical role in UV-triggered cell death. In Unstressed Cells, Daxx Seems to Play an Antiapoptotic Role Daxx may also destabilize p53 by promoting the function of Mdm2, a ubiquitin ligase E3 that facilitates p53 ▶ ubiquitination and degradation (Tang et al. 2006). In unstressed cells, Daxx simultaneously interacts with Mdm2 (heterodimerized with MdmX) and the deubiquitinase HAUSP (herpesassociated ubiquitin-specific protease). In this complex, Daxx can stimulate the stabilizing effect of Hausp on Mdm2, which results in accumulation of Mdm2 in the cell. Moreover, Daxx enhances the intrinsic E3 activity of Mdm2 toward p53 leading to the inhibition of the antiproliferative effects of p53. Upon DNA damage, Daxx, HAUSP, and p53 dissociate from Mdm2, which allows Mdm2–MdmX complex to undergo autoubiquitination and subsequent degradation. The dissociated Daxx and p53 may then form an apoptotic complex with HIPK2 and Axin in the nucleus, in which Daxx and Axin cooperatively promote the HIPK2 kinase activity toward p53 at Serine 46. Therefore, in response to stress signals, Daxx enhances the death-inducing function of p53, suggesting that Daxx in fact may exert opposing roles in controlling p53 activity depending on cellular states. However, the detailed molecular mechanisms as to how Daxx dissociates from the Daxx/Mdm2/HAUSP/Mdmx complex and recycles to form an apoptotic complex upon DNA damage remain to be clarified in the future.

Cross-References ▶ MDM2 ▶ p53 Family ▶ TP53 ▶ Tumor Suppressor Genes ▶ Ubiquitination

Decatenation G2 Checkpoint

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References

Death Receptors Li Q, Wang X, Wu X et al (2007) Daxx cooperates with the Axin/HIPK2/p53 complex to induce cell death. Cancer Res 67:66–74 Salomoni P, Khelifi AF (2006) Daxx: death or survival protein. Trends Cell Biol 16:97–104 Tang J, Qu L-K, Zhang J et al (2006) Critical role for Daxx in regulating Mdm2. Nat Cell Biol 8:855–862 Yang X, Khosravi-Far R, Chang HY et al (1997) Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 89:1067–1076

DCIS ▶ Ductal Carcinoma In Situ

Definition Death receptors belong to the ▶ tumor necrosis factor (TNF) receptor gene superfamily, which is defined by similar, cysteine-rich extracellular domains. The death receptors also contain a homologous cytoplasmic sequence termed the “death domain.” Death domains typically enable death receptors to engage the cell’s apoptotic machinery, but in some instances they mediate functions that are distinct from or even counteract apoptosis. Some molecules that transmit signals from death receptors contain death domains themselves.

Cross-References

DcR3

▶ FLICE-Inhibitory Protein ▶ Tumor Necrosis Factor

▶ Decoy Receptor 3

See Also

DCX

(2012) Death domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1065. doi:10.1007/978-3-642-16483-5_1534

▶ Doublecortin

Death-Associated Protein 6 DDP

▶ Daxx

▶ Cisplatin

Decatenation G2 Checkpoint DDS ▶ Drug Delivery Systems for Cancer Treatment

William K. Kaufmann Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Definition

Death ▶ Necrosis

A surveillance system that ensures that mitosis does not begin when topoisomerase IIa is locked

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in the closed clamp conformation by topoII catalytic inhibitors.

Characteristics During DNA replication, daughter DNA duplexes become catenated or entangled through knots and links. These knots and links must be removed before sister chromatids can be properly segregated during mitosis. Type II DNA ▶ topoisomerases are enzymes that remove knots and links through a concerted set of reactions in which a proteinassociated DNA double-strand break is generated, another segment of DNA is passed through the break, and the protein-associated double-strand break is reversed. The decatenation G2 checkpoint is an active signal transduction pathway that monitors topoisomerase IIa catalytic status and controls the location and/or activity of mitosis-promoting factor to delay the onset of mitosis. Molecular Biology Drugs that inhibit topoisomerase II fall into two groups known as poisons and catalytic inhibitors. Topoisomerase II poisons stabilize the enzyme when in a covalent complex with cleaved DNA strands. Proteolysis of the protein produces frank DNA double-strand breaks which are potentially lethal lesions. Topoisomerase poisons such as doxorubicin and etoposide are among the most widely used and successful of cancer chemotherapeutic drugs. The catalytic inhibitors of topoisomerase II prevent the enzyme from forming protein-associated DNA double-strand breaks; by preventing formation of protein-associated DNA double-strand breaks, catalytic inhibitors are able to block the toxicity of the poisons. There are two type II topoisomerases in mammalian cells. Topoisomerase IIb is a nonessential enzyme that is constitutively expressed in quiescent and proliferating cells. Topoisomerase IIa is an essential enzyme whose level of expression is tightly regulated in the cell division cycle. Maximal expression occurs in G2 and M when topoisomerase II is required to separate linked sister chromatids and condense mitotic chromosomes. Catalytic inhibitors of topoisomerase II include

Decatenation G2 Checkpoint

ICRF-193 and ICRF-187. ICRF-193 causes mammalian cells to delay progression from G2 to mitosis and impedes separation of sister chromatids at anaphase in mitosis. This behavior suggested that cells monitor the state of chromatid catenation in G2 and delay entry to mitosis until sister chromatids are sufficiently decatenated. A test of this hypothesis indicated that chromatid catenations did not induce G2 arrest in human fibroblasts. Further biochemical analysis demonstrated that ICRF-193 inhibited topoisomerase IIa in a conformation with phospho-ser1524 accessible for interaction with MDC1. Binding of MDC1 was required for ICRF-193-induced G2 arrest. As MDC1 binds ATM and ATM is both activated by ICRF-193 and required for ICRF-193-induced G2 arrest, a current model holds that the decatenation G2 checkpoint involves activation of ATM at sites of catalytically inhibited topoisomerase IIa. Both ICRF drugs cause cells to delay progression from G2 to mitosis. The G2 delay was established as an active checkpoint when various loss-of-function mutations or genetic alterations were shown to reverse the delay. The decatenation G2 checkpoint requires signaling by ATM, BRCA1, and WRN to block cellular entry to mitosis. Override of decatenation G2 checkpoint function using caffeine induces chromosome aberrations (breaks and exchanges) as mitotic cells attempt to condense and segregate knotted and linked chromatids. The transition from G2 to mitosis (▶ G2/M Transition) is controlled by mitosis-promoting factor, a ▶ cyclin-dependent kinase made up of a catalytic subunit, CDK1, and a regulatory subunit, cyclin B1. Cyclin B1/CDK1 complexes can initiate all steps of mitosis including nuclear envelope breakdown, chromosome condensation, and formation of the mitotic spindle apparatus. Cyclin B1 is co-regulated with topoisomerase IIa, with peak levels of expression in G2 and mitosis. The activity of mitosis-promoting factor is regulated by phosphorylation/dephosphorylation. Inhibitory phosphates in the enzyme catalytic site are removed at the onset of mitosis by CDC25B/C. Cyclin B1/CDK1 complexes also are actively exported from the nucleus during G2, but at the ▶ G2/M transition this exportation is inhibited, intranuclear

Decay-Accelerating Factor

transport is enhanced, and mitosis-promoting factor accumulates in the nucleus. The decatenation G2 checkpoint blocks cellular progression from G2 to mitosis by preventing the accumulation of mitosispromoting factor in the nucleus. The decatenation G2 checkpoint appears to function by inhibiting the activity of the polo-like kinase PLK1 that stimulates nuclear accumulation of mitosis-promoting factor. Clinical Implications The decatenation G2 checkpoint blocks progression from G2 to M when topoisomerase IIa is inhibited in a closed clamp conformation. Override of decatenation G2 checkpoint function using drugs such as caffeine is expected to cause instability of chromosome numbers and structure (▶ Chromosomal instability) as cells attempt mitosis with intertwined and insufficiently condensed chromatids. Linked chromatids may break under the force of chromatid condensation or segregation producing deletions, amplifications, and rearrangements. Cells with defective decatenation G2 checkpoint function due to defects in ATR, ATM, BRCA1, and WRN display chromosomal instability. The breast cancer susceptibility gene BRCA1 is not only required for decatenation G2 checkpoint function, it also regulates the decatenatory activity of DNA topoisomerase IIa. BRCA1 contains an ubiquitin-ligase activity that is expressed in the presence of the cofactor BARD1. BRCA1 regulates ▶ ubiquitination of topoisomerase IIa, and ubiquitination stimulates decatenatory activity of topoisomerase IIa. Thus, breast cancers with inactivation of BRCA1 may display less chromatid decatenation by topoisomerase IIa. BRCA1 also serves as a mediator in DNA damage and decatenation G2 checkpoints, enhancing phosphorylation by checkpoint kinases of their downstream targets. Breast cancers with defects in BRCA1 are less able to decatenate daughter chromatids and less able to delay mitosis when topoisomerase IIa is inhibited. ▶ Lung cancer lines with defects in decatenation G2 checkpoint function display hypersensitivity to killing by ICRF-187. This suggests that topoisomerase IIa catalytic inhibitors may be clinically useful for selected cancers.

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Cancer stem cells represent immortal clones that drive malignant progression (Stem Cells and Cancer). Mouse embryonic stem cells and human hematopoietic stem cells when grown in cell culture display a defect in the decatenation G2 checkpoint. Because decatenation G2 checkpoint function preserves chromosomal stability during cell division, a checkpoint defect may promote ▶ chromosomal instability in cancer stem cells.

Cross-References ▶ Chromosomal Instability ▶ Cyclin-Dependent Kinases ▶ G2/M Transition ▶ Lung Cancer ▶ Topoisomerases

References Bower JJ, Karaca GF, Zhou Y, Simpson DA et al (2010) Topoisomerase IIa maintains genomic stability through decatenation G(2) checkpoint signaling. Oncogene 29:4784–4799 Damelin M, Sun YE, Sodja VB et al (2005) Decatenation checkpoint deficiency in stem and progenitor cells. Cancer Cell 8:479–484 Downes CS, Clarke DJ, Mullinger AM et al (1994) A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 372:467–470 Lou Z, Minter-Dykhouse K, Chen J (2005) BRCA1 participates in DNA decatenation. Nat Struct Mol Biol 12:589–593 Luo K, Yuan J, Chen J, Lou Z (2009) Topoisomerase IIa controls the decatenation checkpoint. Nat Cell Biol 11:204–210

Decay-Accelerating Factor Burkhard H. Brandt Institute of Clinical Chemistry, University Medical Centre Schleswig-Holstein, Kiel, Germany

Synonyms Antigen of the Cromer blood group; CD55; DAF; Decay-accelerating factor for complement

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Definition Decay-accelerating factor (DAF) participates in the regulation of complement system activity by accelerating the decay of the C3/C5 convertase of the classic as well as of the alternative pathway. The highly polymorphic 50–100 kDa protein facilitates complement regulation by cysteine rich complement control protein repeats (CCP). Most of the DAF isoforms are linked to the cell membrane by a glycosyl phosphatidylinositol (GPI) anchor following the CCPs.

Characteristics DAF has been detected in all mammalians. Physiologically it is expressed in all cells contacting the complement system enclosing cells within the peripheral blood and epithelial as well as endothelial cells. Soluble DAF is detectable in plasma, tears, saliva, and urine, as well as in synovial and cerebrospinal fluids. Besides its function as a regulator of the complement system, DAF inhibits natural killer cells, is a ligand of the CD97 receptor, and is a receptor for viruses and microorganisms. DAF is also involved in the development of spermatozoa and their survival within the female genital tract. DAF is a glycoprotein appearing as different isoforms depending partially on different posttranslational glycosylation patterns and on alternative splicing. The DAF protein possesses four units for the control of complement activation following each other and that are designated as CCP 1–4. Therefore, the molecular weight of the mature protein varies between 50 and 100 kDa in different cell types. DAF has been detected in different malignancies, e.g., CLL, CML, ALL, AML, ▶ colorectal cancer, ▶ gastric cancer, ▶ thyroid cancer, medullary thyroid carcinoma, malignant glioma, ▶ breast cancer, renal cancer, ▶ non-small cell lung cancer, ▶ ovarian cancer, ▶ cervical cancer, and also partially in metastases of colorectal carcinomas. Furthermore, DAF is frequently overexpressed within the stroma of colorectal tumors suggesting that DAF comes from the

Decay-Accelerating Factor

tumor cells and is either cleaved from the cell membrane into the environment or is secreted by the tumor cells as a soluble form. Expression of more than one DAF isoform originated from different glycosylation patterns (colon cancer), or alternative splicing (breast cancer) has been detected. Actions of DAF in Cancer DAF expression in cancer cells is upregulated by interleukins (IL-4, IL-1a, IL-1b), cytokines (TNF-a, IFN-g), growth factors (EGF, bFGF), ▶ prostaglandins (E2), and complement regulatory protein protectin (CD59) (Table 1). DAF decreases complement deposition on tumor cell membranes as well as complementmediated lysis in melanomas, renal tumors, thyroid, lung, squamous cell, and cervical carcinoma as well as in some hematological malignancies. DAF decreases cell adhesion of T-lymphocytes to leukemic cells as well as inhibiting effect on NK cells which could impair immune surveillance of cancer cells. The GPI-anchored and membrane-linked form of DAF is part of a signal transduction cascade. In consequence, tyrosine phosphorylation in malignant tumors functioning within signal transduction goes far beyond immune-modulating effects. For example, the tyrosine kinase p56lck participates in a signal cascade conveying active motility of breast cancer cells. DAF has been identified as ligand of the surface receptor CD97 (EGF-TM7), which belongs to the family of class B seven-span transmembrane (TM7) receptors. Predominantly expressed in hematopoietic cells, CD 97 is ectopic expressed in human thyroid, colorectal, gastric, pancreatic, esophageal, and oral squamous cell carcinomas. CD97 promotes invasive growth, ▶ migration, and ▶ angiogenesis. DAF, being the ligand of CD97, is synthesized and secreted by colorectal carcinoma cells leading to an autocrine stimulation of invasive growth, migration, and ▶ metastasis of these cells. A similar mechanism takes place in breast cancer cells as HER2-positive mammary carcinoma cells showing increased transendothelial invasiveness selectively overexpress and secrete a 45 kDa splice variant of DAF (Table 1).

Decay-Accelerating Factor

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Decay-Accelerating Factor, Table 1 Versatile actions of decay-accelerating factor in cancer Action ▶ Carcinogenesis

Complement inhibition

Inhibition of natural killer (NK) cells Oncogenic tyrosine kinase pathway activation

Angiogenesis Invasive growth, Metastasis

Cell migration

Survival and apoptosis

Mechanisms Upregulation of DAF in cancer and precancerous lesions (factors regulating DAF expression: IL-4, IL-1a, EGF, PGE2, TNFa, IFNg, thrombin, bFGF, VEGF, CD59) Decay acceleration of the C3/C5 convertase leading to decrease in complement deposition and in complement-mediated lysis of cancer cells Downregulation of NK cellmediated cell lysis Signal transduction through GPI-anchored form of DAF (tyrosine phosphorylation of src-kinases, TCR-zeta chain, and ZAP-70) Increase of DAF synthesis by VEGF Increase in DAF expression, ligand binding of DAF to CD97 (autocrine stimulation by tumor-specific DAF isoforms) Ligand binding of DAF to CD97, tyrosine phosphorylation of p56lck Induction of apoptosis through monoclonal SC-1antibody against tumorspecific DAF isoform (upregulation of c-myc, activation of caspase-6 and caspase-8, cleavage of cytokeratin 18)

Perspectives in Cancer Therapy Removal of DAF from the cell membrane or neutralization of the protein increases the intensity of a potential local inflammatory reaction as well as complement-mediated cell lysis and could also possibly improve response to special therapeutic strategies (Table 2). In consequence, DAF was used as a target for molecular cancer therapy. Monoclonal antibody SC-1 binding an isoform of DAF expressed in gastric carcinoma induced ▶ apoptosis of these cells. In first clinical trials,

Decay-Accelerating Factor, Table 2 DAF targeted therapies Targeted cancer Stomach adenocarcinoma of diffuse type

Metastasizing gastric cancer in nude mice

Non-Hodgkin lymphoma

Cervical cancer

Renal cancer

Osteosarcoma of children

Melanoma xenografts in immunodeficient mice

Therapeutic investigational design and benefit Monoclonal antibody SC-1 against a gastric cancerspecific 82 kD isoform of DAF; induced apoptosis in primary tumors as compared to pretreatment biopsy material in up to 90% of the cases, regression of tumor mass up to 50% Monoclonal antibody SC-1 against a gastric cancerspecific 82 kD isoform of DAF; reduce the number of disseminated tumor cells in the bone marrow Rituximab (chimeric antiCD20 monoclonal antibody); enhancement of complement-dependent killing activity of rituximab, by additional application of monoclonal anti-DAF antibody Monoclonal antibody against DAF showed the widest range of specific reactivity Bispecific antibodies binding DAF as well as renal tumor-associated antigen G250; decrease of unwanted side effects DAF as a cancer vaccine additionally applied to myelosuppressive chemotherapy; induction of T-cell proliferation 71% and antigen-specific gammaIFN secretion in 59% of the cases; vaccination was well tolerated Coxsackievirus A21 infection; rapid viral oncolysis

patients with poorly differentiated stomach adenocarcinoma of diffuse type have been treated primarily with the SC-1 antibody followed by gastrectomy and lymphadenectomy. A significant induction of apoptotic activity in primary tumors as compared to pretreatment biopsy material in up

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to 90% of the cases and a significant regression of tumor mass in up to 50% was observed. Application of SC-1 antibody therapy in nude mice with metastasizing gastric cancer reduced the number of disseminated tumor cells in bone marrow. Other studies could show that the complementdependent killing activity of ▶ rituximab, a chimeric anti-CD20 monoclonal antibody, in the treatment of non-Hodgkin lymphoma cells is enhanced by additional application of monoclonal anti-DAF antibody. The DAF is also expressed in many physiological human cells. Therefore, the use of bispecific antibodies binding DAF as well as another tumor-specific antigen like the renal tumor-associated antigen G250 might be a solution to decrease unbeneficial side effects. Additional application of DAF as a cancer vaccine for ▶ osteosarcoma under myelosuppressive chemotherapy induced T-cell proliferation gammaIFN secretion. DAF as a receptor for viruses enhanced the systemic effect on metastatic melanoma cells by an infection with coxsackieA21 viruses (Table 2).

Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Acute Myeloid Leukemia ▶ Angiogenesis ▶ Apoptosis ▶ Breast Cancer ▶ Cancer Vaccines ▶ Carcinogenesis ▶ Cervical Cancers ▶ Chronic Lymphocytic Leukemia ▶ Chronic Myeloid Leukemia ▶ Colorectal Cancer ▶ Complement-Dependent Cytotoxicity ▶ Gastric Cancer ▶ HER-2/neu ▶ Interleukin-4 ▶ Metastasis ▶ Migration ▶ Natural Killer Cell Activation ▶ Non-Small-Cell Lung Cancer

Decay-Accelerating Factor

▶ Osteosarcoma ▶ Ovarian Cancer ▶ Papillary Thyroid Carcinoma ▶ Prostaglandins ▶ Rituximab ▶ Thyroid Carcinogenesis ▶ Tumor Necrosis Factor

References Brandt B, Mikesch JH, Simon R et al (2005) Selective expression of a splice-variant of decay-accelerating factor (DAF) in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness. Biochem Biophys Res Commun 329:319–324 Fishelson Z, Donin N, Zell S et al (2003) Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol 40:109–123 Mikesch JH, Buerger H, Simon R et al (2006) Decayaccelerating factor (CD55): a versatile acting molecule in human malignancies. Biochim Biophys Acta 1766:42–52 (Review) Niehans GA, Cherwitz DL, Staley NA et al (1996) Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin). Am J Pathol 149:129–142 Spendlove I, Li L, Carmichael C et al (1999) Decay accelerating factor (CD55): a target for cancer vaccines? Cancer Res 59:2282–2286

See Also (2012) BFGF. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 388. doi:10.1007/978-3-642-16483-5_596 (2012) EGF. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1824 (2012) Glioma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1557. doi:10.1007/978-3-642-16483-5_2423 (2012) IFN. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1806. doi:10.1007/978-3-642-16483-5_2949 (2012) Interleukin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1892. doi:10.1007/978-3-642-16483-5_3094 (2012) Medullary thyroid carcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2199-2200. doi:10.1007/978-3-642-164835_3600

Decoy Receptor 3 (2012) Non-hodgkin lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4110 (2012) Renal cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3225– 3226. doi:10.1007/978-3-642-16483-5_6575

Decay-Accelerating Factor for Complement ▶ Decay-Accelerating Factor

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Decoy Receptor 3 Shie-Liang Hsieh1 and Wan-Wan Lin2 1 Department of Microbiology and Immunology, National Yang-Ming University, Immunology Research Center, Taipei Veterans General Hospital; Genomics Research Center, Academia Sinica, Taipei, Taiwan 2 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

Synonyms

Decitabine

DcR3

▶ 5-Aza-20 Deoxycytidine

Definition

Decoy Receptor Definition Decoy receptors recognize certain growth factors (such as ▶ vascular endothelial growth factor) or ▶ cytokines with high affinity and specificity but are structurally incapable of signaling or presenting the agonist to signaling receptor complexes. They act as a molecular trap for the agonist and for signaling receptor components. A decoy receptor, or sink receptor, is a receptor that binds a ligand, inhibiting it from binding to its normal receptor. For instance, the receptor VEGFR-1 can prevent ▶ vascular endothelial growth factor (VEGF) from binding to the VEGFR-2.

Cross-References ▶ Cytokine ▶ Decoy Receptor 3 ▶ Vascular Endothelial Growth Factor

DcR3 is a member of the ▶ TNF receptor superfamily (TNFRSF6B), and the DcR3 gene is mapped to chromosome 20q13.3. DcR3 is a glycosylated protein of 300 amino acids and 33 kD. The receptor lacks a transmembrane domain and exists as soluble protein.

Characteristics Tissue Expression and Decoy Function of DcR3 DcR3 is generally undetectable in normal tissues but is highly expressed in several human malignant tissues, such as adenocarcinomas of the esophagus, stomach, colon, rectum, pancreas and lung, glioblastomas, and lymphomas. In addition, high serum levels of DcR3 have been detected in many cancer patients. Clinical data indicate the significance of detecting serum DcR3 as a novel parameter for the diagnosis, treatment, and prognosis of malignancies. Several lines of evidence suggest a significant role for DcR3 in immune suppression and tumor progression. DcR3 has been regarded to function as decoy receptor for

D

1318 Decoy Receptor 3, Fig. 1 This figure shows the biological activities of DcR3. Tumor-secreted DcR3 can neutralize FasL, LIGHT, and TL1A and also bind to HSPG of monocytes to trigger signal cascades, resulting in differentiation of M2 macrophages, osteoclasts, and Th2dominant immune response

Decoy Receptor 3 Th2-prone cytokine production

M2 Macrophage polarization

Th2 Mφ

DC

FasL

Fas-mediated apoptosis Fas

HSPG DcR3

Monocyte

DcR3 Tumor LIGHT

Th1 T cell activation

Osteoclast formation

TL1A

TL1A-induced endothelial cell death

FasL, LIGHT (homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes), and TL1A, which are three cytokine members of the TNF family. FasL and LIGHT are expressed by activated T cells and can induce tumor cell death through signaling pathways mediated by Fas and lymphotoxin b receptor (LTbR), respectively. LIGHT is also a T cell co-stimulator, and this action is mediated by receptor herpesvirus entry mediator (HVEM). Therefore, Fas and LIGHT are two cytokines contributing to the host immune surveillance. TL1A is an angiostatic cytokine releasing from endothelial cells. The cytokine neutralizing actions confer DcR3 function as a tumor molecule to decrease T-cell-mediated immunity and stimulate ▶ angiogenesis. Tumor cells engineered to release a higher amount of DcR3 may escape from FasLinduced apoptotic cell death. Decoy Unrelated Novel Actions of DcR3 In addition to neutralizing bioactive cytokines, DcR3, which is also an effector molecule, directly modulates the activities of many cell types. DcR3 can regulate ▶ dendritic cells

(DCs) differentiation and downregulate several co-stimulatory molecules, leading to Th2 polarization (Fig. 1). Moreover, DcR3 induces actin reorganization and adhesion of monocytes as well as reducing phagocytic activity and proinflammatory cytokine production in ▶ macrophages. Osteoclast formation is promoted by the addition of DcR3 to monocyte/macrophage lineage precursor cells. Finally, DcR3 increases monocyte adhesion to endothelial cells via ▶ NF-kB activation, leading to the transcriptional up-regulation of adhesion molecules and IL-8 in endothelial cells. Thus DcR3 has pleiotropic effects to modulate inflammation and ▶ osteoporosis. As chronic ▶ inflammation has long been associated with tumorigenesis, DcR3induced inflammation might provide a beneficial microenvironment for tumor growth. Increased ▶ osteoclast activity and decreased bone density are observed in DcR3 transgenic mice; therefore, DcR3 might also play an important role in bone erosion and destruction in cancer patients. New Action Mechanisms of DcR3 It is a mystery why DcR3 has such diverse immunomodulatory functions independent of the neutralizing FasL, LIGHT, and TL1A (Fig. 1).

Deferasirox

A glycosaminoglycan-binding domain of DcR3 has been identified as binding and cross-linking heparan sulfate proteoglycans (HSPG), such as syndecan 2 and CD44v3, to induce monocyte adhesion via activating PKC. Recombinant protein, comprising the HSPG-binding domain (HBD) of DcR3 and Fc portion of human IgG1 (HBD.Fc), has a similar effect as DcR3.Fc to modulate the activation and differentiation of DCs, macrophages, and induce osteoclast differentiation. Even though Fc stabilizes dimeric DcR3 and enhances cross-linking activity, transgenic mice overexpressing DcR3 also attenuates Th1 differentiation and enhances osteoclast formation. This indicates that the Fc portion is dispensable, and the biological effects of DcR3 in vivo are not restricted to its neutralizing effects on FasL, LIGHT, and TL1A.

1319 NF-kB-dependent up-regulation of intercellular adhesion molecule-1, VCAM-1, and IL-8 expression. J Immunol 174:1647–1656

See Also (2012) HVEM. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 1766-1767. doi:10.1007/978-3-642-16483-5_2872

D Deferasirox Galit Granot Felsenstein Medical Research Center, Beilinson Hospital, Sackler School of Medicine, Tel Aviv University, Petah Tikva, Israel

Synonyms Cross-References ▶ Angiogenesis ▶ Dendritic Cells ▶ Inflammation ▶ Macrophages ▶ Nuclear Factor-kB ▶ Osteoclast ▶ Osteoporosis ▶ Tumor Necrosis Factor

References Chang YC, Chan YH, Jackson DG et al (2006) The glycosaminoglycan binding domain of decoy receptor 3 is essential for induction of monocyte adhesion. J Immunol 176:173–180 Chang YC, Hsu TL, Lin HH et al (2004) Modulation of macrophage differentiation and activation by decoy receptor 3. J Leukoc Biol 75:486–494 Hsu TL, Chang YC, Chen SJ et al (2002) Modulation of dendritic cell differentiation and maturation by decoy receptor 3. J Immunol 168:4846–4853 Yang CR, Hsieh SL, Teng CM et al (2004) Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A, a cytokine belonging to TNF superfamily and exhibiting angiostatic action. Cancer Res 64:1122–1129 Yang CR, Hsieh SL, Ho FM et al (2005) Decoy receptor 3 increases monocyte adhesion to endothelial cells via

Exjade1; ICL670A

Definition Deferasirox is a rationally designed orally administered iron chelator.

Characteristics Pharmacological Properties Deferasirox is a rationally designed oral iron chelator. Deferasirox is indicated for the treatment of chronic iron overload in patients who are receiving long-term blood transfusions for conditions such as b-thalassemia and other chronic anemias. It was approved by the United States Food and Drug Administration (FDA) in November 2005. It is the first oral medication approved in the USA for this purpose. Its low molecular weight and high lipophilicity allows the drug to be taken orally unlike deferoxamine (DFO) which has to be administered by IV route. The half-life of deferasirox is between 8 and 16 h allowing once a day dosing. Deferasirox is capable of removing iron from cells

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Deferasirox O

O

O OH

OH

+

NH2

SOCl2, pyridine xylenes, reflux, 4 h

N

OH

O

OH O

OH H2N

O

HN OH

, Et3N

EtOH, reflux, 2 h

N

N N HO

OH Defeasirox

Deferasirox, Fig. 1 Synthesis of deferasirox (Tiwari. et al Journal of Analytical Sciences, Methods and InstrumentationVol.3 No.4(2013), Article ID:38319

as well as removing iron from the blood. Deferasirox is a tridentate iron chelator with high affinity for iron as Fe3+; two molecules of deferasirox bind to one Fe3+. This complex is excreted in bile and eliminated primarily via the feces. Tolerability Deferasirox is associated with a clinically manageable tolerability profile. The most common adverse events associated with deferasirox treatment include transient gastrointestinal adverse events (nausea, vomiting, abdominal pain, constipation, and diarrhea) and skin rash. Other adverse events include elevated serum creatinine and, less frequently, elevated ALT levels and visual and auditory disturbances. Regular monitoring and careful management are recommended. Postmarketing cases of renal failure and cytopenia have been reported. Synthesis Deferasirox is prepared from commercially available salicylic acid, salicylamide, and 4hydrazinobenzoic acid in the following two-step synthetic sequence (Fig. 1): The condensation of salicyloyl chloride (formed in situ from salicylic acid and thionyl

chloride) with salicylamide under dehydrating reaction conditions results in formation of 2-(2-hydroxyphenyl)-1,3(4H)-benzoxazin-4-one. This intermediate is isolated and reacted with 4-hydrazinobenzoic acid in the presence of base to give 4-(3,5-bis(2-hydroxyphenyl)-1,2,4triazol-1-yl)benzoic acid (deferasirox). Iron

Iron is essential for life. Hemoglobin and myoglobin both contain iron; therefore, it plays a crucial role in oxygen transport and storage. Iron is equally as vital for processes such as ATP generation, effective DNA synthesis, and cell cycle progression. Iron homeostasis is strictly regulated at the level of intestinal absorption and any deviation from this tight regulation can have deleterious effects. This is best exemplified by the clinical syndrome of iron deficiency anemia, and on the other hand, since the body is incapable of effective iron excretion, excess systemic iron (as in the iron storage disease hereditary hemochromatosis or in iron overload conditions such as b-thalassemia and Friedreich’s ataxia) leads to iron deposition in a number of organs including the heart, pancreas, and liver. This in turn causes irreversible tissue damage and fibrosis through the action of

Deferasirox

▶ reactive oxygen species (ROS) generated by the iron. As such, excess iron has been implicated in a number of diseases including ischemic heart disease, diabetes mellitus, and neurodegenerative disorders. Iron and Cancer

Iron-induced malignant tumors were first reported in 1959 by repeated intramuscular injection of iron dextran complex in rats. About 20 years later, sarcomas were shown to develop after intramuscular injection of iron. At that time, epidemiological reports had also associated increased iron exposure with elevated cancer risk. Although epidemiological studies on the association of iron with cancer remain inconclusive, the majority of these reports support the role of iron in human cancer. There has been data supporting the notion that iron overload is a risk factor for ▶ liver cancer, kidney cancer, lung cancer, ▶ stomach cancers, and ▶ colorectal cancer. Iron Chelation in Cancer Therapy

Tumor cells in a highly proliferative state demonstrate elevated iron uptake and metabolism and have a high density of transferrin receptors. The dependency of tumor cells on high levels of iron and their sensitivity to iron depletion is believed to be the Achilles’ heel of many types of cancers, and thus, it drove researchers to implement Fe chelators, initially developed to treat iron overload, as therapeutic antitumoral agents. It has been shown that iron chelators achieve their anticancer effect by targeting molecules that are critical for the regulation of both ▶ cell cycle and apoptotic processes. Indeed, iron chelation (mainly by DFO) has shown antiproliferative activity against leukemia and ▶ neuroblastoma cells in vitro, in vivo, and in ▶ clinical trials. Transferrin receptor antisense cDNA was shown to reduce transferrin receptor expression and subsequently to inhibit the growth of human ▶ breast carcinoma cells. Monoclonal antibodies against transferrin receptor severely restricted the growth of lymphoma tumors in mice. These data suggest that iron deprivation may be a useful anticancer strategy.

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Deferasirox in Cancer Therapy

Given the apparent association between excess iron and cancer, there is significant interest in the investigation and development of iron-chelating drugs as antineoplastic agents. Over the past few years, the potential for deferasirox to act as a cytotoxic agent has been investigated. At present results are predominantly limited to in vitro and in vivo laboratory studies. Human data currently comprises only small case series and anecdotal case reports. Deferasirox has been shown to decrease cell viability, inhibit DNA replication, and induce DNA fragmentation in the human ▶ hepatoma cell line (HUH7) and human hepatocyte cultures. Interestingly, in contrast to other iron chelators, deferasirox also induces cell cycle blockade during S phase rather than the G1 phase. Importantly, higher concentrations of deferasirox are necessary to induce cytotoxicity in primary hepatocyte cultures compared to hepatoma cells suggesting that malignant cells may be more sensitive to deferasirox than benign ones. Deferasirox was found to significantly reduce esophageal and lung tumor xenograft size with no marked alterations in normal tissue histology. Deferasirox was shown to increase the expression of the metastasis suppressor protein ▶ N-myc downstream-regulated gene 1. In addition, it was shown to upregulate the cyclin-dependent kinase inhibitor ▶ p21 while decreasing ▶ cyclin D1 levels. Moreover, this agent was shown to induce the expression of apoptosis markers, including cleaved caspase-3 and cleaved PARP. Deferasirox and Mantle Cell Lymphoma

The biological and clinical heterogeneity of ▶ mantle cell lymphoma (MCL) is a substantial obstacle in treating and overcoming resistance in this disease. Several studies have represented new therapeutic approaches and strategies to target MCL by exploiting its unique biological features such as the ▶ mTOR and Bruton’s tyrosine kinase (BTK) inhibitors, ▶ everolimus and ibrutinib, respectively. However, the emerging role of cancer metabolism has unveiled new potential targets to treat cancer in general and MCL in particular. As mentioned above, malignant cells exhibit an

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elevated proliferative potential and rapid growth rates and accordingly elevated iron uptake and metabolism. Interestingly, relapsed MCL tumors exhibit increased transferrin receptor mRNA levels when compared to primary tumors. In that concern, the antihuman transferrin receptor monoclonal antibody, A24, was shown to block MCL cell proliferation, to induce cell apoptosis, and to repress tumor growth in mice. These findings not only suggest an essential role of iron in MCL but also point out the vulnerability of MCL cells to iron deprivation making iron deprivation a useful approach for complementing current firstline therapies in this lymphoma. Although currently DFO is considered the gold standard among iron chelation therapeutic approaches, deferasirox has shown promising results in MCL. Deferasirox exhibited antitumoral activity against the MCL cell lines HBL-2, Granta-519, and Jeko-1. Deferasirox was shown to induce apoptosis mediated through caspase-3 activation and decreased cyclin D1 protein levels resulting from increased proteasomal degradation. In addition, exposure to deferasirox led to reduced phosphorylation of RB (Ser780), which resulted in increasing levels of the E2F/RB complex and G1/S arrest. The effect of deferasirox on these MCL cell lines was dependent on its ironchelating ability. These data indicate that deferasirox, by downregulating cyclin D1 and inhibiting its related signals, may constitute a promising adjuvant therapeutic molecule in the strategy for MCL treatment. Deferasirox and the PI3K/AKT/mTOR Pathway

The mTOR pathway is known to demonstrate aberrant activity in a number of cancers including ovarian, breast, colon, brain, lung and MCL. Overactivity of this pathway culminates in deregulation of the cell cycle and increased cellular proliferation. Conversely, inhibition of the mTOR pathway results in cytostatic effects. Deferasirox was shown to represses the mTOR signaling pathway in myeloid leukemia cells by enhancing the expression of REDD1. REDD1 is a stress response gene strongly induced by hypoxia. REDD1 can activate the TSC2 protein which is

Deferasirox

composed of TSC1 and TSC2. It has been shown that the major function of the TSC1/2 complex is to inhibit the checkpoint protein kinase mTOR (a major regulator of cell death and proliferation). mTOR enhances translational initiation in part by phosphorylating two major targets, the eIF4Ebinding protein (4E-BPs) and the ribosomal protein S6 (S6K1 and S6K2) that cooperate to regulate translational initiation rates. Deferasirox and the Wnt Signaling Pathway

Excessive signaling from the Wnt pathway is associated with numerous human cancers, most notably colorectal. In the absence of Wnt, cytoplasmic b-catenin protein is constantly degraded by the Axin complex, which is composed of the Axin protein, the tumor suppressor ▶ adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 b (GSK-3b). CK1 and GSK-3b sequentially phosphorylate b-catenin, resulting in the recognition of b-catenin by the E3 ubiquitin ligase, b-Trcp, leading to b-catenin ubiquitination and subsequent proteasomal degradation. This process prevents b-catenin from reaching the nucleus, enabling the Wnt target genes to be repressed by the DNA-bound T cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins. The Wnt/b-catenin pathway is activated when a Wnt ligand binds to a seven-pass transmembrane Frizzled (Fz) receptor and its co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5) or to LRP6. When this complex recruits the Dishevelled (Dvl) protein, LRP6 is phosphorylated and activated and the Axin complex is recruited to the receptors. These events lead to inhibition of Axinmediated b-catenin phosphorylation and thereby to the stabilization of b-catenin, which accumulates and travels to the nucleus to form complexes with TCF/LEF and activates Wnt target gene expression. Deferasirox has been shown to attenuate Wnt signaling and cell growth in colorectal cancer cell lines with constitutive Wnt signaling through an iron-dependent mechanism. Deferasirox and Nuclear Factor-kB (NF-kB)

NF-kB is a protein known to regulate several fundamental cellular processes such as apoptosis,

Deferasirox

proliferation, differentiation and migration. Incorrect regulation of NF-kB plays a pivotal role in the pathogenesis of a number of cancers. It has been shown to be constitutively active in many tumor cell lines and tissue samples. Deferasirox was found to dramatically reduce NF-kB activity in both leukemia cell lines and blood samples from patients with myelodysplastic disorders. In these experiments, the addition of ferric hydroxyquinoline during incubation did not reinstate NF-kB activity suggesting that deferasirox’s effect may be independent of its iron-chelating abilities. NF-kB pathway inhibition was witnessed solely with deferasirox (it did not occur with DFO or deferiprone). Interesting Case Report

A 73-year-old Japanese man diagnosed with acute monocytic leukemia (AML) was refractory to conventional chemotherapies. After declining further chemotherapy, he started receiving red blood cell transfusion to maintain a pretransfusional hemoglobin level. In parallel, iron chelation therapy with deferasirox was initiated. Less than 2 years later, bone marrow aspiration and biopsy revealed hematological and cytogenetic complete remission; the bone marrow was normocellular without leukemic monoblasts and myelofibrosis; cytogenetic abnormalities had disappeared in conventional karyotype analysis. Till the time when the paper was written (November 2010), normal blood cell counts have been maintained without transfusion. Remarkably, complete remission was achieved after iron chelation therapy with deferasirox in this AML patient, suggesting that deferasirox may have an antileukemic effect in the clinical setting. Conclusion Malignant cells exhibit an elevated proliferative potential and rapid growth rates and accordingly elevated iron uptake and metabolism. Therefore, most tumor cells are dependent on high levels of iron and are consequently sensitive to iron depletion. Deferasirox is an emerging drug that features a variety of significant biochemical, pharmaceutical and clinical advantages over other traditional iron chelators and is considered an effective

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antitumor agent against solid tumors, leukemias and lymphomas.

Cross-References ▶ Acute Myeloid Leukemia ▶ Cell Cycle Checkpoint ▶ Clinical Trial ▶ Colorectal Cancer ▶ Cullin Ubiquitin E3 Ligases ▶ Everolimus ▶ Gastric Cancer ▶ Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention ▶ Mantle Cell Lymphoma ▶ Neuroblastoma ▶ N-myc Downstream-Regulated Gene ▶ P21 ▶ Reactive Oxygen Species

References Bedford MR, Ford SJ, Horniblow RD, Iqbal TH, Tselepis C (2013) Iron chelation in the treatment of cancer: a new role for deferasirox? J Clin Pharmacol 53:885–891 Chantrel-Groussard K, Gaboriau F, Pasdeloup N, Havouis R, Nick H, Pierre JL, Brissot P, Lescoat G (2006) The new orally active iron chelator ICL670A exhibits a higher antiproliferative effect in human hepatocyte cultures than O-trensox. Eur J Pharmacol 541:129–137 Fukushima T, Kawabata H, Nakamura T, Iwao H, Nakajima A, Miki M, Sakai T, Sawaki T, Fujita Y, Tanaka M, Masaki Y, Hirose Y, Umehara H (2011) Iron chelation therapy with deferasirox induced complete remission in a patient with chemotherapy-resistant acute monocytic leukemia. Anticancer Res 31:1741–1744 Huang X (2003) Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal. Mutat Res 533:153–171 Vazana-Barad L, Granot G, Mor-Tzuntz R, Levi I, Dreyling M, Nathan I, Shpilberg O (2013) Mechanism of the antitumoral activity of deferasirox, an iron chelation agent, on mantle cell lymphoma. Leuk Lymphoma 54:851–859

See Also (2001) Hepatoma. In: Schwab M (ed) Encyclopedic reference of cancer. Springer, Berlin/Heidelberg, p 401. doi:10.1007/3-540-30683-8_738

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1324 (2001) Reactive oxygen species. In: Schwab M (ed) Encyclopedic reference of cancer. Springer, Berlin/Heidelberg, pp 755–756. doi:10.1007/3-540-30683-8_1440 (2004) Retinoblastoma (Rb) gene. In: Offermanns S, Rosenthal W (eds) Encyclopedic reference of molecular pharmacology. Springer, Berlin/Heidelberg, p 815. doi:10.1007/3-540-29832-0_1404 (2005) Transferrin receptor. In: Vohr HW (ed) Encyclopedic reference of immunotoxicology. Springer, Berlin/ Heidelberg, p 658. doi:10.1007/3-540-27806-0_1499 (2006) Adenomatous polyposis coli. In: Ganten D et al (eds) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, p 20. doi:10.1007/3-540-29623-9_6056 (2006) Apoptosis. In: Ganten D et al (eds) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, pp 84–85. doi:10.1007/3-540-29623-9_6191 (2006) Cyclin D. In: Ganten D et al (eds) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, p 363. doi:10.1007/3-540-29623-9_6677 (2006) Dishevelled. In: Ganten D et al (eds) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, p 409. doi:10.1007/3-540-29623-9_6774 (2008) Btk (Bruton’s tyrosine kinase). In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics. Springer, Netherlands, p 241. doi:10.1007/978-1-4020-6754-9_2087 (2008) Frizzled. In: Offermanns S, Rosenthal W (eds) Encyclopedia of molecular pharmacology, 2nd edn. Springer, Berlin/Heidelberg, p 511. doi:10.1007/9783-540-38918-7_5730 (2008) GSK3 (glycogen synthase kinase 3b). In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics. Springer, Netherlands, pp 827–828. doi:10.1007/978-1-4020-6754-9_7189 (2008) Iron Chelator. In: Offermanns S, Rosenthal W (eds) Encyclopedia of molecular pharmacology, 2nd edn. Springer, Berlin/Heidelberg, p 665. doi:10.1007/9783-540-38918-7_6014 (2008) Myelofibrosis. In: Baert AL (ed) Encyclopedia of diagnostic imaging. Springer, Berlin/Heidelberg, p 1183. doi:10.1007/978-3-540-35280-8_1626 (2008) Myoglobin. In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics. Springer, Netherlands, p 1314. doi:10.1007/978-14020-6754-9_11113 (2008) S6K1. In: Offermanns S, Rosenthal W (eds) Encyclopedia of molecular pharmacology, 2nd edn. Springer, Berlin/Heidelberg, p 1101. doi:10.1007/9783-540-38918-7_6664 (2009) Iron. In: Manutchehr-Danai M (ed) Dictionary of gems and gemology. Springer, Berlin/Heidelberg, p 473. doi:10.1007/978-3-540-72816-0_11678 (2012) Anemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 178. doi:10.1007/978-3-642-16483-5_269

Deferasirox (2012) Axin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 324. doi:10.1007/978-3-642-16483-5_496 (2012) bTrCP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3777. doi:10.1007/978-3-642-16483-5_5963 (2012) Beta-Catenin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 385. doi:10.1007/978-3-642-16483-5_889 (2012) Caspase-3. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 675. doi:10.1007/978-3-642-16483-5_874 (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) E3 Ubiquitin ligase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1184. doi:10.1007/978-3-642-16483-5_1771 (2012) Hemoglobin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1646. doi:10.1007/978-3-642-16483-5_2632 (2012) Hepatocyte. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1677. doi:10.1007/978-3-642-16483-5_2665 (2012) Hereditary hemochromatosis. In: Chen H (ed) Atlas of genetic diagnosis and counseling. Springer, pp 1025–1031. doi:10.1007/978-1-4614-1037-9_116 (2012) Leukemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2005. doi:10.1007/978-3-642-16483-5_3322 (2012) Liver Cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2063. doi:10.1007/978-3-642-16483-5_3393 (2012) LRP5/6. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2077. doi:10.1007/978-3-642-16483-5_3425 (2012) Lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2124. doi:10.1007/978-3-642-16483-5_3463 (2012) Medullary breast carcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2199. doi:10.1007/978-3-642-164835_3599 (2012) MTOR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2384. doi:10.1007/978-3-642-16483-5_3867 (2012) Poly(ADP-Ribose) Polymerase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2935. doi:10.1007/978-3-64216483-5_4655 (2012) Renal Cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3225–3226. doi:10.1007/978-3-642-16483-5_6575 (2012) Sarcoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3335. doi:10.1007/978-3-642-16483-5_5161 (2012) TCF/LEF. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3625. doi:10.1007/978-3-642-16483-5_5705

Deleted in Malignant Brain Tumors 1 (2012) Wnt. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3953. doi:10.1007/978-3-642-16483-5_6255 (2013) Diabetes Mellitus. In: Gebhart GF, Schmidt RF (eds) Encyclopedia of pain, 2nd edn. Springer, Berlin/Heidelberg, p 954. doi:10.1007/978-3-642-28753-4_200571 (2013) Ischemic heart disease. In: Gebhart GF, Schmidt RF (eds) Encyclopedia of pain, 2nd edn. Springer, Berlin/ Heidelberg, pp 1680–1681. doi:10.1007/978-3-64228753-4_201097 Angelini C (2009) Neurodegenerative disorders. In: Lang F (ed) Encyclopedia of molecular mechanisms of disease. Springer, Berlin/Heidelberg, pp 1453–1455. doi:10.1007/978-3-540-29676-8_248 Galanello R, Origa R (2010) Beta-thalassemia. Orphanet J Rare Dis 5:11. doi:10.1186/1750-1172-5-11 Gilbert P (1996) Friedrich’s ataxia. In: Gilbert P (ed) The A-Z reference book of syndromes and inherited disorders, 2nd edn. Springer, pp 123–126. doi:10.1007/9781-4899-6918-7_32

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homozygous deletions in malignant ▶ brain tumors. It codes for an extracellular glycoprotein of the evolutionary highly conserved group of scavenger receptor cysteine-rich (SRCR) proteins, and it is widely expressed in human tissues with strongest expression in epithelia and associated glands. Based on genomic alterations and loss of expression in several cancer types, DMBT1 has been proposed to play a role in tumorigenesis. The currently available data suggests two physiological functions for DMBT1: a function in innate immunity/mucosal protection and a function in epithelial/stem cell differentiation and regenerative processes.

Characteristics

Deleted in Liver Cancer 1 ▶ DLC1

Deleted in Malignant Brain Tumors 1 Caroline End1, Marcus Renner1, Jan Mollenhauer2 and Annemarie Poustka1 1 Division of Molecular Genome Analysis, DKFZ, Heidelberg, Germany 2 Molecular Oncology Group, University of Southern Denmark, Odense, Denmark

Synonyms Apactin (mouse); CRP-ductin (mouse); DMBT1; Ebnerin (rat); Glycoprotein-340 (gp-340 human); H3 (rhesus monkey); Hensin (rabbit); Muclin (mouse); Salivary agglutinin (SAG, human); Vomeroglandin (mouse)

Definition DMBT1 located at chromosome 10q26.13 2 was initially identified as a gene that shows frequent

The human DMBT1-locus is built up by tandemarrayed repeats with extensive homologies in the coding and noncoding region. The locus spans a region of about 103 kb with 55 exons with the last exon coding for a putative transmembrane domain (TMD). In contrast to the DMBT1 homologues in mouse and rat, the TMD exon has not yet been found to be contained in human transcripts. DMBT1 contains a signal peptide at the N-terminus and ▶ immunohistochemistry demonstrated that it is expressed and secreted mainly by epithelial cells and glands. The mode of secretion is thought to determine its functions: lumenally secreted DMBT1 is part of the mucus (the protective layer of epithelial surfaces) and is assumed to play a role in pathogen defense, while DMBT1 secreted to the extracellular matrix (ECM) may trigger epithelial and stem cell differentiation. The largest (wild-type) variant of DMBT1 contains 13 highly homologous (87–100% identical) SRCR domains. The SRCR domains are separated by so-called SRCR-interspersed domains (SIDs) harboring potential sites for O-▶ glycosylation (Fig. 1). The SRCR domains have been shown to function in pathogen binding and in mediating interactions with endogenous proteins involved in pathogen defense. Functions or ligands for the two C1r/C1s Uegf Bmp1 (CUB) domains and the 14th SRCR

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Deleted in Malignant Brain Tumors 1, Fig. 1 Genomic organization and domain structure of DMBT1. Topline represents scale in kilobases followed by the exon–intron structure of the DMBT1 gene consisting of 55 exons. The exons are colored according to the domain that they code for (see below). The presence of a further exon (black box) with coding potential for a transmembrane domain and a short cytoplasmic tail is predicted by homology searches with the cDNA sequences of the rodent homologues. The bottom two lines depict the domain organization of the wild-type protein (DMBT1/

8 kb.2) isolated from human adult trachea and one of the 6 kb transcripts isolated from human fetal lung (DMBT1/ 6 kb.1). The two variants could represent alternative splice products, but simultaneously also correspond to the largest and the smallest allelic variants identified so far. Pink triangle, signal peptide; blue box, unknown motif; red circles, SRCR domains; orange circles, SIDs, threonineand threonine–serine–proline-rich domain, respectively; purple boxes, CUB domains; green circle, ZP domain; green letters, ebnerin homologous domain

domain, which shares less homology to the 13 C-terminal ones, have not been identified so far. The second CUB domain is followed by a so-called zona pellucida (ZP) domain (Fig. 1). This type of domain mediates the formation of protein oligomers in other molecules, which is likely the case for DMBT1 as well. Genetic polymorphisms give rise to DMBT1 alleles with copy number variations resulting in a reduced number of SRCR domains and SIDs. Several DMBT1 alleles were identified coding for transcripts with sizes ranging from 6 to 8 kb (Fig. 1). A hemizygous deletion of SRCR4-SRCR7 was found in approximately 25% of the normal population.

bacteria-agglutinating activity. However, several hints have also suggested an involvement of DMBT1-attached glycosyl residues in pathogen interactions. Furthermore, DMBT1 binds to viruses like HIV-1 and influenza A and is able to inhibit viral infection in vitro. Besides its interactions with microorganisms, DMBT1 is known to interact with mucosal and circulating defense factors among these surfactant proteins A and D, (s) IgA, ▶ trefoil factor 2, lactoferrin, and complement component C1q. This suggests a potential cooperative action of DMBT1 and its endogenous ligands in host defense. Further DMBT1 may activate the complement cascade and ▶ macrophage chemokinesis and regulate neutrophil respiratory burst response in vitro. Upregulation of DMBT1 has been reported in inflammatory processes, for example, following tissue injury or infection. Except for a role in innate mucosal protection, DMBT1 expression in lymphoid organs may point to a participation in the regulation of the acquired immune system, as has been reported for other proteins of the SRCR superfamily.

Functional Characteristics DMBT1 and Innate Immunity/Mucosal Protection

There is increasing evidence that DMBT1 is part of the innate immune system against bacteria and viruses. The protein is present at most mucosal surfaces and binds to a broad range of bacteria including ▶ Helicobacter pylori, a human pathogen, which is linked to the development of peptic ulcer and ▶ gastric cancer. Evidence that the SRCR domains could mediate the binding to bacteria has been provided by the identification of a small peptide sequence present in these domains, which exerts a broad bacteria-binding and

DMBT1 and Epithelial/Stem Cell Differentiation and Regenerative Processes

Functional studies in vitro and in vivo suggest a role for DMBT1 and its rodent homologues in

Deleted in Pancreatic Carcinoma Locus 4

epithelial/stem cell differentiation. The rabbit homologue of DMBT1 (hensin) was found to be responsible for the switch of cell polarity and the induction of terminal differentiation of intercalated epithelial cells in the kidney. These processes are triggered by the deposition and polymerization of hensin in the extracellular matrix. Localization to the ECM in human adult multilayered and fetal developing epithelia, and expression along the proliferation–differentiation axis in the intestine, may support such function. Rabbit DMBT1 has further been reported to trigger in vitro the differentiation of mouse embryonic stem cells toward columnar epithelial cells. DMBT1-deficient mice (termed hensin/ mice) have been reported to display early embryonic lethality. Based on the upregulation of rat DMBT1 in liver stem cells during liver regeneration, a participation in stem cell-mediated regenerative processes has been proposed. DMBT1 and Tumorigenesis

Genomic alterations in DMBT1 and a loss or a reduction of its expression in several cancer types have led to the proposal that DMBT1 might represent a ▶ tumor suppressor gene. Inactivating point mutations have not been found within DMBT1. Homozygous deletions (deletions concerning both copies of the gene) of/or within DMBT1 initially have been observed in a substantial number of malignant ▶ brain tumors, ▶ lung cancer, and ▶ esophageal cancer. Due to the finding that DMBT1 is a highly polymorphic gene with frequent intragenic deletions within the repetitive SRCR/SID exon-containing region, however, it has been proposed that the majority of the previously identified deletions may represent preexisting polymorphisms unmasked by a loss of heterozygosity of the wild-type allele. Hence, inactivation of DMBT1 at the transcriptional/posttranscriptional level rather than genomic alterations may represent the predominant mechanism. Evidence has been gained that ▶ epigenetic silencing by DNA ▶ methylation may account for DMBT1 silencing in a smaller subset of tumors. Contrary to other cancer types, prostate cancer and pancreatic cancer display highly elevated

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DMBT1 expression levels, so that DMBT1 has been discussed as potential biomarker for these cancer types. A 29 amino acid C-terminal peptide of DMBT1 has further been found at high levels in pancreatic cancer and has been suggested as tumor biomarker. These data have led to the suggestion of a complex involvement of DMBT1 in tumorigenesis. Through protection from carcinogenic agents and/or pathogens and/or via regulation of the inflammatory response, DMBT1 may counteract tumor initiation and/or progression. Alternatively or additionally, loss of DMBT1 function may interfere with processes of differentiation and promote tumorigenesis.

References Kang W, Reid KBM (2003) DMBT1 a regulator of mucosal homeostasis through the linking of mucosal defence and regeneration? FEBS Lett 540(1–3):21–25 Mollenhauer J, Wiemann S, Scheurlen W et al (1997) DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-q26.1 is deleted in malignant brain tumours. Nat Genet 17:32–39 Mollenhauer J, Holmskov U, Wiemann S et al (1999) The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability. Oncogene 18:6233–6240 Mollenhauer J, Helmke B, Muller H et al (2002) An integrative model on the role of DMBT1 in epithelial cancer. Cancer Detect Prev 26(4):266–274 Vijayakumar S, Takito J, Gao X et al (2006) Differentiation of columnar epithelia: the hensin pathway. J Cell Sci 119(Pt 23):4797–4801

Deleted in Pancreatic Carcinoma Locus 4 Stephan A. Hahn University of Bochum, Bochum, Germany

Synonyms MADH4; Mother against decapentaplegic, drosophila, homolog of 4; SMA- and MAD-related protein 4; SMAD4; DPC4

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Deleted in Pancreatic Carcinoma Locus 4

Definition Deleted in pancreatic carcinoma locus 4 (DPC4) belongs to the class of tumor suppressor genes (▶ tumor suppressor genes). It was identified within chromosomal band 18q21.1, which is frequently deleted in pancreatic carcinoma. DPC4 is a component of the transcription complex (transcriptional complex) that mediates cell surface signals to the nucleus which are initiated by transforming growth factor b [TGF-b]-related growth and differentiation factors.

Characteristics The open reading frame of DPC4 spans 1,656 nucleotides and comprises 11 exons that code for

a 60 kD protein (552 amino acids). DPC4 belongs to the highly conserved family of Smad genes that has been identified by protein sequence homology studies. The founding member Mad (Mother against decapentaplegic) was identified in Drosophila melanogaster. Mad and its homologs mediate signals from cell surface receptors to the cell nucleus (▶ Smad proteins in TGFb signaling). Involved in this signaling cascade are serine/threonine kinase receptors of the TGFb family that become activated upon binding of polypeptides of the TGFb cytokine family (Fig. 1). At least 25 different cytokines from various species are currently known. They include TGF-b, activin, inhibin, bone morphogenic protein (BMP), and Müllerianinhibiting substance and control, among others, important biological functions such as embryonic

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Deleted in Pancreatic Carcinoma Locus 4, Fig. 1 A model for DPC4 signaling. In response to ligand binding to the TGFb receptor complex, receptor-regulated Smads (also called R-Smads) become C-terminally phosphorylated through the receptor kinase. The phosphorylated Smads change their folding pattern and form a heterodimeric or trimeric complex with DPC4. The newly formed Smad complex is then translocated into the

nucleus. In the nucleus, the Smad complex will make contact to transcription factors as well as bind directly to DNA through the Smad-binding element (SBE), thus stabilizing the higher order DNA-binding complex. In addition, transcriptional coactivators or corepressors may be recruited into the complex ultimately leading to either activation or repression of target gene expression

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development, cell growth and cell differentiation, modulation of immune responses, and bone formation. The number of Smad genes identified in humans has grown to a total number of eight (Table 1). Common to all of them is a characteristic threedomain structure (Fig. 2): a highly conserved region, the Mad homology domain 1 (MH1), is located at the amino (N)-terminal end. Next to it is a poorly conserved, proline-rich linker, region which is followed by a second highly conserved

Deleted in Pancreatic Carcinoma Locus 4, Table 1 Summary of functional classes of human Smads Classes of smad proteins Receptor-regulated 9 Smad 1 = Smad 5 BMP ; Smad 8  Smad 2 TGF b, Activin Smad 3

Common DPC 4/Smad 4

TGFb-Smad Signaling Cascade The Smad signaling cascade involves three different classes of Smad proteins: the receptorregulated Smads (R-Smads), the commonmediator Smad (Co-Smad), and the inhibitory Smads (I-Smads).

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domain (MH2) located at the carboxy (C)-terminal end of the protein. A link between the DPC4 protein and TGFb signaling cascade was initially established by sequence homology studies of the proteins DPC4 and Mad. At the time of DPC4 discovery, its potential tumor suppressor function was hypothesized; it was believed that the loss of DPC4 function in tumors is the reason for the observed resistance towards TGFb-mediated growth inhibition of many tumor types. Although this hypothesis has only been partly proved, a wealth of information regarding the signaling pathway has been collected. This gave rise to a model of DPC4 protein function within the TGFb signaling pathway.

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Deleted in Pancreatic Carcinoma Locus 4, Fig. 2 Functional domains and sites of identified DPC4 mutations. In addition to the mad homology domains MH1 and MH2, DPC4 carries a nuclear localization signal (NLS) domain and a nuclear export signal (NES) domain responsible for constant shuttling of DPC4 between nucleus and cytoplasm, thus helping the cell to constantly

sense the TGFb receptor activation state. A number of candidate phosphorylation target sites (P) for kinase pathways such as the MAPK (▶ MAP-Kinase) pathway have been described within the linker region which for example may modify nuclear accumulation rates of DPC4. The numbered squares forming the schematic DPC4 molecule also depict the 11 known exons within the DPC4 transcript

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Upon ligand induced TGFb receptor stimulation, R-Smads can transiently interact with the type I receptor. They become C-terminally phosphorylated by the receptor kinase and, once phosphorylated, are able to form a heterodimeric or trimeric complex with the “common-mediator” DPC4/Smad4 which then translocates into the nucleus. Here it can either up- or downregulate the transcription levels of target genes by interacting with other nuclear factors and by recruiting transcriptional coactivators or corepressors. This signaling cascade can be negatively regulated by the I-Smads (Smad6 and Smad7). Whereas Smad7 acts as a more general inhibitor of TGFb family signaling, Smad6 seems to preferentially block BMP signaling. I-Smads can compete with R-Smads for type I receptor binding and can therefore prevent the phosphorylationdependent activation of R-Smads. Furthermore, Smad7 can interact with the E3 ubiquitin ligases Smurf 1 and 2. Once the Smad7/Smurf complex is bound to the TGFb receptor, it induces TGFb receptor degradation. Direct binding of I-Smads to R-Smads has also been shown, yielding R-Smads inactive. The expression of I-Smads appears to be regulated by TGFb and BMP via an autoregulatory feedback loop. Furthermore, it has been shown that Interferon-g (interferon-g) (IFN-g) via the Jak1/STAT1 pathway, and tumor necrosis factor alpha (TNFa) and interleukin 1 through NFkB (▶ Nuclear factor kB) /RelA can induce the expression of I-Smads to antagonize TGFb signaling. Transcriptional Regulation Through Smads Since Smad proteins have no intrinsic enzymatic activity, they exert their effector function as transcriptional regulators either directly by binding to specific promoter consensus sequences termed Smad-binding elements (SBE) and/or indirectly by associating with transcription factors already bound to the promoter. Therefore, many but not all Smad responsive promoters have two adjacent DNA sequences. One provides the binding site for transcription factors that are cooperating with the Smad complex; the other allows direct binding of

Deleted in Pancreatic Carcinoma Locus 4

the Smad complex to the DNA. While R-Smads provide the interface for the binding to transcription factors, DPC4 makes the contact to SBE elements. DPC4 thereby stabilizes the formation of a higher order DNA-binding complex which is able to recruit transcriptional coactivators or corepressors. Many of the factors that cooperate with the Smad complex are regulated independently by other signaling cascades. The function of an active Smad complex can therefore be described as a co-modulator of transcription. It can modulate gene expression positively as well as negatively by integrating various incoming signals, including those mediated by the TGFb ligand family. Therefore, it is not surprising that currently more than 1,000 genes are described to be either directly or indirectly regulated by DPC4. In addition, many of these DPC4 target genes can only be found in a certain cell type and growth state, again illustrating how much the cellular differentiation and signaling state determines the net gene expression regulation pattern of a DPC4 containing transcription complex. What Makes DPC4/Smad4 Unique Among the Other Smad Family Members? • DPC4 is the only human Co-Smad that is currently known. • It seems particular to DPC4 that it is, almost without exception, essential for the establishment of a functional active Smad complex, a fact that emphasizes its role as a “master switch” in the regulation of TGFb-like signals. • Most somatic and all germ line mutations in human Smad genes identified to date target DPC4. Only very few somatic mutations were found in the human Smad2 gene; none were found in the other members of the human Smad gene family. Which Human Tumors Show Alterations of the DPC4 Gene? Changes, resulting in the inactivation of the DPC4 gene, were found in approximately 50% of pancreatic carcinomas (▶ pancreas cancer). Research carried out in a variety of other cancer types

Deleted in Pancreatic Carcinoma Locus 4

suggested that DPC4 may contribute primarily to the formation of pancreatic neoplasia and to a lesser extent to ▶ colorectal cancer, ▶ cervical cancer, and biliary cancer (▶ bile duct neoplasia) as well as the induction of nonproducing ▶ neuroendocrine tumors. However, such changes appear to play only a minor role in the development of other tumor types such as head and neck cancer, ▶ lung cancer, ▶ ovarian cancer, ▶ breast cancer, and ▶ bladder cancer. The frequency of DPC4 mutations is markedly increased in metastatic colorectal carcinoma (35%) compared to nonmetastatic colorectal carcinomas (7%). Furthermore, during pancreatic carcinoma development, a high incidence of biallelic DPC4inactivation is generally not present before the carcinoma-in-situ stage, suggesting the loss of DPC4 function is critical for the tumor cell to develop characteristics such as the ability to invade into the surrounding tissue and to form metastasis. In addition, germ line mutations of the DPC4 gene have been identified in patients with familial juvenile polyposis, an autosomal dominant disorder that is characterized by a predisposition to hamartomatous polyps as well as an increased risk for gastrointestinal carcinomas. How Are Naturally Occurring DPC4 Mutations Interfering with the Smad Signaling Cascade Most DPC4 mutations identified to date are located within the C-terminal MH2 domain. Functional studies identified the MH2 domain as providing the binding properties to R-Smads, the latter being important for a functionally active Smad complex. It is therefore likely that compromising mutations of the MH2 domain structure restrict the formation of a functional Smad complex, thus preventing signal transduction to downstream components. In addition, a few mutations have been identified within the N-terminal MH1 domain which was shown to mediate the direct binding of DPC4 to DNA promoter sequences. Such mutations might interfere with Smad signaling by rendering the formation of the higher order SmadDNA complex unstable. Furthermore, some DPC4 missense mutations targeting the MH1

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domain result in an instable protein due to a mutation-induced poly-ubiquitination of DPC4 and its subsequent proteasomal degradation. Does DPC4 Contribute to the Familial Risk for Pancreatic Cancer? Although the DPC4 gene is most frequently altered in sporadic pancreatic carcinoma, to date no germ line mutations were found in families with an increased risk of this type of carcinoma. DPC4 is therefore unlikely to play an important role as a heritable genetic risk factor in pancreatic carcinoma. How Does DPC4 Contribute to Tumor Formation? Although Smad signaling (including DPC4/ Smad4) is regarded as central to the TGFb pathway, there are now numerous examples illustrating that DPC4 inactivation is not simply abolishing TGFb responsiveness and thus providing the cell a growth advantage. This can partly be explained by the ability of TGFb to modulate also Smad-independent pathways such as the Ras (▶ RAS)-ERK, PI3K (▶ PI3K Signaling)–AKT (▶ AKT Signal Transduction Pathway), and Rac/Rho pathways. Thus, loss of DPC4 function is not able to completely abrogate TGFb signaling rather than shifting the balance between DPC4/ Smad-dependent and DPC4/Smad-independent TGFb signaling pathways towards the DPC4/ Smad-independent pathways. The output of the latter is dependent on the successful activation of the latent form of TGFb ligands and intactness of the TGFb receptors. The cellular context will further modulate the signaling state of the DPC4/ Smad-independent pathways through regulating the activity status of their pathway target genes by integrating signals from other signaling pathways. Thus, loss of DPC4 function has been shown in a cell specific manner to be involved in altering a number of different cell behaviors relevant to tumor formation such as, cell growth rate by modulating the cell cycle and/or the rate of ▶ apoptosis, altering the extracellular matrix components (▶ extracellular matrix remodeling), the cell adhesion (▶ adhesion) properties, and

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supporting epithelial to mesenchymal transition, thereby facilitating tumor ▶ invasion and ▶ metastasis. Furthermore, other experiments provided evidence that loss of DPC4 function might promote tumor ▶ angiogenesis by causing an increase in the concentration of angiogenic factors and/or a decrease in its corresponding inhibitors. Additional insight of DPC4 function was provided by targeted mutagenesis in mice. Mice with two mutated alleles for DPC4 die at embryonic day 7.5, a result that underlines the importance of DPC4 in early embryonic development. DPC4 heterozygous mice develop gastric and duodenal polyps which resemble human juvenile polyps. Furthermore, knockout mice experiments have demonstrated a functional cooperation between the DPC4 and the APC (APC) (adenomatous polyposis coli) gene. In mice that were carrying defect copies of both genes, compared to mice carrying only the mutated APC gene, the induced colonic tumors displayed a much more aggressive phenotype. Lastly, data from primary human tumors and from mice experiments provided evidence that haploinsufficiency of the DPC4 locus may also contribute to progression of cancer. These data clearly support the importance of DPC4 in the suppression of tumorigenesis.

Cross-References ▶ Neuroendocrine Neoplasms

References Alberici P, Jagmohan-Changur S, De Pater E et al (2005) Smad4 haploinsufficiency in mouse models for intestinal cancer. Oncogene 25:1841–1851 Hahn SA, Schutte M, Hoque AT et al (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350–353 Howe JR, Roth S, Ringold JC et al (1998) Mutations in the smad/dpc4 gene in juvenile polyposis. Science 280:1086–1088 Miyaki M, Iijima T, Konishi M et al (1999) Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis. Oncogene 18:3098–3103 Takaku K, Oshima M, Miyoshi H et al (1998) Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell 92:645–656

Dendritic Cells

Dendritic Cells Nathalie Cools, Viggo Van Tendeloo and Zwi Berneman Vaccine and Infections Disease Institute (VAXINFECTIO) Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium

Definition Dendritic cells are a special subset of leukocytes that form a complex network of antigen-presenting cells (APC) throughout the body. They play a principal role in the initiation of immune responses to invading microorganisms (bacteria, fungi, and viruses), malignant cells, and allografts by activating naïve lymphocytes, by interaction with innate cells, and by the secretion of cytokines. At certain developmental stages they grow branched projections, the dendrites, hence the cell’s name.

Characteristics Origin and Function Dendritic cells (DC) were characterized for the first time by Steinman in 1973 based on their distinct morphology with different cytoplasmic extensions, such as dendrites, pseudopodia, and lamellipodia, which give the cell its star-shaped feature. Due to their pronounced morphology, DC have a large surface, ensuring close contact with neighboring cells. Variations among the tissue distribution of DC and differences in their phenotype and function indicate the existence of heterogenous populations of DC. DC originate from different hematopoietic lineages in the bone marrow (Table 1). A myeloid progenitor cell can differentiate in vivo to different DC populations: Langerhans cells that migrate to the skin epidermis and interstitial DC that migrate to the skin dermis and various other tissues (airways, liver, and intestine). Circulating, or migrating, DC are

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Dendritic Cells, Table 1 Different subsets of dendritic cells CD34+ hematopoietic stem cell Myeloid progenitor cell Monocyte-derived DC Phenotype CD11c + CD1a  CD123 – Birbeck granules – Factor XIIIa  Function Endocytosis + IL-10* + IL-12* + IFN-a* –

Langerhans cells

Interstitial DC

Lymphoid progenitor cell Plasmacytoid DC

+ + – + –

+ – – – +

– – + – –

+ + – –

+ + + –

+ + + +

found in the blood and in the afferent lymphatics, respectively (the latter called veiled cells). Interdigitating DC are found in the paracortex of lymph nodes in close proximity with T cells. In addition, monocytes represent an abundant source of DC precursors during physiological stress. Another subset of DC, plasmacytoid DC (pDC) originate from a lymphoid progenitor cell in lymphoid organs. By contrast, follicular DC (FDC) are probably not of hematopoietic origin, despite similar morphology and function to the abovementioned subsets of DC. FDC are APC of the B cell follicles in lymph nodes and central players in humoral immunity. After application of danger signals, DC express several different types of membrane molecules that determine their phenotypic and functional characteristics: 1. DC display a high surface density of antigenpresenting molecules, such as CD1a, major histocompatibility complex (MHC) class I and class II molecules. The level of expression of these molecules is 10- to 100-fold higher compared to other APC (e.g., B cells). 2. In addition, mature DC have high expression levels of costimulatory and adhesion molecules: CD40, ICAM-1/CD54, ICAM-3/CD50, LFA-3/CD58, B7-1/CD80, and B7-2/CD86. Binding of these molecules with their respective receptors on T cells results in T cell activation and subsequently stimulates the

expression of cytokines, cytokine receptors, and genes for cell survival. 3. Several members of the integrin family are expressed by DC. Cadherins contribute to the generation of cell contacts and selectins are important for the motility of DC. 4. DC also express pathogen-recognition receptors, e.g., DEC-205, a macrophage-mannose receptor capable of binding bacterial carbohydrates and ▶ toll-like receptors (TLR), recognizing a variety of pathogen-associated molecular patterns (PAMP), such as carbohydrates, nucleic acids, peptidoglycans, and lipoteichoic acids. 5. Cytokine and chemokine receptors are also important for DC function, since growth, differentiation, and migration of DC as well as antigen processing and presentation are tightly regulated by cytokines and/or chemokines. The widespread distribution of DC and their expression of a variety of membrane molecules underline their sentinel function: they patrol the body to capture invading pathogens and certain malignant cells in order to induce efficient antimicrobial or anti-tumor ▶ T cell responses. In their in vivo steady-state condition, immature DC are specialized in capturing antigens, i.e., they efficiently take up pathogens, apoptotic cells, and antigens from the environment by phagocytosis, macropinocytosis, or ▶ endocytosis. However, immature DC remain tissue resident, expressing

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only small amounts of (MHC) class II and of costimulatory molecules, which leads to T cell unresponsiveness. After encounter of a “danger” signal (e.g., TLR ligand) immature DC mature and migrate to the secondary lymphoid organs. Mature DC are considered to be immunogenic, mainly due to the marked upregulation of MHC class II and costimulatory molecules. This maturation step is believed to be a crucial event to regulate DC function and makes DC potent inducers of T cell immunity. Dendritic Cell-Based Immunotherapy Despite our immune system’s function to protect us from malignant cells, tumor cells grow undisturbed and, unless treated, are fatal to the host. The reasons for the failure to eliminate tumor burden in a majority of patients can be the consequence of different tumor escape mechanisms. For example, tumor-derived inhibitory factors (e.g., PD-L1/2 IL-10 and/or TGF-b) or tumor cell-induced T regulatory cells (Treg) might be involved in downregulating or altering immune function. The goal of cancer ▶ immunotherapy is to resolve or circumvent these problems and generate tumor-specific immune responses. It is important to realize that immunotherapies will likely only be successful after reducing tumor mass via primary therapies: surgery and radioand/or chemotherapy, i.e., in a ▶ minimal residual disease (MRD) setting. Because of their pivotal immune-stimulatory capacity and their ability to activate naïve tumorspecific T cells, DC-based ▶ cancer vaccines could have important applications in the future treatment of cancer. For this, it was necessary to cultivate DC with high yields. Several cultivation protocols were developed for in vitro generation of DC. First, DC can be differentiated from CD34+ hematopoietic progenitor cells using granulocyte-monocyte colony stimulating factor (GM-CSF), tumor necrosis factor (TNF-a), stem cell factor (SCF), interleukin (IL)-3, and ▶ interleukin-6. Second, DC can be generated starting from monocytes using GM-CSF and ▶ Interleukin-4. Finally, DC can be directly

Dendritic Cells

harvested from the peripheral blood of a patient, where they reside at low percentages (0.1%). Next, cultivated DC can be loaded with the tumor antigen of importance in different ways: 1. DC can be grown in vitro in the presence of tumor-associated antigens (TAA). This technique is called peptide pulsing and results in direct binding of the immunodominant epitope on an empty MHC class I molecule on the DC membrane. This circumvents the need for antigen uptake and processing and ensures the stimulation of tumor-specific cell-mediated cytotoxicity. However, the number of known TAA is still restricted and highly dependent on the human leukocyte antigen (HLA) haplotype of the patient. 2. DC can also be fused with the patient’s tumor cells in vitro or pulsed with tumor cell lysates. The former method combines sustained tumor antigen expression with the antigen-presenting and immunostimulatory capacities of DC. DC-tumor cell hybrids will also stimulate an active antitumoral immune response. 3. Tumor antigen can also be loaded on DC using plasmid DNA transfection or ▶ viral vector mediated gene transfer. The former method results in only low transfection efficiencies. On the other hand, viral transduction, for example by using adenoviral or lentiviral, vectors is very effective with regard to transfection efficiency. However, the immunogenic character of the viral vector itself is a serious disadvantage. In both cases, DC will transcribe and process the tumor antigen. This will result in a cytotoxic immune response, necessary for immunological defense against cancer cells. 4. It is also possible to transfect DC using in vitro transcribed mRNA coding for tumor antigens or total tumor RNA. It has been shown that electroporation of RNA is the most effective nonviral transfection method for DC (▶ Nonviral Vector for Cancer Therapy). mRNA is brought directly into the cytoplasm and the cell’s metabolism will translate mRNA

Dendritic Cells

into proteins, which can be presented onto MHC class I molecules after processing. This will guarantee a specific cell-mediated antitumoral immune response. In a clinical context, in vitro cultured and activated DC loaded with appropriate tumor antigens could be administered to cancer patients in a therapeutic setting (active specific immunotherapy). The aimed generation of anti-tumor immunity, mediated by DC, could be of importance for both treatment (as adjuvant to conventional therapies) and to prevent relapse in an MRD setting. On the other hand, tumor antigen-loaded DC can also be used for the ex vivo generation of tumor-specific cytotoxic T lymphocytes (CTL) in an autologous system. These tumor-specific CTL can, in their turn, be administered to the patient to exert a direct cytotoxic effect on the patient’s cancer cells (passive or adoptive immunotherapy). The impact of a DC-based cancer vaccine is clear: an antigen-specific anti-tumor vaccine would influence both morbidity and mortality of various cancers. Currently, several phase I–II or III ▶ clinical trials using TAA-loaded DC are ongoing worldwide in order to stimulate the patient’s immune system against tumor antigens. A number of these trials demonstrated some clinical and immunological responses (as evidenced by T cell proliferation, IFN-g ELISPOT, and delayed type hypersensitivity [DTH] reaction) without any significant toxicity. However, despite the presence of expanded antigen-specific T cells in patients after vaccination, only a minor population of these patients showed a beneficial biologically relevant clinical response, i.e., tumor regression and increased disease-free survival. Clinical trials using DC have shown moderate success. To date, the combination of a targeted therapy exploiting the capacity of DC to stimulate the patient’s own immune system against cancer with so-called immune checkpoint inhibitors is being examined in ongoing and future trials in order to eliminate tumor burden in patients.

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Dendritic Cells in Cancers DC can also infiltrate human tumors where they are involved in the induction of anti-tumor immune responses. It is likely that the establishment of tumor-specific immune responses depends on the migratory capacity of DC from the tumor microenvironment to the draining lymph nodes, where tumor antigen presentation to T cells takes place. Moreover, by their expression of costimulatory molecules and several cytokines, such as IFN-a and IL-12, DC also mediate T cell survival by preventing T cell ▶ apoptosis. In addition, mature DC have been reported to cause direct lysis, apoptosis, as well as cell cycle arrest of cancer cells through the secretion of soluble factors. As a consequence, the presence of a high number of DC in the tumoral or peritumoral area, as well as in the draining lymph nodes of various human tumors, has been shown to correlate with patients’ survival and a better prognosis. Decreased numbers or dysfunction (e.g., decreased expression of costimulatory molecules) of DC is reported in poor-prognosis tumors. Furthermore, tumor cells can secrete certain factors (e.g., IL-10 and TGF-b) that counteract DC maturation and migration and thus actively contribute to DC dysfunction. Occasionally, neoplasms of accessory immune cells (antigen-presenting cells, dendritic cells) can occur. These are primarily found in lymph nodes and extranodal lymphoid tissues (lymph node interdigitating cell sarcoma), but are also reported from other sites such as the skin (▶ Langerhans Cell Histiocytosis). The incidence of dendritic cell tumors is very rare: until now, only a few dozens of cases have been reported in literature.

Cross-References ▶ Apoptosis ▶ Cancer Vaccines ▶ Clinical Trial ▶ Endocytosis ▶ Immunotherapy ▶ Interleukin-4

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▶ Interleukin-6 ▶ Langerhans Cell Histiocytosis ▶ Macrophages ▶ Minimal Residual Disease ▶ Nonviral Vector for Cancer Therapy ▶ T-Cell Response ▶ Toll-Like Receptors ▶ Viral Vector-Mediated Gene Transfer

References Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252 Gilboa E (2007) DC-based cancer vaccines. J Clin Invest 117:1195–1203 Lotze MT, Thomson AW (2001) Dendritic cells, 2nd edn. Academic, London Ponsaerts P, Van Tendeloo VF, Berneman ZN (2003) Cancer immunotherapy using RNA-loaded dendritic cells. Clin Exp Immunol 134:378–384 Van Tendeloo VF, Van Broeckhoven C, Berneman ZW (2001) Gene-based cancer vaccines: an ex vivo approach. Leukemia 15:545–558

Dental Pulp Neoplasms (2012) Treg. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3782. doi:10.1007/978-3-642-16483-5_5967 (2012) Tumor-associated antigen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3807–3808. doi:10.1007/978-3-64216483-5_6017

Dental Pulp Neoplasms Klaus W. Neuhaus School of Dental Medicine, Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland

Definition Are tumors that are located in the dental pulp.

Characteristics See Also (2012) Adhesion molecules. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 66. doi:10.1007/978-3-642-16483-5_96 (2012) Antigen-presenting cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 209–210. doi:10.1007/978-3-642-164835_321 (2012) Cadherins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p pp 581–582. doi:10.1007/978-3-642-16483-5_770 (2012) Delayed type hypersensitivity reaction. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1073. doi:10.1007/978-3-64216483-5_1550 (2012) ELISPOT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1217. doi:10.1007/978-3-642-16483-5_1850 (2012) Lamellipodia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1971. doi:10.1007/978-3-642-16483-5_3267 (2012) Langerhans cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1975. doi:10.1007/978-3-642-16483-5_3272 (2012) Leukocytes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2028. doi:10.1007/978-3-642-16483-5_3330 (2012) Major histocompatibility complex. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2137. doi:10.1007/978-3-642-164835_3500

Dental pulp neoplasms (DPNs) are rare tumors of the dental pulp tissue which is not exposed to the oral cavity. Two types of DPNs can be distinguished: Type 1 originates from the dental pulp itself (primary DPN) and type 2 originates from tissue outside of the tooth (secondary DPN). Most DPNs are somewhat incidental findings in patients with a known tumor anamnesis. Therefore the number of histologic examples of DPNs is rather limited, and one also has to take into account articles from old literature in order to draw a complete clinical picture. History In the late nineteenth century, when systematic dental care and oral hygiene in general were considerably more deficient than today, dentists encountered numerous teeth with deep caries and sometimes massive exposed pulp tissue. This phenomenon was called “pulpitis chronica sarcomatosa,” a chronically inflamed dental pulp supposedly caused by a sarcoma. Later it was found out that this pulpal alteration was in fact nothing to do with a sarcoma but rather was the

Dental Pulp Neoplasms

result of colonization of the exposed dental pulp by free epithelium cells of the gums. This entity is a so-called dental pulp polyp. However, the first true description of a type 1 DPN was made in 1904 by V. A. Latham from Rogers Park, Illinois. He presented the case of a 56-year-old woman presenting with an upper right canine with a greenish-white tinge. The tooth was vital, symptomless, and caries-free, i.e., the dental pulp was not exposed to the oral environment. After tooth extraction (for prosthodontic reasons) and histological processing, this canine proved to have an epithelioma of the pulp. The extraction socket was curetted and subsequently cleaned with iodine and carbolic acid. According to his report, Latham thus seems to have cured the patient from a tumor by simply extracting the tooth. Until today, descriptions of type 1 DPNs are very rare. First descriptions of type 2 DPNs also date back to the early twentieth century where reports of involvement of dental pulps in patients with ▶ breast cancer, lymphoma, or neuroma have been given. Three to thirty percent of tumors of the head and neck region (HNR) are associated with involvement of the dental pulp. Carcinomas are more likely to be associated with DPNs than sarcomas or any other type of tumors of the HNR. The maximum incidence of DPNs lies between the fifth and sixth decade of life.

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inflammation of the dental pulp in slowly progressing caries may lead to the calcification of parts of the pulpal tissue via secondary odontoblasts. These particular cells are differentiations of former pulpoblasts. Pulpoblast differentiation can be modified by bone morphogenetic protein (BMP) 2, 4, and 11, GDF, TGF-b, or high calcium concentrations, all of which are present in dentin. It can also be modified by certain medicaments, which originally were only used in periodontal regenerative therapy but have now also been introduced in endodontic therapy as well as dental traumatology as a means of direct pulp capping.

Inflammatory Pulp Reactions

Radiotherapy As an additional point of discussion the possibility of therapeutically induced DPNs by radiotherapy has to be mentioned. It is a given fact that one of the risks of radiotherapy of the HNR consists in radiation-induced tumors (▶ radiation-induced sarcomas after radiotherapy). Establishing a causal connection is often difficult due to a latency period of several years. However, teeth after radiotherapy sometimes show calcifications of the dental pulps, which can be detected in postirradiation radiographs. Since there has not been a study distinguishing between bacterial and abacterial calcifications as signs of chronic inflammations of the dental pulp in postirradiated cases, no predication can be given about a higher risk of DPN after radiotherapy.

A DPN causes inflammatory reactions (▶ inflammation) in the dental pulp. Chronic inflammation of the pulp may either lead to calcification of parts of the dental pulp tissue or to resorption of the surrounding hard tissue, i.e., dentin. Calcifications – as regularly observed histological findings in dental pulps with a neoplasm – can be explained by the behavior of primary and secondary odontoblasts. These cells are determined to secrete dental hard substance. If a bacterial impact is directed toward the pulp (as is the case with dental caries), the primary odontoblasts immediately start to produce tertiary dentin in the targeted area. Thus increasing the distance between the bacteria and the pulp, an early opening of the pulp chamber in the course of the carious process is evaded. Meanwhile, the chronic

Animal Investigations As to DPN-cases, the pulp tissue reaction with respect to calcifications seems to be the same as in cases of dentin caries. At this point, observations made in experimental animal models become of interest: After several days calcification of the dental pulp tissue (with a simultaneous breakdown of the odontoblast layer) can be detected when inoculating the dental pulps of rodents with virulent sarcoma cells. Regular findings in these studies consist in massive development of intrapulpal dental hard substance like denticles, osteoids, or pulp stones. Also destruction of pulpal cells, particularly of the odontoblasts, by tumor tissue has been described in an animal study. In none of these investigations do the dental pulps survive longer

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than 3 weeks. Nevertheless, it is a matter of speculation whether this effect is really due to the sarcoma cells or rather to the increased extravasal pressure of the inflamed pulpal tissue. In these animal models, the sarcomata are able to infiltrate the dental pulps and to proliferate to adjacent tissue like periodontium, mandibular bone, and masseteric muscle. In later stages, ▶ metastasis in the regional lymph nodes as well as in the sublingual, submandibulary, and parotid glands can be found. The fact that rodent teeth are substantially different from human teeth must not be neglected. While rodent teeth are growing lifelong and have a largely open apex, human tooth formation literally comes to an endpoint in a constriction at the tip of the root(s). Clinical Relevance Since systematic autopsies of the jaws are no longer common, the entity of DPNs have somewhat moved out of the focus of scientific attention. Type 1 DPNs are certainly of small clinical relevance. In the dental pulp, fibroblasts, subodontoblastic progenitor cells, pericytes, stem cells, and, occasionally, Malassez epithelium remainders of the Hertwig root sheath are cells with mitotic competence and thus are able to undergo neoplastic alteration. A relatively high grade of differentiation of the pulpal tissue limits further differentiation of purported neoplasms. Due to the restricted anatomical macroenvironment of a tooth and possibly further due to microenvironmental interactions, a type 1 DPN is more or less self-limited. Concerning the formation of a DPN, the capability of the dental pulp to regularly form calcifications under certain circumstances as well as the fact that one encounters a terminal blood supply in the pulp plays a crucial role. Growth of a neoplasm will increase extravasal pressure within the dental pulp and thus stimulate secondary odontoblasts to secrete irritation dentin. A large amount of irritation dentin might influence the blood supply of the dental pulp and thus will probably lead to a hemorrhagic infarct. A growing tumor in the root canal

Dental Pulp Neoplasms

system will contribute to this effect. While becoming necrotic in such a way, the dental pulp does not necessarily have to show clinical symptoms (such as tooth ache). Teeth with necrotic pulps will normally receive endodontic treatment (i.e., root canal therapy) or they will be “cured” by tooth extraction. It can be acclaimed that the specialty about a type 1 DPN lies in its possibility to be removed successfully and in a relatively easy way. The anatomic prerequisite of the root canal system presents the unique fact that while growing a tumor is already limiting its further existence. The risk of metastasis of a DPN is not given in normal-sized teeth. The volume of the dental pulp chamber and the root canal system do not provide sufficient space for a tumor to gain a critical cell mass in order to disseminate clonal cells. Only teeth with incomplete root formation (as in children or adolescents) or taurodonts, i.e., teeth with an abnormally large crown and roots, might provide enough space allowing a tumor to gain a critical cell mass. Large animal teeth, whose pulp chambers can surely provide enough space for a tumor (for instance in large mammalians), are not systematically screened for dental pulp diseases. Type 2 DPNs seem to be mere incidental findings in patients with tumors mainly of the HNR. This leads to the assumption that DPNs are normally symptomless and of relatively small clinical relevance. Nevertheless, type 2 DPNs may also lead to tooth-related symptoms (pain) as has been described in single case reports. Far more often (and clinically more important) is the opposite case when seemingly healthy teeth with no sign of caries, filling, or a positive trauma history mimic toothache. The projected toothache is thus drawing off the attention of a true HNR tumor, which often leads to unnecessary root canal treatment or tooth extraction. Therefore, apart from regular or ofacial neuropathic or nociceptive pain conditions, differential diagnosis therefore should always consider a neoplasm in the HNR. In common classifications of dental pulp diseases, inflammation of the dental pulp due to

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neoplasms are neglected. However, animal tumor models (▶ mouse models) should be reinvestigated for changes within the dental pulp.

(2012) TGF-β. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3661. doi:10.1007/978-3-642-16483-5_5753

Cross-References

20 -Deoxy-5-azacytidine

▶ Breast Cancer ▶ Inflammation ▶ Metastasis ▶ Mouse Models ▶ Radiation-Induced Sarcomas Radiotherapy ▶ Transforming Growth Factor-Beta

▶ 5-Aza-20 Deoxycytidine

After

Deoxyazacytidine ▶ 5-Aza-20 Deoxycytidine

References Neuhaus KW (2007) Teeth: malignant neoplasms in the dental pulp? Lancet Oncol 8:75–78 Stewart EE, Stafne EC (1955) Involvement of the dental pulp by malignant tumors of the oral cavity. Oral Surg Oral Med Oral Pathol 8:842–855 Zajewloschin MN, Libin SI (1934) Histologische Untersuchungen der Zähne bei Neubildungen der Kiefer. Virchows Arch Pathol Anat Physiol Klin Med 293:365–380

See Also (2012) BMP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 441. doi:10.1007/978-3-642-16483-5_675 (2012) Caries. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 666. doi:10.1007/978-3-642-16483-5_861 (2012) Dentin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1087. doi:10.1007/978-3-642-16483-5_1560 (2012) GDF. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1516. doi:10.1007/978-3-642-16483-5_2349 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720 (2012) Odontoblasts. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2598. doi:10.1007/978-3-642-16483-5_4193 (2012) Periodontium. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2815. doi:10.1007/978-3-642-16483-5_4449 (2012) Taurodont. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3614. doi:10.1007/978-3-642-16483-5_5685

Dephosphorylating Enzyme ▶ Phosphatase

DES ▶ Diethylstilbestrol

Designer Foods ▶ Nutraceuticals

Desmoglein-2 Masakazu Yashiro Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan

Synonyms Dsg2

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Desmoglein-2

Definition Dsg2 is one of the calcium-binding transmembrane glycoprotein components of the cell-cell ▶ adhesion molecules of the ▶ desmosomes. Dsg2 is one of the cadherin cell adhesion molecule superfamily in vertebrate epithelial cells.

the intermediate filament cytoskeleton. Desmosomes are essential adhesion structures in most epithelia that link the intermediate filament network of one cell to its neighbor, thereby forming a strong bond. Desmosomes contain the desmosomal cadherins, desmoglein (Dsg) and desmocollin (Dsc), that are linked to the intermediate filament cytoskeleton through interactions with plakoglobin and desmoplakin (Fig. 2).

Characteristics Cell Junctions Epithelial cell-cell junctions consist of four junctions: ▶ tight junctions, ▶ adherens junctions, ▶ desmosomes, and ▶ gap junctions (Fig. 1). Two adhering-type junctions, the adherens junctions and the desmosomes, are responsible for strong cell-cell adhesion. Each of these junctions consists of a transmembrane cadherin and a complex cytoplasmic plaque that serve to link cadherin to actin microfilaments or the intermediate filament cytoskeleton. Desmosome

Intercellular junctions known as desmosomes are multimolecular membrane domains that provide intercellular adhesion and membrane anchors for

Desmoglein and Cancer Epithelial cell-cell adhesion is important in tumor development. Dsgs are transmembrane glycoproteins of the desmosome, a cell-cell adhesive structure prominent in epithelial tissues, which have been reported to be associated with tumor development. cDNA and protein studies have revealed that there are subfamilies of Dsg (types 1, 2, and 3) and Dsc (types 1, 2, and 3) (Buxton et al. 1993). Dsg2 and Dsc2 are widely expressed and are found together in desmosomes of the basal layer of stratified epithelia, simple epithelia, and nonepithelial cells such as in the myocardium of the heart and lymph node follicles, whereas Dsg3/Dsc3 and Dsg1/Dsc1 are more restricted to complex epithelial tissues. Although considerable overlap is exhibited in the distribution of these isoforms in

Desmoglein-2, Fig. 1 Cell junctions. Epithelial cell-cell junctions consist of four junctions: tight junctions, adherens junctions, desmosomes, and gap junctions

Tight junctions: occuldin, claudin, ZO-1, ZO-2, ZO-3, ZO-6

Adherens junctions: cadherin, β-catenin, plakoglobin, α-actinin, vinculin, ZO-1, actin filaments

Desmosomes: desmoglein1, 2, 3, desmocollin1, 2, 3, plakoglobin, desmoplakin, intermediate filament cytoskeleton

Gap junctions: connexons

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Desmoglein-2, Fig. 2 Desmosomes. Desmosomes contain the desmosomal cadherins, desmoglein and desmocollin, that are linked to the intermediate filament cytoskeleton through interactions with plakoglobin and desmoplakin

Desmoglein Desmocollin

D Plakoglobin Desmoplakin

stratified tissues, their expression is clearly differentiation dependent. Dsg2, but not Dsg1 or Dsg3, is expressed in stomach epithelia. ▶ Gastric cancers have been classified into two histological types: intestinal type and diffuse type. Diffusetype gastric cancers show decreased cell-cell adhesion, which is associated with metastatic potential. These histological features indicate that a decrease in adhesive junctions may be involved in the emergence of diffuse-type gastric cancers. A decrease in E-cadherin has been reported to be one cause of the decrease in adhesive junctions, but not all diffuse-type gastric cancers show such a decrease. Decreased expression of Dsg2 is associated with diffuse-type gastric cancers and poor prognosis in gastric carcinoma. Adherens Junctions

The adherens junction is composed of a classic cadherin (e.g., E-, P-, or N-cadherin) linked to b-catenin or plakoglobin (Yashiro et al. 2006). Thus, plakoglobin is found in both adherens junctions and desmosomes, while b-catenin is restricted to the adherens junction. Alpha-catenin links the cadherin/catenin complex to the actin cytoskeleton through interactions with a-actinin, vinculin, ZO-1, and actin filaments. Lost or reduced plakoglobin expression has been observed in tumor tissues and metastatic lesions and has been linked to poor prognosis in a variety of tumors (Rieger-Christ et al. 2005; Holen et al. 2012; Pantel et al. 1998).

Intermediate filament cytoskeleton

Cross-References ▶ APC/b-Catenin Pathway ▶ E-Cadherin

References Buxton RS, Cowin P, Franke WW et al (1993) Nomenclature of the desmosomal cadherins. J Cell Biol 121:481–483 Holen I, Whitworth J, Nutter F, et al (2012) Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo. Breast Cancer Res 14:R86 Jou TS, Stewart DB, Stappert J et al (1995) Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex. Proc Natl Acad Sci U S A 92:5067–5071 Pantel K, Passlick B, Vogt J, et al (1998) Reduced expression of plakoglobin indicates an unfavorable prognosis in subsets of patients with non-small-cell lung cancer. J Clin Oncol 16:1407–1413 Rieger-Christ KM, Ng L, Hanley RS, et al (2005) Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential. Br J Cancer 92:2153–2159 Tselepis C, Chidgey M, North A et al (1998) Desmosomal adhesion inhibits invasive behavior. Proc Natl Acad Sci U S A 95:8064–8069 Wahl JK, Nieset JE, Sacco-Bubulya PA et al (2000) The amino- and carboxyl-terminal tails of (beta)-catenin reduce its affinity for desmoglein 2. J Cell Sci 113(Pt 10):1737–1745 Yashiro M, Nishioka N, Hirakawa K (2006) Decreased expression of the adhesion molecule desmoglein-2 is associated with diffuse-type gastric carcinoma. Eur J Cancer 42:2397–2403

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Desmoid Tumor Sue Clark Imperial College London, London, UK

Synonyms Aggressive fibromatosis; Gardner syndrome; Mesenteric fibromatosis

Definition Desmoid (meaning tendon-like) tumors are a heterogeneous group of rare connective tissue neoplasms, which can occur at almost any anatomical location. Desmoids have been classified as fibromatoses, along with pathologies such as palmar fasciitis, which are due to proliferation of well-differentiated fibroblasts and are locally infiltrative and tend to recur after excision but do not metastasize.

Characteristics Desmoids are rare, accounting for less than 0.1% of all tumors, and have an annual incidence of two to four per million. While most occur sporadically, 2% are associated with ▶ familial adenomatous polyposis (FAP), an autosomal dominantly inherited cancer predisposition syndrome due to mutation of the tumor suppressor gene APC (▶ APC gene in familial adenomatous polyposis). Desmoids are over 1,000 times more common in individuals with FAP than in the population in general, occurring in about 10–20% of them, and are an important cause of death in this group. It is useful to classify desmoid tumors as being either sporadic or FAP associated and by their

Desmoid Tumor

location into intra-abdominal, abdominal wall, or extra-abdominal. Pathology Both sporadic and FAP-associated desmoids have been shown to be clonal proliferations of myofibroblasts. Those associated with FAP result from acquired mutations in the wild-type copy of APC. Somatic loss of the b-catenin gene has been described in sporadic desmoids, and APC mutation has also been identified in some cases ▶ APC/b-catenin pathway. Thus abnormal activation of the Wnt pathway seems to have an important role in desmoid tumorigenesis ▶ Wnt Signaling. A variety of complex chromosomal abnormalities, including trisomy 8 and gain of 1q21, has also been found in some tumors. There is no true capsule, and the desmoid compresses and infiltrates surrounding tissues as it grows. Desmoids range in size from a few centimeters to large masses weighing several kilograms. A photograph of a mesenteric desmoid tumor taken at surgery can be found in ▶ APC gene in familial adenomatous polyposis. Growth rates are very variable. There have been reports of spontaneous resolution, and some desmoids grow relentlessly. The majority, however, either display cycles of growth and resolution or stabilize. The cut surface is usually pale and whorled. There may be central hemorrhage, necrosis, or cystic degeneration. Histologically desmoids consist of mature, highly differentiated spindle shaped fibroblasts in an abundant collagen matrix. The histological appearances are not necessarily diagnostic and need to be interpreted in the light of the macroscopic findings. Etiology Trauma, sex hormones, and genetics have all been implicated in their etiology. Many sporadic abdominal wall desmoids seem to arise in women in pregnancy, perhaps as a result of low-grade trauma of stretching, coupled with high levels of female sex hormones. There have been numerous reports of desmoids arising at sites of surgical wounds, although many, particularly at

Desmoid Tumor

extra-abdominal sites, seem to occur in the absence of any previous trauma. The higher incidence of desmoids in females, association with pregnancy, presence of estrogen receptors, and results of some experimental studies on desmoid cell lines all suggest that estrogens may have a role in stimulating desmoid development. Desmoids are very much more common in individuals with FAP. Within this group some familial clustering has been observed, in part explained by a genotype-phenotype correlation in which families with an APC mutation 30 of codon 1444 have an attenuated colorectal phenotype but a high risk of desmoid development. There is also evidence of the influence of as yet unidentified modifier genes. Clinical Features Desmoids most commonly occur in young adults (mean age of onset around 30 years) but have been described in children and even babies. Sporadic desmoids are more frequent in women than men (reported gender ratio 2–5:1) but in FAP there is a less marked gender difference. Sporadic desmoids are found predominantly in the abdominal wall (50%) and at extra-abdominal sites (40%), whereas about 80% of desmoids associated with FAP are within the abdomen, mostly in the small bowel mesentery. It is not uncommon for an individual to develop desmoids at multiple sites. Intra-abdominal desmoids characteristically arise in the small bowel mesentery. Potential “desmoid precursor lesions,” consisting of small plaques of peritoneal thickening, have been observed in patients with FAP. It is thought that these enlarge, causing a diffuse thickening and puckering of the mesentery which can be seen on CT scans. In some cases, a frank desmoid mass develops. Most extra-abdominal desmoids cause symptoms because of their bulk and resulting mechanical effects. At some sites, for example in the neck, they can compress nerves and blood vessels. The overlying skin may ulcerate and abdominal wall desmoids occasionally adhere to and erode abdominal organs. Intra-

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abdominal desmoids can cause major morbidity and even death, usually due to ureteric obstruction, bowel obstruction, or perforation, either due to direct erosion or to compromise of the vascular supply. CT and MRI are the most useful imaging modalities, showing both tumor size and relationship to neighboring structures. Signal intensity on T2 weighted MRI may reflect cellularity and is correlated with tumor growth. Treatment The treatment of desmoids is difficult and controversial. There are numerous case reports and small uncontrolled series in the literature, but these are difficult to interpret, particularly as the natural history of these tumors is so variable. The drugs most widely used are ▶ nonsteroidal anti-inflammatory drugs (NSAIDs) (particularly sulindac) and antiestrogens (▶ tamoxifen or toremifene). Overall the response rates to a variety of drugs in these classes are claimed to be in the region of 50% but in reality is likely to be considerably less than this. There have been a handful of reports of acute desmoid necrosis, with abscess formation or bowel perforation in some, occurring in the weeks after initiation of drug treatment. As NSAIDs have little in the way of adverse effects they are often used as first-line treatment. The mechanism of action in this setting is not clear, but there is some evidence that Cox-2 inhibition may inhibit desmoid growth, ▶ celecoxib, and ▶ cyclooxygenase-2 in colorectal cancer. There have been no trials of Cox-2 inhibitors used therapeutically. Antiestrogens can be used alone or in combination with NSAIDs. Surgery is widely accepted as the first-line treatment for extra-abdominal and abdominal wall tumors. Recurrence rates are high (20–80%) but unaffected by use of prosthetic mesh in reconstruction. Serious morbidity and mortality rates are generally very low, although some sites, such as the neck, pose particular challenges. There are some reports suggesting that radiotherapy given postoperatively might reduce recurrence rates.

D

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Excision of intra-abdominal desmoids is also associated with frequent recurrence but carries a substantial risk of perioperative mortality or major morbidity. The commonest reason for this is that the tumors lie close to or encase the superior mesenteric blood vessels, so that the blood supply of a large part of the intestine may be damaged or deliberately sacrificed during surgery. This may result in the need for lifelong parenteral nutrition and in a handful of cases small bowel transplantation has been performed in these circumstances. Careful case selection, using CT angiography and multiplanar reconstruction, together with accumulation of expertise in a specialized institution has been shown to produce better surgical results in the last 10 years. Generally, however, major resection of intra-abdominal desmoids should be avoided. Ureteric obstruction can be successfully overcome by stenting, and intestinal obstruction or fistulation may be managed in many cases, at least acutely, by defunctioning. Cytotoxic chemotherapy has been used to treat life-threatening desmoids. Response rates of 50% have been obtained using doxorubicin and dacarbazine in combination and also with a less toxic regimen of methotrexate and vinblastine. In view of the potential toxicity of this type of treatment, it should probably be reserved for progressive, inoperable desmoid tumors in which other treatments have failed.

Desmoplasia

Desmoplasia A. Kate Sasser and Brett M. Hall Department of Pediatrics, Columbus Children’s Research Institute, The Ohio State University, Columbus, OH, USA

Synonyms Scirrhous; Stroma; Stromal cell response

Definition Desmoplasia is the formation of fibrous connective tissue by proliferation of fibroblasts. Desmoplasia is a key component of solid tumor stroma (Fig. 1).

Characteristics Tumors have many parallels to wounds, including similar inflammatory and desmoplastic responses, and fibroblasts are the key cellular component in the development of desmoplasia. Fibroblasts are recruited into the wound or tumor, secrete and remodel extracellular matrix (ECM) (▶ extracellular matrix remodeling), and serve as scaffolding for other cell types in connective

Cross-References ▶ Aggressive Fibromatosis in Children

References Clark SK, Phillips RKS (1996) Desmoids in familial adenomatous polyposis. Br J Surg 83:1494–1504 Hosalkar HS, Fox EJ, Delaney T et al (2006) Desmoid tumours and current status of management. Orthop Clin North Am 37:53–63 Okuno S (2006) The enigma of desmoid tumours. Curr Treat Options Oncol 7:438–443 Reitamo JJ, Scheinin TM, Hayry P (1986) The desmoid syndrome. New aspects in the cause, pathogenesis and treatment of the desmoid tumour. Am J Surg 151:230–237 Sturt NJH, Clark SK (2006) Current ideas in desmoid tumours. Fam Cancer 5:275–285

Desmoplasia, Fig. 1 Hematoxylin and eosin (H&E) stain. Tumor fibroblasts (i.e., desmoplasia) appear pink

Desmoplasia

tissue. As fibroblasts incorporate into a tumor environment, they undergo a phenotypic change and acquire an “activated fibroblast” appearance, which are also known as myofibroblasts or tumorassociated fibroblasts. Myofibroblasts have similar markers to fibroblasts, but myofibroblasts upregulate proteins such as a-smooth muscle actin (a-SMA), fibroblast activation protein (FAP-1), and ▶ fibronectin fibrils. During wound repair, the number of myofibroblasts returns to a normal level upon wound resolution. In contrast to wound repair, ▶ tumor microenvironments simulate a chronic wound in many ways. Thus, local fibroblasts and those that were recruited into the expanding stroma are continuously exposed to activation signals. Activated fibroblasts expand and contribute to an increased stromal response known as desmoplasia. Desmoplasia can be associated with increased tumor stage and poor prognosis in ▶ breast cancer patients, but it is unclear whether fibroblasts are active inducers or passive participants in cancer progression. It is clear, however, that activated fibroblasts play a large role in the expanding tumor stroma (▶ stromagenesis). Fibroblastic stromal cells and desmoplasia have been linked to several activities that promote cancer growth and ▶ metastasis (▶ semaphorin) including ▶ angiogenesis, ▶ epithelial to mesenchymal transition (EMT), and progressive genetic instability. Additionally, fibroblastic stromal cells can dysregulate antitumor immune responses, as exemplified by experiments demonstrating that allogeneic murine tumor cells, when co-injected with fibroblastic stromal cells, can engraft across immunologic barriers. Together, these studies suggest that tissue-specific fibroblasts are influential players in progression of metastatic cancer. However, with the exception of promoting epithelial to mesenchymal transition, the direct biological impact on cancer cells themselves has been difficult to distinguish from indirect mechanisms such as enhanced support for angiogenesis or recruitment of inflammatory cells. The origins of desmoplastic fibroblasts are not fully understood. Some studies have suggested that stromal cell fibroblasts are recruited to the expanding tumor mass from local tissue fibroblasts. However, other experimental evidences

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support that additional tumor-associated fibroblasts can be recruited from peripheral fibroblast pools, such as bone marrow-derived mesenchymal stem cells (MSC) or fibrocytes. It has been shown that once fibroblasts are recruited into the expanding stroma, they change their phenotype and may also undergo selective genetic alterations, which may drive additional tumor growth. Desmoplastic tumor fibroblasts have also been shown to carry unique genetic lesions when compared to those found in expanding tumor cells. These observations offer an additional insight into potential mechanisms for how genetic lesions can induce tumor cell expansion. The mechanism for recruitment of desmoplastic fibroblasts into a developing tumor remains poorly defined. Yet several groups have shown that ▶ platelet-derived growth factor (PDGF) can contribute to the formation of desmoplasia. In a xenograft model using the human breast carcinoma cell line MCF-7 expressing the cellular oncogene, c-ras, investigators demonstrated that blocking tumor PDGF inhibited the formation of desmoplasia. Others have shown that blocking TGF-a, TGF-b, IGF-I, and IGF-II had no effect on the desmoplastic response. Since these models used murine xenografts, it remains unclear whether PDGF is as critical for the development of desmoplasia in human carcinomas (▶ epithelial tumorigenesis). One important way that desmoplastic fibroblasts can contribute to tumor growth and metastasis is through the production of multiple growth factors (▶ fibroblast growth factors). Paracrine growth factors such as the stroma-derived factor 1 (SDF-1/CXCL12) (▶ angiogenesis), ▶ vascular endothelial growth factor (VEGF) (angiogenesis), ▶ fibroblast growth factor (FGF) family, hepatocyte growth factor (HGF), ▶ transforming growth factor beta (TGF-b) family, ▶ interleukin-6 (IL-6), and epidermal growth factor (EGF) have all been linked to increased tumor growth. Desmoplastic fibroblasts also contribute to tumor stroma through the production of fibrous connective tissues and extracellular matrix proteins (▶ fibronectin) (▶ focal adhesion kinase (FAK)). Collagen production is a hallmark feature of desmoplasia. As fibroblasts convert to

D

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Desmoplasia

Desmoplasia, Fig. 2 The tumor microenvironment is composed of many cell types that support tumor cell growth and survival

myofibroblasts or tumor-associated fibroblasts, parallel increases in production of collagen are observed. A pathologist can readily visualize increased levels of tumor collagen using standard histology procedures (▶ pathology), and collagen types I and IV are the most prevalent forms of collagen found within most desmoplastic reactions. Collagen bundles interact with extracellular matrix and cell surface proteins such as integrins (▶ cell adhesion molecules) (focal adhesion kinase (FAK)) to influence the stiffness of a given tumor microenvironment. Desmoplasia varies extensively between tumors and even within the same tumor. Some studies have suggested that desmoplasia is a defensive mechanism used to wall off the expanding tumor, but other data demonstrate that desmoplasia is associated with increased tumor growth, invasion, and metastasis. It is unclear, however, which underlying mechanisms determine the extent to which desmoplasia may promote tumor progression. As investigators continue to recognize the importance of the

tumor microenvironment (Fig. 2), more detailed studies will allow clarification of the biological impact of desmoplasia in tumor development, survival, and metastasis.

Cross-References ▶ Angiogenesis ▶ Breast Cancer ▶ Cell Adhesion Molecules ▶ Cutaneous Desmoplastic Melanoma ▶ Epithelial-to-Mesenchymal Transition ▶ Epithelial Tumorigenesis ▶ Extracellular Matrix Remodeling ▶ Fibroblast Growth Factors ▶ Fibronectin ▶ Focal Adhesion Kinase ▶ Interleukin-6 ▶ Metastasis ▶ Pathology ▶ Platelet-Derived Growth Factor ▶ Semaphorin

Desmoplastic Small Round Cell Tumor

▶ Stem Cell Plasticity ▶ Stromagenesis ▶ Transforming Growth Factor-Beta ▶ Tumor Microenvironment ▶ Vascular Endothelial Growth Factor

References Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432:332–337 Kunz-Schughart LA, Knuechel R (2002) Tumorassociated fibroblasts (part I): active stromal participants in tumor development and progression? Histol Histopathol 17(2):599–621 Mahadevan D, Von Hoff DD (2007) Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther 6(4):1186–1197 Walker RA (2001) The complexities of breast cancer desmoplasia. Breast Cancer Res 3:143–145 Zipori D (2006) The mesenchyme in cancer therapy as a target tumor component, effector cell modality and cytokine expression vehicle. Cancer Metastasis Rev 25:459–467

See Also (2012) Allogeneic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 138. doi:10.1007/978-3-642-16483-5_194 (2012) Bone Marrow-Derived Mesenchymal Stem Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 446. doi:10.1007/978-3642-16483-5_681 (2012) Collagen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 895. doi:10.1007/978-3-642-16483-5_1260 (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Fibroblasts. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1398. doi:10.1007/978-3-642-16483-5_2176 (2012) Genetic Instability. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1527–1528. doi:10.1007/978-3-642-16483-5_2380 (2012) HGF In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1693. doi:10.1007/978-3-642-16483-5_2710 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720

1347 (2012) Myofibroblasts. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2440–2441. doi:10.1007/978-3-642-16483-5_3944 (2012) Paracrine. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2783. doi:10.1007/978-3-642-16483-5_4380 (2012) Xenograft. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3967. doi:10.1007/978-3-642-16483-5_6278

D Desmoplastic Melanoma ▶ Cutaneous Desmoplastic Melanoma

Desmoplastic Small Round Cell Tumor Sean Bong Lee Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA, USA

Synonyms Malignancy of small round blue cell type; Small round cell tumor

Definition DSRCT is a rare and highly aggressive tumor occurring mostly in the abdominal peritoneal cavity of adolescents and young adults. In rare cases, the tumors can also be found in other sites such as pleural cavity, pelvis, bone, and head and neck region. DSRCT belongs to a group of undifferentiated small round cell tumors, which include ▶ Ewing sarcoma/primitive peripheral neuroectodermal tumor (PNET)/Askin’s tumor and ▶ rhabdomyosarcoma. DSRCT is invariably defined by a ▶ chromosomal translocation involving chromosomes 11 and 22, t(11;22)(p13;q12),

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leading to a fusion of two unrelated genes, EWS and WT1, into a single chimeric gene.

Characteristics Clinical and Pathological Features DSRCT was first described in 1989 and is a poorly understood cancer that primarily affects young adults in their second and third decades of life. DSRCT occurs predominantly in males than females, but the reason for this is unknown. Symptoms of DSRCT are usually associated with abdominal pain or pain in the primary site of tumor involvement, distention, and palpable mass. Local invasion or metastasis to the liver, lungs, and bone is commonly found at diagnosis. DSRCT displays distinct histological and immunological features. Most of DSRCT cases are presented as tumors in the serosal surface of abdominal cavity, displaying nests of tumor cells surrounded by dense stromal components (hence the term desmoplastic) containing spindle-shaped fibroblasts and hyperplastic blood vessels. Though rare, the primary tumors in sites other than abdominal region have been documented. The tumors are positive for various cell lineage markers, such as epithelial membrane antigen, keratin (epithelial), desmin (muscle), and neuron-specific enolase (neural). Thus, the tumor cell origin of DSRCT is not known. DSRCT is a clinically aggressive tumor with a high risk of recurrence and an overall poor prognosis. A report on the comparison of different treatments of DSRCT patients suggests that compared to patients who received conventional treatments, a multimodal therapy, which include highdose multiagent chemotherapy, aggressive debulking surgery, and radiotherapy, can prolong overall survival at 3 years (55%) and may provide a possibility of achieving a long-term survival, albeit at a low rate. The two key elements of the multimodal approach are the use of high-dose polychemotherapy, so-called P6 protocol, and greater than 90% removal of tumor by surgery. P6 protocol consists of seven courses of high-dose alkylating agents ▶ cyclophosphamide, doxorubicin, vincristine, ifosfamide, and ▶ etoposide.

Desmoplastic Small Round Cell Tumor

This is followed by aggressive debulking surgery, which was shown to be the major determinant in patient survival. Postoperative radiotherapy also contributed to improved survival. Although the multimodal therapy can improve survival at 3 and 5 years, the prognosis of DSRCT still remains extremely low (median survival of 2.5 years). Molecular Diagnosis Although clinical, histological, and immunological features of DSRCT are distinct, a definitive diagnosis of DSRCT can be provided by genetic techniques. FISH technique, using fluorescently labeled genomic DNA probes derived from EWS and WT1, can be used to identify the specific t(11;22)(p13;q12) translocation of DSRCT. Alternatively, a definitive DSRCT diagnosis can be made with the use of reverse transcriptasepolymerase chain reaction (RT-PCR) technique to amplify and detect the DSRCT-specific EWS/WT1 hybrid mRNA transcripts using DNA primers specific for EWS and WT1 genes. This is an extremely sensitive detection method that can provide accurate diagnosis with limiting tumor materials. Molecular Genetics Molecular genetic studies revealed that all cases of DSRCT harbor a balanced reciprocal chromosomal translocation, t(11;22)(p13;q12) (reciprocal translocation) (Fig. 1). The breakpoint in chromosome 22 has been mapped to the intron 7 of Ewing sarcoma gene, EWS (breakpoints in other sites of EWS, such as in introns 8 and 10, have also been observed in rare cases), while the other breakpoint in chromosome 11 has been invariably mapped to the intron 7 of ▶ Wilms’ tumor gene WT1. This DSRCT-specific chromosomal translocation between EWS and WT1 results in a fusion of the N-terminal domain (NTD) of EWS to the C-terminal DNA-binding domain of WT1. EWS gene was first isolated from the Ewing sarcoma chromosomal breakpoint, where the translocation generates a fusion between EWS and an ETS-family transcription factor gene FLI-1. EWS encodes a putative RNA-binding protein with presumptive roles in transcription and

Desmoplastic Small Round Cell Tumor Chr 22q12

1349 Chr 11p13

EWS Breakpoint

Exon 1 2

3

4

5

6

7

8

9

6

WT1

Breakpoint

KTS

7

9

8

10

Chromosomal translocation

D EWS/WT1

t(11;22)(p13;q12)

KTS DSRCT 1 2

3

4

5

EWS: Transactivation domain WT1: DNA dinding domain

6

7

8

9

10

Two fusion protein products KTS

EWS/WT1(−KTS)

EWS/WT1(+KTS)

Desmoplastic Small Round Cell Tumor, Fig. 1 Schematic representation of DSRCT-specific chromosomal translocation. A reciprocal balanced chromosomal translocation that results in the fusion of EWS gene to WT1 gene is shown. The arrow indicates the promoter of

EWS which drives the transcription of the fusion gene and the boxes mark the exons. Alternative KTS splicing (gray box, KTS) within the exon 9 of WT1 is shown. Two isoforms of the fusion product are shown separately. See text for details

splicing. The NTD of EWS mediates potent transcriptional activation when fused to a heterologous DNA-binding domain, while its C-terminal domain, which is lost in the translocation gene product, is involved in RNA recognition. WT1 encodes a transcription factor which is mutated in a subset of Wilms’ tumor, a childhood kidney cancer. WT1 encodes four Cys2-His2 zinc fingers in the C terminus that mediate sequence-specific DNA binding and the NTD containing both transcriptional activation and repression domains. WT1 is subjected to two alternative RNA splicing events, one of which involves the usage of two alternative splice donor sites at the end of exon 9, leading to inclusion or exclusion of three amino acids, lysine, threonine, and serine (termed KTS), between the zinc fingers 3 and 4 (Fig. 1). The KTS insertion leads to a markedly decreased DNA-binding affinity of WT1. In all EWS/WT1

translocations examined, only the last 3 exons of WT1 (exons 8–10) encoding the last three zinc fingers are fused to the NTD of EWS (Fig. 1), while the first zinc finger of WT1 is invariably lost. The alternative KTS splicing of WT1, however, is preserved. As a result, EWS/WT1 produces two isoforms, EWS/WT1(KTS) and (+KTS), that differ in the DNA-binding affinity and specificity (Fig. 1). In vitro study has shown that only the EWS/WT1(KTS) isoform, but not the EWS/WT1(+KTS), possesses the oncogenic activity in NIH3T3 transformation assay. DSRCT is a rare disease and has been recognized as a distinct cancer type. Therefore, not much is known about the mechanisms of DSRCT, but molecular details are starting to emerge. The novel fusion protein EWS/WT1(KTS) acts as an aberrant transcription factor to presumably initiate the oncogenic process. To

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date, a number of direct transcriptional targets of EWS/WT1(KTS) have been identified, which include PDGF-A (platelet-derived growth-factor A), IGFR1 (insulin-like growth-factor receptor 1), IL2RB (interleukin 2 receptor beta), BAIAP3 (BAI1-associated protein 3), a potential regulator of growth-factor release, and TALLA-1 (T-cell acute lymphoblastic leukemia-associated antigen 1), a gene encoding a tetraspanin-family protein. There is only one target gene identified for EWS/WT1(+KTS), which is LRRC15 (leucinerich repeat containing 15), a gene implicated in cell invasion. All of these target genes are not transcribed by the native WT1 and thus represent EWS/WT1-specific transcripts. Identification of the EWS/WT1 target genes may provide clues to the molecular and cellular pathways that are central to DSRCT. For example, the expression of IGFR1 and IL2RB can promote proliferation and survival of the tumor cells, while the expression of PDGF-A and BAIAP3 by the tumor cells can enhance recruitment and proliferation of surrounding fibroblasts and stromal tissues, which may further enhance the growth of the tumor cells and may explain the dense stroma (desmoplastic feature) associated with DSRCT. Some of these target genes may also have diagnostic and therapeutic values, but it will require further evaluation.

Cross-References ▶ Chromosomal Translocations ▶ Cyclophosphamide ▶ Etoposide ▶ Ewing Sarcoma ▶ Fusion Genes ▶ Rhabdomyosarcoma ▶ Wilms’ Tumor

References Gerald WL, Haber DA (2005) The EWS-WT1 gene fusion in desmoplastic small round cell tumor. Semin Cancer Biol 15:197–205

Desmoplastic Small Round Cell Tumor Gerald WL, Rosai J (1989) Desmoplastic small round cell tumor with divergent differentiation. Pediatr Pathol 9:177–183 Gerald WL, Ladanyi M, de Alava E et al (1998) Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round cell tumor and its variants. J Clin Oncol 16:3028–3036 Ladanyi M, Gerald WL (1994) Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res 54:2013–2840 Lal DR, Su WT, Wolden SL et al (2005) Results of multimodal treatment for desmoplastic small round cell tumors. J Pediatr Surg 40:251–255

See Also (2012) Alternative RNA splicing. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 148. doi:10.1007/978-3-642-16483-5_212 (2012) Cytoreductive surgery. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1057. doi:10.1007/978-3-642-16483-5_1489 (2012) Desmoplastic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1095. doi:10.1007/978-3-642-16483-5_1581 (2012) Doxorubicin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1159. doi:10.1007/978-3-642-16483-5_1722 (2012) EWS. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1352. doi:10.1007/978-3-642-16483-5_2045 (2012) NIH-3T3 cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2520. doi:10.1007/978-3-642-16483-5_4084 (2012) P6 protocol. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2752. doi:10.1007/978-3-642-16483-5_4319 (2012) Reciprocal translocation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3204. doi:10.1007/978-3-642-16483-5_4989 (2012) RT-PCR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3322. doi:10.1007/978-3-642-16483-5_5129 (2012) Surgical debulking. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3575. doi:10.1007/978-3-642-16483-5_5598 (2012) Transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3757–3758. doi:10.1007/978-3-642-16483-5_5913 (2012) Translocation reciprocal. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5945 (2012) Vincristine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3908. doi:10.1007/978-3-642-16483-5_6188 (2012) WT1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3958. doi:10.1007/978-3-642-16483-5_6265

Desmosomes

Desmoplastic Tumor Microenvironment ▶ Stromagenesis

Desmosomes Martyn A. Chidgey School of Cancer Sciences, University of Birmingham, Birmingham, UK

Synonyms Macula adherens; Maculae adherentes

Definition Desmosomes are intercellular junctions that mediate cellular ▶ adhesion and maintain tissue integrity. They are found in epithelial cells, myocardial and Purkinje fiber cells of the heart, arachnoid cells of brain meninges, and follicular dendritic cells of lymph nodes.

Characteristics Desmosomes are localized at sites of close cellcell contact (Fig. 1a). They are less than 1 mm in diameter, have a highly organized structure at the ultrastructural level, and act as anchoring points for intermediate filaments of the cell cytoskeleton (Fig. 1b). By linking intermediate filaments of adjacent cells, desmosomes confer structural continuity and mechanical strength on tissues. Desmosomes are particularly prevalent in tissues, such as the epidermis and heart, that experience mechanical stress. The proteins that form desmosomes belong to three families, the desmosomal cadherins, the armadillo family, and the plakin family of cytolinkers.

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Desmosomal Cadherins The desmosomal cadherins are the membrane spanning cell adhesion molecules of desmosomes. In humans, there are seven, four desmogleins (Dsg1–4) and three desmocollins (Dsc1–3). Each desmoglein and desmocollin is encoded by a distinct gene that is located in the desmosomal cadherin gene cluster on chromosome 18q21. All three desmocollin genes encode a pair of proteins, a larger “a” protein and a smaller “b” protein, that are generated by alternative splicing of mRNA. All desmosomes contain at least one desmoglein and one desmocollin and both are required for adhesion. The desmosomal cadherins show tissuespecific patterns of expression with Dsg2 (desmoglein-2 adhesion molecule) and Dsc2 ubiquitously expressed in tissues that produce desmosomes and the others largely restricted to stratified epithelial tissues. The extracellular domains of desmosomal cadherins produced by adjacent cells interact in the intercellular space. Within the cell desmosomal cadherin, cytoplasmic domains associate with armadillo proteins (Fig. 1c). Armadillo Family Armadillo proteins that are found in desmosomes include plakoglobin (gamma-catenin) and plakophilins. Plakoglobin is indispensable for desmosome function and interacts with desmosomal cadherins, plakophilins, and desmoplakin. Plakoglobin is also found in adherens junctions where it is interchangeable with a closely related armadillo protein, beta-catenin. In addition to its structural role in adherens junctions, beta-catenin acts as a signaling molecule in the APC/betacatenin pathway. There is a strong possibility that plakoglobin also has a signaling function in this pathway although its role has yet to be fully defined. There are three plakophilins (PKP1–3), each of which is encoded by a distinct gene. Two PKP1 and two PKP2 isoforms are known; in each case, a shorter “a” variant and a longer “b” variant are generated by alternative splicing. The plakophilins exhibit complex tissue-specific patterns of expression, and all three show dual localization in desmosomes and in the nucleus. The

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Desmosomes

a

b

Light microscopy

Electron microscopy IF

ICS PM

c IF

ICS

Dsg

PM

PM

Dsc

PG

PKP

DP

Desmosomes, Fig. 1 Appearance of desmosomes by light and electron microscopy, and a schematic representation of desmosome structure. a) By light microscopy desmosomes have a punctate appearance and delineate the borders of adjacent cells. In this image desmosomes are highlighted by staining for the cytoplasmic desmosomal protein desmoplakin (green) and nuclei are stained blue. b) Desmosomes act as anchoring sites for intermediate filaments and at high magnification appear to rivet

together the plasma membranes of adjacent cells. c) The minimum complement of proteins required for normal desmosomal adhesion include a desmoglein (Dsg), a desmocollin (Dsc), plakoglobin (PG), a plakophilin (PKP) and the intermediate filament (IF) binding protein desmoplakin (DP). For simplicity the Dsc ‘b’ protein and DPII are not shown. ICS, intercellular space; PM, plasma membrane. [Electron micrograph courtesy of M.Berika and D.Garrod, Manchester, UK]

plakophilins have an important structural role in desmosomes, and because of their nuclear localization and similarity to other armadillo proteins, it is possible that they act as signaling molecules.

desmoplakin is obligatory for normal desmosomal adhesion. It is a dumbbell-shaped molecule with two globular domains separated by a coiledcoil rod domain and is thought to exist as a homodimer. The desmoplakin gene encodes two proteins (DPI and DPII) that are generated by alternative splicing and differ only in the length of their central rod domain; the role of DPII, the smaller of these proteins, is unclear. The N-terminal end of desmoplakin binds to

Plakin Family Plakin family proteins bind intermediate filaments and several, including desmoplakin, plectin, envoplakin and periplakin, localize to desmosomes. Of these, only the presence of

Desmosomes

plakoglobin and plakophilins, whereas its C-terminal end binds to intermediate filaments. In epithelial tissues, desmoplakin anchors keratin intermediate filaments to the membrane, but in myocardial and Purkinje fiber cells, it interacts with desmin intermediate filaments, and in arachnoid and follicular dendritic cells, it associates with vimentin intermediate filaments. Null Mutations in Mice Genetic ablation studies in mice have shown the importance of desmosomes for embryonic development and normal tissue biology. Knock-out mice of either Dsg2, Dsc3, or desmoplakin display early embryonic lethality at around implantation or before. Mice without either plakoglobin or PKP2 survive longer but die during mid-gestation as a result of heart defects. Embryonic survival is not affected by the absence of either Dsg3, Dsg4, Dsc1, or PKP3, but loss of these molecules does result in defects in keratinocyte adhesion and skin and hair abnormalities. Clinical Relevance Loss of desmosomal adhesion can result in skin blistering diseases. Pemphigus is an autoimmune blistering disease that is caused by pathogenic autoantibodies against desmogleins. Staphylococcal scalded skin syndrome is caused by toxins with serine protease activity that are released by the bacterium Staphylococcus aureus and specifically cleave Dsg1. Mutations in DNA encoding the desmosomal proteins Dsg2, Dsc2, plakoglobin, PKP2, and desmoplakin can result in arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. Mutations in desmosomal genes can also result in skin disorders such as palmoplantar keratoderma and hair loss. Mutations in plakoglobin, concomitant with strong nuclear accumulation, have been linked to the pathogenesis of prostate cancer. Nuclear accumulation and improper activation of transcriptional targets as a result of a failure to degrade cytoplasmic b-catenin have been implicated in FAP (▶ APC gene in familial adenomatous polyposis), a familial syndrome that predisposes to ▶ colon cancer and sporadic colon cancer. It

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remains to be seen whether plakoglobin has, in common with b-catenin, pro-proliferative effects in cancer. In many cancers, loss of expression of plakoglobin has been observed, and it may be that plakoglobin is antiproliferative in some cell types. There is little doubt that plakoglobin plays a role in cancer, but whether this is related to its participation in desmosomes remains unclear. To date, no mutations in desmosomal cadherin, plakophilin, or desmoplakin genes have been found in cancer. However, many reports have documented altered levels of expression of desmosomal proteins in carcinogenesis. Loss of expression of desmosomal cadherins, plakophilins, and desmoplakin has been reported in some types of cancer. Perhaps surprisingly, elevated expression of desmosomal cadherins and plakophilins has also been reported in certain cancers. It is difficult to come to any sort of firm conclusion about the role of desmosomes in cancer at present. However, it may be that the importance of desmosomes in cancer is twofold. Firstly, as mediators of cell-cell adhesion, reduced expression of desmosomal constituents could lead to loss of cell-cell adhesion, epithelial-mesenchymal transition, increased invasiveness, and metastasis. Secondly, desmosomes may act as signaling centers, and variations in expression levels of desmosomal proteins could trigger intracellular signaling cascades that contribute to cancer pathogenesis.

Cross-References ▶ Adherens junctions ▶ Adhesion ▶ APC/b-catenin pathway ▶ APC gene in Familial Adenomatous Polyposis ▶ Cell adhesion molecules ▶ Colorectal Cancer Clinical Oncology ▶ Cytoskeleton ▶ Desmoglein-2

References Chidgey M, Dawson C (2007) Desmosomes: a role in cancer? Br J Cancer 96:1783–1787

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1354 Garrod D, Chidgey M (2008) Desmosome structure, composition and function. Biochim Biophys Acta 1778:572–587 Getsios S, Huen AC, Green KJ (2004) Working out the strength and flexibility of desmosomes. Nat Rev Mol Cell Biol 5:271–281 Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127:2499–2515 Kottke MD, Delva E, Kowalczyk AP (2006) The desmosome: cell science lessons from human diseases. J Cell Sci 119:797–806

Detachment-Induced Cell Death

Definition Metabolic and transport processes used to chemically inactivate noxious compounds and eliminate them from cells for subsequent excretion from the body.

Characteristics See Also (2012) Epithelial Cell. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin Heidelberg, pp 1291–1292. doi:10.1007/978-3-642-16483-5_1958 (2012) Plakin Family. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin Heidelberg, p 2899. doi:10.1007/978-3-642-16483-5_4592

Detachment-Induced Cell Death ▶ Anoikis

Determination of Tumor Extent and Spread ▶ Staging of Tumors

Detoxication ▶ Detoxification

Detoxification John D. Hayes Medical Research Institute, Jacqui Wood Cancer Centre, University of Dundee, Dundee, UK

Synonyms Carcinogen metabolism; Detoxication; Drug metabolism; Xenobiotic biotransformation; Xenobiotic metabolism

Humans are continuously exposed to foreign chemicals (▶ xenobiotics) through administration of medicines, the consumption of food and drink, and air breathed. Protection against the detrimental effects of xenobiotics is achieved by the concerted actions of a battery of proteins that metabolize, transport, and ultimately pump out of cells modified forms of the compounds originally encountered. This process is called detoxification or detoxication (in instances where no toxicity occurs). Although detoxication occurs primarily in the liver, all cells possess some capacity to metabolize and eliminate unwanted chemicals. The xenobiotics subject to this process are numerous and include mycotoxins, phytoalexins, pesticides, herbicides, environmental pollutants, cytotoxic anticancer agents, and many pharmacologically active drugs. Detoxication processes also confer protection against harmful compounds of endogenous origin, many of which arise as a consequence of interaction with reactive oxygen species, such as the superoxide anion, produced normally in the body. Detoxication is achieved in two distinct stages, the first involving metabolism of the xenobiotic and the second involving energy-dependent efflux of the xenobiotic from the cell. Historically, description of xenobiotic biotransformation has been divided into phase 1 and phase 2 metabolism, and consequently efflux of xenobiotics is referred to as phase 3 of detoxication. • Phase 1 drug metabolism involves an initial chemical modification of the xenobiotic that results in the introduction, or exposure, of a functional chemical group (e.g., –OH, –NH2, –SH, –COOH) into the compound. This usually entails enzyme-catalyzed oxidation

Detoxification

reactions by ▶ cytochrome P450 (CYP) or flavin monooxygenase. • Phase 2 drug metabolism often involves a second chemical alteration of the xenobiotic, usually at the same region of the molecule where the functional group was introduced. This is performed by enzymes catalyzing conjugation reactions (such as ▶ glutathione S-transferase (GST), N-acetyltransferase (NAT), ▶ sulfotransferase (SULT), and UDP-glucuronosyltransferase (UGT)). It should be noted that the use of the terms phase 1 and phase 2 to define the detoxication enzymes is somewhat arbitrary and does not necessarily reflect the pathway of biotransformation of all chemicals. Thus, a number of xenobiotics are subject to several modifications by the phase 1 CYP isoenzymes before serving as substrates for the phase 2 enzymes. Alternatively, some xenobiotics do not require modification by phase 1 enzymes before metabolism by phase 2 enzymes, and others are subject to modification by more than one phase 2 drugmetabolizing enzyme. As a result of differences in drug metabolism, the group of enzymes catalyzing reduction of hydrolysis reactions (e.g., ▶ aldehyde dehydrogenase (ADH), aldo-keto reductase (AKR), epoxide hydrolase (EPHX), and NAD(P)H-quinone oxidoreductase (NQO)) are variously referred to as phase 1 or phase 2 detoxication, depending on the individual xenobiotic being considered and the preferences of research workers. Clearly, these enzymes provide a highly flexible metabolic defense that has evolved to protect against a diverse spectrum of chemicals. • Finally, phase 3 of detoxication involves ATP-dependent elimination of the parent compound or modified xenobiotic by proteins that are drug efflux pumps (e.g., multidrug resistance protein (MDR) and multidrug resistanceassociated protein (multidrug resistance protein) (MRP)). As a consequence of the combined actions of phase 1 and phase 2 enzymes, a diverse spectrum of xenobiotics acquires a limited number of molecular “tags” (i.e., acetate, glutathione, glucuronide, or sulfate moieties) that are recognized by the MRP transmembrane pumps. Furthermore, the

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xenobiotic metabolites produced by phase 1 and phase 2 are usually more soluble, and easily excreted, than the parent compound. While the ability of CYP to oxidize xenobiotics is generally desirable, as it facilitates further metabolism and elimination of harmful chemicals, it can sometimes result in the generation of highly reactive products that may not be readily detoxified. In such instances, modification of intracellular macromolecules will occur resulting in necrosis, ▶ apoptosis, or malignant transformation. As an example of the interplay between toxification and detoxification reactions, a scheme depicting metabolism of ▶ aflatoxin B1 (AFB1), modification of macromolecules by AFB1 metabolites, and efflux of the AFB1glutathione conjugate from a cell is shown in the illustration (Fig. 1).

Genetic Variation Numerous proteins have evolved that detoxify drugs, and certain of the families listed above comprise over twenty genes. In total, the human probably possesses between 100 and 150 genes encoding detoxication proteins. Substantial variation can occur in the levels of these proteins in tissues from different individuals, and this can result in increased sensitivity of cells to chemical insult. In part, this interindividual variation is due to genetic polymorphisms. By definition, such differences must be present in at least 1% of the population in order to be considered a genetic polymorphism. In some instances, the variation involves deletion of detoxication genes with complete loss of specific functions, whereas in other instances, point mutations result in alteration of protein structure, causing only a modest attenuation of activity. In other cases, mutations alter the regulatory regions of genes, causing altered expression of normal protein. Detoxication genes that are polymorphic in the human include those for the enzymes CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP2E1, AKR1C4, GSTM1, GSTP1, GSTT1, NAT2, SULT1A1, SULT1E1, SULT2A1, UGT1A1, UGT1A4, UGT1A6 and UGT2B7, EPHX and NQO1, as well as the

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Detoxification, Fig. 1 Detoxification pathways for aflatoxin B1. The mycotoxin is converted to the ultimate carcinogen AFB1-8,9-epoxide, by the actions of the hepatic phase 1 CYP enzyme system. The epoxidated AFB1 is highly reactive, and if it is not detoxified, it will form DNA adducts that may cause hepatocarcinogenesis. The phase 2 GST enzymes can achieve detoxification of this unstable intermediate, and the resulting AFB1-

Detoxification

glutathione conjugate is eliminated from the liver cell by MRP. In addition, AFB1-8,9-epoxide can rearrange to form a dialdehyde-containing metabolite which will covalently modify proteins by forming Schiff’s bases. The dialdehyde can be reduced by phase 2 AKR to yield a dialcohol that may be a substrate for SULT or UGT before being transported out of the cell, presumably by MRP

Development Lymph Vessel

MRP2 efflux pump. It is clear additional polymorphisms remain to be identified. Cellular Regulation In addition to genetic polymorphisms, induction of detoxication proteins by xenobiotics and environmental agents is a further mechanism that can cause interindividual differences in detoxification capacity. Induction of detoxication proteins represents an adaptive response to chemical and ▶ oxidative stress, which can be brought about by synthetic drugs or by naturally occurring compounds such as coumarins, indoles, and isothiocyanates that are found in edible plants. Increased expression provides short-term resistance to toxic xenobiotics. Enzyme induction also results in increased metabolism of therapeutic drugs. Many of the enzymes and pumps such as CYP, GST, ADH, AKR, NQO, and MRP are inducible, often by transcriptional activation of genes encoding the proteins. The promoters of these genes contain enhancers that enable a transcriptional response to a diverse spectrum of chemical agents. The enhancers that are involved in induction of detoxication proteins include ▶ AP-1 binding sites, the antioxidant responsive element, the xenobiotic responsive element, the phenobarbital-responsive enhancer module, progesterone X receptor, and peroxisome proliferator-activated receptor enhancer. Clinical Relevance It is apparent from studies into the mechanisms of selective toxicity between species that variation in the activity of detoxication proteins influences sensitivity to chemical insult. Increasing evidence suggests that genetic polymorphisms in detoxication enzymes can confer an inherited predisposition to a number of malignant diseases that are influenced by environmental factors (e.g., lung and colorectal cancer). They may also confer a predisposition to adverse drug reactions. Induction of some phase 2 detoxication systems is believed to represent a major mechanism of cancer ▶ chemoprevention and is thought to explain in part the epidemiological data

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suggesting that consumption of diets rich in fruit and vegetables protects against certain malignant diseases. Acquired ▶ drug resistance to chemotherapy is a major problem in the treatment of many cancers. There is overwhelming evidence that the overexpression of several detoxication proteins, particularly GST, MDR, and MRP, contributes to the drug-resistant phenotype.

References Borst P, Evers R, Kool M et al (2000) A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 92:1295–1302 Dinkova-Kostova AT, Massiah MA, Bozak RE et al (2001) Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci U S A 98:3404–3409 Guengerich FP, Shimada T (1991) Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol 4:391–407 Hayes JD, McLellan LI (1999) Glutathione and glutathione-dependent enzymes represent a coordinately regulated defence against oxidative stress. Free Radic Res 31:273–300 Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: regulation of GST and contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30:445–600 Klaassen CD (ed), Amdur MO, Doull J (eds emeriti) (1996) Casarett and Doull’s toxicology: the basic science of poisons. McGraw-Hill, New York

Deubiquitinating Enzymes (DUBs) ▶ Herpesvirus-Associated Protease De-ubiquitinase

Ubiquitin-Specific

Development Lymph Vessel ▶ Lymphangiogenesis

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Development of New Lymphatic Vessels

Definition

Development of New Lymphatic Vessels ▶ Lymphangiogenesis

Diabody is a noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavychain variable (VH) and light-chain variable (VL) regions connected by a small peptide linker. Another form of diabody is single-chain (Fv)2 in which two scFv fragments are covalently linked to each other.

Dezocitidine ▶ 5-Aza-20 Deoxycytidine

D-factor ▶ Leukemia Inhibitory Factor

dFdC ▶ Gemcitabine

DIA ▶ Leukemia Inhibitory Factor

Diabody Shuji Ozaki Department of Hematology, Tokushima Prefectural Central Hospital, Tokushima, Japan

Synonyms Engineered antibody; Multimeric antibody fragments; Single-chain Fv dimer

Characteristics Advances in antibody technology are enabling the design of antibody-based reagents for specific purposes in cancer diagnosis and monoclonal antibody therapy. First, to minimize the immunogenicity and enhance the efficacy in human use, mouse monoclonal antibodies are engineered to chimeric antibodies or humanized antibodies by grafting to the human constant region or framework. Moreover, fully human antibodies are developed by the use of transgenic mice (see ▶ Transgenic Mouse) or phage display technology. Second, monoclonal antibodies are designed as immunoconjugates to deliver the cytotoxic agents such as chemotherapeutic drugs, toxins, enzymes, and radioisotopes. These therapeutic antibodies have emerged as potent agents and are used worldwide for cancer therapy. Engineered antibody fragments have been investigated as alternative reagents because of their unique properties resulting from the structure. Structure A variety of antibody fragments are developed including single VH domain, Fab, scFv, and multimeric formats such as multivalent scFvs (diabody, triabody, and tetrabody), bispecific scFv, and minibody (scFv–CH3 dimer) (Fig. 1). In scFv fragments, the VH domain binds to its attached VL domain when the linker is flexible and long enough (a length of at least 12 amino acids). For example, the linker sequence of (Gly4Ser)3 provides sufficient flexibility for the VH and VL domain to form Fv comparable to the parent antibody. In contrast, when the linker is shortened to less than 12 residues (e.g., five

Diabody

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VL VH

CH1 CL

Fab (∼55 KDa)

Fv (∼30 KDa)

scFv (∼30 KDa)

C H2

D

CH3 Intact IgG (∼150 KDa)

Minibody (∼75 KDa)

(scFv)2 (∼60 KDa)

(scFv)2 (∼60 KDa)

Bispecific (scFv)2 (∼60 KDa)

Triabody (∼90 KDa)

Tetrabody (∼120 KDa)

Bispecific sc(Fv)2 (∼60 KDa)

Diabody, Fig. 1 Schematic structure of intact IgG antibody and engineered antibody fragments. The variable regions of heavy (VH) and light chains (VL) contribute to the antigen binding. The VH and VL domains can be connected by a peptide linker to form single-chain Fv (scFv). The scFv fragments can form multimers such as

(scFv)2 diabody, triabody, and tetrabody depending on the linker length and the V-domain orientation. The bivalent sc (Fv)2 can be generated by connecting two scFvs covalently. Bispecific diabodies can also be engineered by using two different Fv domains

amino acids of (Gly4Ser)), the VH and VL domains are unable to bind each other, and instead the scFv fragment forms a noncovalent dimer by another scFv molecule [(scFv)2 diabody]. Shortening of the linker length between the VH and VL domains (less than three residues) promotes the assembly of trimeric or tetrameric structures (triabody or tetrabody). However, this multimer formation also depends on the V-domain orientation either VH–VL or reverse VL–VH orientation in the scFv constructs. The bivalent Fv fragment can also be designed by linking two scFv domains covalently as a single-chain version [sc(Fv)2 diabody]. The sc(Fv)2 version is more stable than (scFv)2, and this structure may form a noncovalent dimer [sc(Fv)2]2. The capacity of multivalent binding of these fragments offers a significant opportunity to design multifunctional antibody reagents. The diabody structure is used to form ▶ bispecific antibodies by linking different VH and VL domains of two

antibodies (e.g., VHA–VLB and VHB–VLA). However, when two different polypeptides are produced within a single cell, purification steps are necessary to obtain the active heterodimeric antibody among the inactive homodimers. Therefore, bispecific sc(Fv)2 version is developed by connecting two different scFv domains with the middle-length linker (e.g., VHA–VLB–VHB–VLA or VHA–VLA–VHB–VLB). Pharmacokinetics and Distribution The pharmacokinetics of these antibody fragments is markedly different from intact IgG antibodies that exhibit prolonged circulation (t1/2 of up to 3 weeks). The lower molecular weight constructs (below than 60 kDa) are subject to be excreted by renal clearance, resulting in a shorter serum half-life than intact IgG. In most cases, the t1/2 values of scFv and diabody are extremely short such as 2 and 6 h, respectively. This rapid pharmacokinetics is the most favorable for

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imaging applications and ▶ radioimmunotherapy because of the lower background levels in normal tissues. Drug biodistribution studies of radiolabeled scFv and sc(Fv)2 have shown high tumor-to-blood ratios in xenograft models compared with intact IgG antibodies. The fast blood clearance of antibody fragments contributes to avoid undesired toxicity, and these fragments have the remarkable advantage for ▶ targeted drug delivery of toxins or radioisotopes. In addition, antibody fragments show better penetration into the tumor mass, but these smaller constructs have shorter retention to tumor cells at the same time. Thus, the valance of penetration and retention of antibodies is an important factor for therapeutic use, especially in solid tumors. The Fab and scFv fragments are monovalent and exhibit poor retention on target cells, but multivalent forms of these fragments such as diabody, triabody, and tetrabody exhibit dramatically increased affinity and high tumor retention compared with the parent scFv. The ideal tumortargeting reagents are intermediate-sized multivalent antibodies such as bivalent diabodies that show a longer half-life as well. In another approach, the Fc portion is fused to antibody fragments to control the serum levels of the antibody. The scFv–Fc or scFv–CH3 fusion antibodies (minibodies) are expected to have a more prolonged half-life and increased tumor accumulation in vivo. The serum half-life of antibody fragments can also be extended by modification such as linkage to polyethylene glycol (PEG). Agonistic Activity In terms of mechanism of action, intact IgG antibodies kill tumor cells mainly by Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and ▶ complement-dependent cytotoxicity. In contrast, antibody fragments have the compact structures without the Fc portion and have unique characteristics for using cancer treatment. The two binding sites of diabodies are located at a distance of about 70 Å less than half for those of intact IgG antibodies. Therefore, diabodies can place the antigens more closely to each other

Diabody

than by the parent IgG antibodies, which efficiently induces the ligation of target molecules on the cell surface. When the targets are functional receptors, the diabody can mediate a direct effect or signal transduction in tumor cells, including the stimulation of ▶ apoptosis or cell death. For example, we and our collaborators have generated (scFv)2 and sc(Fv)2 diabodies that recognize CD47 or ▶ HLA class I molecules. These diabodies can cross-link the target antigens and show the enhanced cytotoxic activities against hematological malignancies such as leukemia, lymphoma, and myeloma cells when compared with the original IgG antibodies. Thus, enhancement of the cross-linking potential is one of the important bioactivities of antibody fragments. Application of Diabodies A variety of target antigens have been evaluated for therapeutic purposes including CD19, CD20, CD22, epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), HER2, MUC1, and ▶ carcinoembryonic antigen (CEA). Several types of diabodies and minibodies are engineered for targeting these candidate antigens on tumor cells. Immunotoxins are also constructed to deliver the cytotoxic agents, radioisotopes, enzymes, cytokines, and liposomes by using antibody fragments. Previous studies have shown the effectiveness of these reagents in preclinical and clinical trials. ▶ Bispecific antibodies that comprise two different binding specificities have been studied extensively in cancer diagnosis and therapy. Most of the bispecific reagents are designed for the retargeting of effector cells such as cytotoxic T lymphocytes and NK cells. Recombinant bispecific diabodies such as anti-CD19 x antiCD3, anti-Ep-CAM x anti-CD3, and anti-HER2 x anti-CD3 have been used in the immunotherapy of ▶ B-cell lymphoma, Breast (see ▶ Breast Cancer), ovarian (see ▶ Ovarian Cancer) and colorectal cancer (see ▶ Colorectal Cancer Clinical Oncology). Another strategy of bispecific antibodies is the recruitment of effector molecules including toxins, drugs, prodrugs, cytokines, and radionuclides in vivo. First, the tumor cells are targeted by the tumor-specific binding site of the diabody. After

Diet

the unbound diabody is cleared from the serum, cytotoxic drugs or radiolabeled hapten is administered to be captured by another binding site of the bound diabody. Future Directions In principle, selection of target molecules and modification of antibody constructs are key issues of antibody-based strategies in clinical utility. Based on the properties of pharmacokinetics, biodistribution, and manufacturing production, engineered antibody fragments have been investigated as alternative reagents to target cancer cells. As more small-sized antibody than diabody, single-domain antigen-binding fragments from camelid heavy-chain antibodies (called VHH or nanobody, 15 kDa) are also used. Although the efficacy of these antibody fragments needs to be evaluated in clinical settings, the drastic potential of agonistic activity or multivalent activity of these reagents will provide new promises for development of the next generation of antibody drugs in cancer diagnosis and treatment.

Cross-References ▶ Apoptosis ▶ B-cell Lymphoma ▶ Bispecific Antibodies ▶ BRMS1 ▶ Carcinoembryonic Antigen ▶ Colorectal Cancer Clinical Oncology ▶ Complement-Dependent Cytotoxicity ▶ HLA Class I ▶ Ovarian Cancer ▶ Radioimmunotherapy ▶ Targeted Drug Delivery ▶ Transgenic Mouse

1361 Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136 Jain M, Kamal N, Batra SK (2007) Engineering antibodies for clinical applications. Trends Biotechnol 25:307–316

See Also (2009) Antibody-dependent cell mediated cytotoxicity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 188. doi: 0.1007/9783-540-47648-1_313 (2012) Chimeric antibodies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 806. doi:10.1007/978-3-64216483-5_1091 (2012) Cytokine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1051. doi:10.1007/978-3-642-16483-5_1473 (2012) Drug biodistribution. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1160. doi:10.1007/978-3-64216483-5_1732 (2012) Humanized antibodies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1760. doi:10.1007/978-3-64216483-5_2863 (2012) Monoclonal antibody therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2367–2368. doi:10.1007/978-3642-16483-5_3823 (2012) Pharmacokinetics. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2845. doi:10.1007/978-3-642-16483-5_4500

Diagnostic Pathology ▶ Pathology

Dibasic Processing Enzyme ▶ Furin

References Batra SK, Jain M, Wittel UA et al (2002) Pharmacokinetics and biodistribution of genetically engineered antibodies. Curr Opin Biotechnol 13:603–608 Beckman RA, Weiner LM, Davis HM (2007) Antibody constructs in cancer therapy. Cancer 109:170–179

Diet ▶ Colorectal Cancer Nutritional Carcinogenesis

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Dietary Carcinogens ▶ Food-Borne Carcinogens

Dietary Essential Minerals ▶ Mineral Nutrients

Diethylstilbestrol Rosemarie A. Ungarelli and Carol L. Rosenberg Boston Medical Center and Boston University School of Medicine, Boston, MA, USA

Synonyms DES

Definition Diethylstilbestrol is a synthetic nonsteroidal estrogen with biological properties similar to endogenous estrogens such as estradiol-17-beta and estrone (▶ Estradiol).

Characteristics Pharmacology Diethylstilbestrol is administered orally, is lipidsoluble, and readily absorbed from the proximal gastrointestinal tract. It is metabolized via the hepatic microsomal system to dienestrol and quinone and epoxide intermediates. It crosses the placenta and is thought to be metabolized by the fetus. Initial Use and Early Epidemiologic Studies Diethylstilbestrol (DES), first manufactured by Dodds and associates in London in 1938, was

Dietary Carcinogens

used to treat several gynecologic conditions. In particular, it was prescribed for the treatment of frequent or threatened miscarriages. As early as 1953, Dieckmann and colleagues demonstrated that DES did not improve pregnancy outcomes. (In fact, in a later reanalysis of these data in 1978, Brackbill and Berendes showed that women exposed to DES had higher risks of premature births, perinatal death, and miscarriages than women who were given placebo.) Other studies also found DES to be ineffective in preventing adverse pregnancy outcomes, but physicians continued to prescribe the drug to try to maintain high-risk pregnancies because its use seemed logical and it was well established. It was administered to approximately 5–10 million pregnant women in the United States between 1940 and 1971. It remained in use in Europe until the early 1980s. In 1971, Herbst and colleagues established a strong connection between DES exposure in utero and subsequent development of clear-cell adenocarcinoma of the vagina and cervix in young women, aged 14–21 years. The incidence of this cancer in women whose mothers had been administered DES during pregnancy (DES daughters) is estimated to range from 1.4 cases per 1,000 exposed to one case per 10,000 exposed persons (▶ Cervical cancer). Previously, clear-cell adenocarcinoma of the vagina and cervix had been observed only rarely, primarily in postmenopausal women (over age 50) not exposed to DES. Consequently, the US Food and Drug Administration issued a drug bulletin recognizing DES as a ▶ transplacental carcinogen and banned its use during pregnancy. Since then, DES exposure has been observed to cause a range of teratogenic and neoplastic changes in humans and animals. It is used now mainly for treatment of a small subset of hormonally responsive refractory cancers. However, DES exposure can serve as a model for evaluating the potential effects of xenoestrogens (▶ Hormonal carcinogenesis; ▶ estrogenic hormones). Therefore, its investigation remains important and should not be limited to the study of the consequences of a specific, unintentionally deleterious administration.

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Diethylstilbestrol, Table 1 Effects of diethylstilbestrol exposure DES mothers DES daughters DES sons

Nonneoplastic effects Increased risk of • Adverse pregnancy outcomes

Neoplastic effects Increased risk of • Breast cancer

Increased risk of • Adverse pregnancy outcomes • Infertility • Reproductive tract structural abnormalities • Vaginal adenosis Increased risk of • Epididymal cysts • Cryptorchidism • Testicular hypoplasia • Semen and sperm abnormalities

Increased risk of • Clear-cell adenocarcinoma of the vagina and cervix • Breast cancer over age 40 No increased risk of hormone-related cancers

Animal Studies DES has been studied extensively in animal models. In the Syrian hamster model, exposure to DES induces neoplasms of the liver and kidney, as well as aneuploidy (particularly chromosome gains) in the renal neoplasms (▶ Aneuploidy, chromosome instability). DES exposure in rats elicits tumors of the reproductive tract and pituitary and mammary glands. In addition, Green and colleagues have observed that DES metabolites produce DNA adducts (▶ Adducts to DNA) and, ultimately, cancer in the breast of female ACI rats. Tumors of the reproductive tract and mammary glands are seen in the murine model as well, along with alterations in the genetic pathways governing uterine differentiation. Data from Newbold and colleagues suggest that an increased susceptibility to tumor formation is transmitted along the maternal lineage to subsequent generations, both male and female. Thus, not only is the developing organism sensitive to the endocrine-disrupting chemical, but transgenerational effects are plausible as well. Neoplastic Effects in Humans The only increased risk of hormone-dependent cancers observed in women who took DES during pregnancy (DES mothers) is ▶ breast cancer (Table 1). Hatch and colleagues found that DES mothers had a 30% increased rate of breast cancer compared to the general population. In contrast, DES daughters have an increased risk of two types of hormone-dependent cancers, clear-cell adenocarcinoma of the vagina and cervix (mentioned above) and breast cancer (▶ Breast cancer). Clearcell adenocarcinoma of the vagina and cervix generally presents in these women when they are in their teens and twenties; however, because

some women have been diagnosed in their thirties and forties, concerns have arisen about whether another increase in risk will occur as DES daughters approach the age at which this type of cancer is seen in the general population (the postmenopausal period). There was a paucity of information on breast effects in DES daughters. However, the National Cancer Institute’s Continuation of Follow-Up of DES-Exposed Cohorts has proven a rich source of information to study the long-term effects of in utero estrogen exposure in humans. These cohorts include over 4,000 women who had documented in utero exposure to DES and more than 2,000 unexposed women from the same record sources; all have been followed from 1994 or earlier. As women exposed in utero to DES have begun to reach the ages at which breast cancer is more common, it appears that these women have an increased risk. In the data from the National Cancer Institute collaborative follow-up study of DES health effects (Continuation of Follow-Up of DES-Exposed Cohorts), Palmer and colleagues found that at ages 40 and older, DES daughters had double the risk of breast cancer of unexposed women; no association was seen prior to age 40. To date, males exposed in utero to DES (DES sons) do not exhibit an increase risk of developing hormone-related cancers. However, DES sons do display nonneoplastic abnormalities (see below) that place them at increased risk of developing testicular cancer regardless of DES exposure (▶ Testicular cancer). Nonneoplastic Effects in Humans In utero exposure to DES has been shown to elicit a variety of nonneoplastic reproductive tract abnormalities, including structural cervical,

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vaginal, or uterine abnormalities, in addition to changes in the vaginal epithelium such as adenosis (see Table 1). DES dosage and the stage of pregnancy during which the drug was administered appears to be directly linked to the severity of the adenosis, with the most severe manifestations seen in DES daughters whose mothers took the drug during their first trimesters. It is unclear whether these areas of adenosis progress to vaginal clear-cell adenocarcinoma. DES daughters have an increased risk of poor pregnancy outcomes, such as ectopic pregnancy or miscarriage, and also have a higher incidence of infertility than the general population. DES sons are more likely than unexposed men to exhibit genital abnormalities such as epididymal cysts, cryptorchidism, and testicular hypoplasia. Although DES sons show an increased incidence of semen and sperm abnormalities, they have not demonstrated an increased risk of infertility, but this is still under investigation. Mechanism of Toxicity The mechanism by which estrogens in general, and DES in particular, exert their toxic and carcinogenic effects is not fully understood. Both proliferative and genotoxic mechanisms have been postulated. Classically, estrogen exerts its effects through interaction with the estrogen receptor a (ERa), which stimulates cell proliferation and inhibits apoptosis (▶ Estrogen receptor). ERa may also interact with other receptors (such as ERb, insulin-like growth factor 1 receptor, and epidermal growth factor receptor) to influence proliferation or may act through non-genomic pathways, since it is found in nonnuclear subcellular fractions such as the plasma membrane and the mitochondria. An alternative or additional mechanism that may mediate estrogen’s and perhaps DES’ toxic effects is through the metabolites’ genotoxic capacity. Estrogens, and specifically DES, can be oxidatively metabolized into potentially genotoxic intermediates. Estrogen and its metabolites have been reported to induce DNA damage (▶ DNA damage), manifesting as ▶ allele imbalance and DNA amplification in

Diethylstilbestrol

human breast epithelial cells in vitro (▶ Amplification). DES metabolites have been reported to produce DNA adducts and cancer in mammary glands of female rats. DES is a strong mitotic inhibitor in cell lines, blocking equatorial plate formation, tubulin polymerization, and spindle assembly. This may, in turn, induce ▶ aneuploidy. Tumors associated with DES exposure exhibit genetic instability, such as whole and partial chromosome gains in vitro and in vivo, in the Syrian hamster model. ▶ Microsatellite instability has been reported in human vaginal clear-cell adenocarcinomas associated with in utero DES exposure, as well as in murine endometrial carcinomas after DES treatment. In contrast to the substantial microsatellite instability seen in human vaginal clear-cell adenocarcinomas associated with in utero DES exposure, breast neoplasms in DES daughters do not exhibit an increased amount of microsatellite instability. Breast tumors of DES mothers have not been investigated. In fact, little microsatellite instability was observed in breast tumors in both exposed and unexposed women, which is consistent with previous results from unselected human breast cancers, confirming that microsatellite instability is unusual in human breast cancers and suggesting that prenatal DES exposure does not affect DNA mismatch repair mechanisms in the breast. Similarly, equivalent amounts of allele imbalance have been observed in breast tissue regardless of exposure, which differs from findings in animal models and in vitro systems. Therefore, the effect of in utero DES exposure may be tissue, timing, and/or species specific, as is the case with other hormonal agents such as ▶ tamoxifen, which has variable effects on human endometrium and mammary tissue. It remains under investigation as to whether the potential effects of in utero DES exposure on human breast carcinogenesis are mediated by enhanced proliferation, by alternative genotoxic effects, or by other pathways entirely. Summary The unfortunate consequences of DES administration to pregnant women have yielded clinical

Diffuse Intrinsic Pontine Glioma

and scientific insights into estrogen’s effects on developing and mature tissues. Its continued investigation should provide additional clinical and mechanistic information about these effects and have relevance to understanding the effects of exposure to xenoestrogens.

Cross-References ▶ Adducts to DNA ▶ Allele Imbalance ▶ Amplification ▶ Aneuploidy ▶ Breast Cancer ▶ Cervical Cancers ▶ Chromosomal Instability ▶ DNA Damage ▶ Estradiol ▶ Estradiol ▶ Estrogen Receptor ▶ Estrogenic Hormones ▶ Hormonal Carcinogenesis ▶ Microsatellite Instability ▶ Tamoxifen ▶ Testicular Cancer ▶ Transplacental Carcinogenesis

References Giusti RM, Iwamoto K, Hatch EE (1995) Diethylstilbestrol revisited: a review of the long-term health effects. Ann Int Med 122:778–788 Larson PS, Ungarelli RA, De Las Morenas A et al (2006) In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium. Cancer 107(9): 2122–2126 Newbold RR, Padilla-Banks E, Jefferson WN (2006) Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations. Endocrinology 147(Suppl 6): S11–S17 Schrager S, Potter BE (2004) Diethylstilbestrol exposure. Am Fam Physician 69:2395–2402 Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast cancer. New Eng J Med 354:270–282

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See Also (2012) Carcinogen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 644. doi:10.1007/978-3-642-16483-5_839 (2012) DNA amplification. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1129. doi:10.1007/978-3-642-16483-5_1665 (2012) DNA mismatch repair mechanism. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1140. doi:10.1007/978-3-642-164835_1684 (2012) Estrogens. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1333. doi:10.1007/978-3-642-16483-5_2019 (2012) Genetic instability. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 1527-1528. doi:10.1007/978-3-642-16483-5_2380

Diferuloylmethane ▶ Curcumin

Differentiation-Inducing Factor ▶ Tumor Necrosis Factor

Diffuse ▶ Mantle Cell Lymphoma

Diffuse Astrocytoma ▶ Astrocytoma

Diffuse Intrinsic Pontine Glioma ▶ Astrocytoma

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Diffuse Large B-Cell Lymphoma Ken H. Young1 and Michael B. Møller2 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Pathology, Odense University Hospital, Odense, Denmark

Synonyms KIEL classification: Centroblastic, B-immunoblastic, B-anaplastic; WHO: Diffuse large cell; Working Formulation: Diffuse large cell, Large cell immunoblastic, Diffuse mixed small and large

Definition Diffuse large B-cell lymphoma is an entity of non-Hodgkin lymphoma composed of malignant large lymphoid cells with blastic morphologic features, expression of B-cell markers, and a diffuse growth pattern. The postulated cells of origin are germinal or post germinal center B-cells. This lymphoma entity is morphologically, clinically, and molecularly heterogeneous.

Characteristics Diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma comprising 30–40% of non-Hodgkin lymphomas in adult and approximately 20% in childhood and adolescence. Diffuse large B-cell lymphoma can be seen in all age groups, but the incidence increases with age. The median age at diagnosis is approximately 65 years. There is a slight male preponderance (1.2:1). In most patients, the tumor resides in lymph nodes, but 30–40% of patients have primary extranodal disease. Approximately 70% of patients have at least one and 30% have multiple extranodal involvements. Virtually any extranodal site may be involved, but the most

Diffuse Large B-Cell Lymphoma

frequently involved organs include the gastrointestinal tract, soft tissue, thyroid, skin, central nervous system, testis, liver, bladder, bone, gonads, breast, ovary, cervix, kidney, pancreas, lung, and salivary glands. Transformed diffuse large B-cell lymphomas arise from indolent lymphomas such as ▶ chronic lymphocytic leukemia/ small lymphocytic lymphoma, ▶ marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma and ▶ follicular lymphoma. The etiology of most diffuse large B-cell lymphoma cases is unclear. However, patients with immunodeficiency such as patients with human immunodeficiency virus, Epstein-Barr virus, or human herpes virus 8 infection; patients receiving immunosuppressive therapy; and those having chronic inflammatory disease are at increased risk of developing lymphoma or high-grade transformation. Diagnosis The typical clinical presentation of diffuse large B-cell lymphoma patients with nodal disease is enlarged lymph nodes. Patients with extranodal presentation of the disease often have a mass with rapid growth or symptoms related to dysfunction of the involved organ(s). One third of the patients have B symptoms. Approximately half of the patients have localized lymphoma, i.e., Ann Arbor stage I or II, and the remainder have disseminated or high stage disease. The morphologic diagnosis is based on the World Health Organization Classification. Diffuse large B-cell lymphoma typically consists of a diffuse proliferation of medium to large neoplastic B-lymphoid cells with a nucleus at least twice the size of a normal lymphocyte or similar to and exceeding the size of the macrophage nuclei. These large cells are a mixture of cells that resemble either the centroblasts or the immunoblasts that normally reside in reactive germinal centers. The diffuse large B-cell lymphoma entity is morphologically heterogeneous with several morphologic variants. The two most common variants are the centroblastic variant which is dominated by centroblasts and the immunoblastic variant with >90% being immunoblasts. In the T-cell/ histiocyte rich variant, the majority of cells are small T-cells and histiocytes and less than 10% of

Diffuse Large B-Cell Lymphoma

the large cells are large neoplastic B-cells. An anaplastic variant of diffuse large B-cell lymphoma is recognized, which has a similar morphology and CD30 expression as the T-cell lymphoma anaplastic large cell lymphoma. However, anaplastic diffuse large B-cell lymphoma is clinically and genetically unrelated to anaplastic large cell lymphoma. Other rare variants include plasmablastic diffuse large B-cell lymphoma and diffuse large B-cell lymphoma with expression of full-length ALK, CD30 and IRF4 rearrangement. Diffuse large B-cell lymphoma cells usually express CD45 and B-lymphoid markers such as CD19, CD20, CD22, CD79a, and PAX5. The proliferation rate is high with most cases expressing the proliferation associated marker Ki-67 in >40% of the tumor cells. In some tumors, 95–100% of the malignant cells express Ki-67. The prognosis is variable with a 5-year overall survival rate of 45–50% for all patients. The International Prognostic Index is widely used for prognostication of diffuse large B-cell lymphoma. It consists of five clinical factors (age, stage, performance score, serum lactate dehydrogenase, number of extranodal sites involved), each with independent prognostic value regarding overall survival. The index allocates 35–40% of the patients to the low risk group with a 5-year overall survival rate of >70%, while 15–20% of the patients have high risk lymphoma and a 5-year overall survival rate of 90%), especially to albumin, a1-acid glycoprotein, and lipoproteins. In addition, peak plasma concentrations generally exceed levels required to induce relevant biologic effects in vitro. Limited information is available about the distribution of docetaxel in humans. Immediately after treatment, tissue uptake of radioactivity is highest in the liver, bile, and intestines, a finding that is consistent with substantial hepatobiliary extraction and excretion. High levels of radioactivity are also found in the stomach, which indicates the possibility of gastric excretion, as well as in the spleen, bone marrow, myocardium, and pancreas. Docetaxel has hepatic metabolism, and biliary excretion and urinary excretion account only 2%. Approximately 80% of the administered dose of total radioactivity is excreted in the feces within 7 days after treatment, with the majority of excretion occurring in the first 48 h. In the hepatic

Docetaxel

▶ cytochrome P450-mixed function, oxidases are responsible for the bulk of drug metabolism, and CYP3A, CYP2B, and CYP1A isoforms may play major roles in biotransformation. The main metabolic pathway consists of oxidation of the tertiary butyl group on the side chain at the C-13 position of the taxane ring as well as cyclization of the side chain. Toxicity ▶ Neutropenia is the principal toxicity of docetaxel. At dose of 100 mg/m2, neutrophil count nadirs are 65–80% of all gastric cancer cases worldwide), but it also exerts synergistic effect with other pro-carcinogenic factors, such as low-fiber diet and nitrosamine, it is of paramount importance to understand how H. pylori contributes to the pathogenesis of gastrointestinal diseases particularly gastric cancer and how this culprit bacterium can be eradicated.

in Western countries have H. pylori infection, and up to 88.4–99.3% of the people in the Eastern countries may be H. pylori infected.

Epidemiology and the Pathogenic Role of H. pylori By far, H. pylori infection is the most important risk factor for the development of gastric cancer, especially noncardiac gastric cancer. It has been reported that more than 60% of the world’s population has H. pylori infection, even though most of the H. pylori-infected individuals have no symptoms. H. pylori are often contracted during childhood, and the prevalence of H. pylori infection varies with geographic regions, age, socioeconomic status, education level, living environment, and occupation. For example, about 5% of European children become H. pylori infected by the age of 20, whereas up to 50% of Polynesian children of the same age group are H. pylori infected. In some parts of the world where the incidence of gastric cancer is significantly high (e.g., in Gansu Province of China, where the reported incidence of gastric cancer was 63.8/100,000), up to 70–80% of the population is H. pylori infected. Those who live in crowded conditions or live with someone who has already been infected with H. pylori and people who lack a reliable supply of clean water generally have an increased incidence of H. pylori. Overall, the vast majority of H. pylori infection occurs in the developing countries where as many as 80% of the middle-aged adults may be H. pylori infected. On the other hand, about 75.4–86.2% of gastric cancer patients

Characteristics H. pylori is a helix-shaped, microaerophilic, gram-negative bacterium which grows in the mucus layer of stomach. H. pylori contains a hydrogenase which is used to obtain energy by oxidizing molecular hydrogen produced by intestinal bacteria. To survive the harsh acidic environment of the gastric lumen, H. pylori uses its flagella to burrow into the mucus lining of the stomach to reach the epithelial cells underneath, where the pH is more neutral. H. pylori is able to sense the pH gradient in the mucus and move toward the less acidic region (chemotaxis). This also keeps the bacteria from being swept away into the lumen. Currently, there is no unified standard for the classification of H. pylori. It has been reported that if H. pylori grows in disadvantageous environments, such as in gastric juice, bile, and lysozyme, in the presence of antibiotics, and in a specific environment with a significant change of the oxygen concentration, it could transform to Helicobacter pylori “L” type (Fig. 1). Compared to the classic type of H. pylori, the L-type H. pylori is more adhesive, more aggressive, and more difficult to eliminate. H. pylori can also be classified according to its gene expression patterns. For example, based on the expression patterns of VacA and CagA genes, H. pylori strains could be divided into two types: CagA positive (type I) and CagA negative (type II). CagA-positive H. pylori is more aggressive and is responsible for most gastric diseases. Mechanisms of H. pylori-Induced Gastric Carcinogenesis H. pylori-Induced Inflammation in the Gastric Epithelium

Development of gastric cancer occurs over an extended period of time and involves multiple steps. H. pylori infection and the associated

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tissues and adjacent H. pylori-infected gastric epithelium than the normal gastric tissues without H. pylori infection. Increased intracellular level of NOS/RNS can lead to the point mutations and loss of function of tumor suppressor genes. H. pylori-Induced Methylation and Loss of Function of Key Genes in Gastric Epithelium

Helicobacter Pylori in the Pathogenesis of Gastric Cancer, Fig. 1 “L”-type H. pylori identified within gallbladder

inflammation play a key role in gastric carcinogenesis. H. pylori renders the normal gastric mucosa (gastric epithelium) to undergo numerous cycles of cellular damage regeneration, and this may lead to the glandular atrophy of the gastric mucosa, followed by intestinal metaplasia, atypical hyperplasia, and subsequently gastric carcinoma. H. pylori-Induced Oxidative Stress and DNA Damage in the Gastric Epithelium

Other proposed mechanisms by which H. pylori induce the development and progression of gastric cancer include oxidative stress and associated DNA damage, abnormal immune response, and dysregulated key signaling pathways. It has been well established that H. pylori can induce oxidative stress in the gastric epithelial cells and subsequent DNA damage. A significantly higher level of 8-hydroxydeoxyguanosine (a marker for oxidative stress-induced DNA damage) is present in the H. pylori-induced gastric cancer

H. pylori can induce hypermethylation in many key genes such as chromodomain helicase DNA-binding protein 1 (CHD1), cell adhesionand cell growth control-associated genes (e.g., p16, p14, and APC), DNA repair genes (e.g., hMLH, BRCA1, and MGMT), tumor suppressor genes (e.g., RUNX3), and genes related to gastrointestinal mucosa defense mechanisms (e.g., TFF2) (Fig. 2); all these are associated with gastric carcinogenesis. In addition, H. pylori-induced local inflammatory response is considered to be the important mechanism in the mediation of inflammation and DNA methylation, for example, TNF-a, IL-1b, and COX-2 have all been shown to significantly promote the level of DNA methylation. H. pylori Infection Induces Aberrant Activities of the Signaling Pathways in the Gastric Epithelium

H. pylori infection can lead to the hyperplasia of gastric epithelial cells. In vitro experiments have confirmed that H. pylori could significantly increase proliferative capacity of gastric cancer cells, especially during the process of chronic infection. Cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) are the two major pathogenic elements produced by H. pylori. CagA can be directly released into gastric epithelial cells, triggering a series of downstream molecular abnormalities such as altered cell proliferation and apoptosis. VacA facilitates H. pylori insertion into the cell membrane and eventually leads to cell vacuolation and cellular damage (Fig. 3). Additionally, H. pylori infection could stimulate the expression of macrophage migration inhibitory factor (MIF) and intestinal metaplasia of gastric mucosa and increased expression of MMP9 and VEGF. All these

Helicobacter Pylori in the Pathogenesis of Gastric Cancer

2007

Helicobacter Pylori in the Pathogenesis of Gastric Cancer, Fig. 2 Methylated DNA

VacA facilitates H. pylori insertion into the cell membrane and eventually leads to cells vacuolation and cellular damage

CagA is directly released into gastric mucosa epithelial cells, triggering a series of downstream events and promoting disease progression

CagA

VacA

Gastric mucosa epithelial cells Helicobacter Pylori in the Pathogenesis of Gastric Cancer, Fig. 3 CagA and VacA are the two major pathogenic factors generated by H. pylori

changes are closely carcinogenesis.

linked

to

gastric

Detection and Treatment for H. pylori Infection H. pylori infection can be detected by blood and/or stool antibody test. As revealed by a meta-analysis of 22 studies involving 2,499 patients, monoclonal antibodies have a high accuracy both for initial and posttreatment diagnosis of H. pylori. In clinical practice, H. pylori can be detected more sensitively, accurately, and perhaps more conveniently by C13-urea breath test

(RAPID-13 kit). However, the most reliable method for diagnosing H. pylori infection is by endoscopic biopsy and histological examination. The combination of H. pylori serology and serum pepsinogen I/II ratio may constitute a promising noninvasive approach for the detection of premalignant conditions. Although H. pylori is sensitive to many antibiotics in vitro, no single agent is effective in vivo, possibly because the bacterium resides below the gastric mucus and is adherent to the gastric epithelium, making them less accessible to drugs. In addition, many H. pylori strains may have

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acquired resistance to the commonly available antimicrobial agents. Hence, eradication of H. pylori requires a minimum of two antibiotics in combination with gastric acid inhibitors (usually proton pump inhibitors, PPIs). The most commonly used regimen or so-called “first-line” therapy is termed triple therapy, in which patients are treated for 7 days with the combination of amoxicillin (1 g twice daily), clarithromycin (500 mg twice daily), and either esomeprazole (20 mg twice daily) or omeprazole (20 mg twice daily). Metronidazole was previously used (and is still being used in some countries) in the place of clarithromycin, but studies have suggested that, in some countries, H. pylori can easily develop resistance to metronidazole. The above triple regimen can achieve an eradication rate of greater than 90% with good tolerability. In case the above first-line therapy fails (most commonly because of the clarithromycin resistance), patients can be treated with the “secondline” therapy termed quadruple therapy, in which patients are given omeprazole (20 mg, once daily), bismuth subsalicylate (120 mg, four times daily), metronidazole (400 mg, three times daily), and tetracycline (500 mg, four times daily) for 7 ~ 14 days.

gastric epithelium during chronic Helicobacter pylori infection. Nat Commun 5:4497 Kuipers EJ, Thijs JC, Festen HP (1995) The prevalence of Helicobacter pylori in peptic ulcer disease. Aliment Pharmacol Ther 9:59–69 Kusters JG, van Vliet AH, Kuipers EJ (2006) Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 19:449–490 Lin LL, Huang HC, Ogihara S, Wang JT, Wu MC, McNeil PL, Chen CN, Juan HF (2012) Helicobacter pylori disrupts host cell membranes, initiating a repair response and cell proliferation. Int J Mol Sci 13:10176–10192 Malfertheiner P, Megraud F, O’Morain C, Bazzoli F, El-Omar E, Graham D, Hunt R, Rokkas T, Vakil N, Kuipers EJ (2007) Current concepts in the management of Helicobacter pylori infection: the Maastricht III consensus report. Gut 56:772–781 Matsuo T, Ito M, Takata S, Tanaka S, Yoshihara M, Chayama K (2011) Low prevalence of Helicobacter pylori-negative gastric cancer among Japanese. Helicobacter 16:415–419 Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, Murata M, Kawanishi S (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013:387014 Ou Y, Kang M, Zhou L, Cheng Z, Tang S, Yu D (2014) Infection with L-form of Helicobacter pylori and expressions of MIF, MMP9 and VEGF in gastric carcinoma. J South Med Univ 34:180–187 Peterson AJ, Menheniott TR, O’Connor L, Walduck AK, Fox JG, Kawakami K, Minamoto T, Ong EK, Wang TC, Judd LM, Giraud AS (2010) Helicobacter pylori infection promotes methylation and silencing of Trefoil Factor 2, leading to gastric tumor development in mice and humans. Gastroenterology 139:2005–2017 Sepulveda AR, Yao Y, Yan W, Park DI, Kim JJ, Gooding W, Abudayyeh S, Graham DY (2010) Cpg methylation and reduced expression of O6-methylguanine DNA methyltransferase is associated with Helicobacter pylori infection. Gastroenterology 138:1836–1844 Take S, Mizuno M, Ishiki K, Nagahara Y, Yoshida T, Yokota K, Oguma K, Okada H, Shiratori Y (2005) The effect of eradicating Helicobacter pylori on the development of gastric cancer in patients with peptic ulcer disease. Am J Gastroenterol 100:1037–1042 Wang AY, Peura DA (2011) The prevalence and incidence of Helicobacter pylori-associated peptic ulcer disease and upper gastrointestinal bleeding throughout the world. Gastrointest Endosc Clin N Am 21:613–635 Yoon H, Kim N, Lee HS, Shin CM, Park YS, Lee DH, Jung HC, Song IS (2011) Helicobacter pylori-negative gastric cancer in South Korea: incidence and clinicopathologic characteristics. Helicobacter 16:382–388

References Amieva MR, El-Omar EM (2008) Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 134:306–323 Gisbert JP, de la Morena F, Abraira V (2006) Accuracy of monoclonal stool antigen test for the diagnosis of H. pylori infection: a systematic review and metaanalysis. Am J Gastroenterol 101:1921–1930 Hur K, Niwa T, Toyoda T, Tsukamoto T, Tatematsu M, Yang HK, Ushijima T (2011) Insufficient role of cell proliferation in aberrant dna methylation induction and involvement of specific types of inflammation. Carcinogenesis 32:35–41 Iammarino NK, Gribble G (2009) The intercultural cancer council’s fact sheet series: a tool for public and professional education. J Cancer Educ 24:S7–S9 Kiga K, Mimuro H, Suzuki M, Shinozaki-Ushiku A, Kobayashi T, Sanada T, Kim M, Ogawa M, Iwasaki YW, Kayo H, Fukuda-Yuzawa Y, Yashiro M, Fukayama M, Fukao T, Sasakawa C (2014) Epigenetic silencing of mir-210 increases the proliferation of

Hematological Malignancies, Leukemias, and Lymphomas

Hematological Malignancies, Leukemias, and Lymphomas Jesper Jurlander Department of Hematology, Rigshospitalet, Copenhagen, Denmark

2009

side). Platelets, erythrocytes, granulocytes, monocytes, and ▶ dendritic cells are derived from the myeloid lineage. T- and B-lymphocytes, plasma cells, and NK-cells are derived from the lymphoid lineage. The lineages, and the maturation steps involved, can be recognized using morphologic, cytochemical, and immunophenotypic features. Similarly, hematologic malignancies are categorized according to such features.

Definition Hematological malignancies are a collective term for neoplastic diseases of the hematopoietic and lymphoid tissues, with clinical presentation as leukemia, lymphoma, or myeloma.

Characteristics The Hematopoietic System The blood contains a number of different cell types, which can be divided into cells with and without a nucleus. The unnucleated cells are red blood cells (erythrocytes) and platelets (thrombocytes). The nucleated white blood cells consist of three major subclasses: granulocytes, lymphocytes, and monocytes, which can be further categorized as eosinophil, basophil or neutrophil granulocytes, T-lymphocytes, B-lymphocytes, and ▶ Natural Killer Cell Activation (NK-cells). These cells are all derived from a common ancestor, the pluripotent progenitor cell in the bone marrow. In the bone marrow, different lineages are derived from this pluripotent progenitor. Precursor cells proceed through specific maturation steps before they, as mature circulating cells, leave the bone marrow in order to circulate in the bloodstream. Figure 1 shows a simplified schematic representation of these processes. The pluripotent progenitor cell is shown in black in the middle, cells which are normally found only in the bone marrow (or thymus) are in blue, cells normally circulating in the blood are in red, and cells found in tissues are in green. Two major lineages can be defined: The myeloid lineage (shown in green on the left side) and the lymphoid lineage (shown in blue on the right

The WHO Classification The World Health Organization (WHO) has published a unified classification of neoplastic diseases of the hematopoietic and lymphoid tissues. In relation to the development of hematopoietic cells, the two major WHO classes can be viewed as disease “domains”, based on the cell types primarily affected in each class and specific subclasses. The principles of this classification are shown in Fig. 2. The domains of the myeloid neoplasms are on the left side, rare histiocytic, dendritic, or mast cell neoplasms are at the bottom and the domains of lymphoid neoplasms are on the right side. The WHO classification adopts the REAL (Revised European-American Lymphoma) classification of lymphoid neoplasms, whereas the classification of myeloid neoplasms has been revised in several categories compared to the FAB (French-American-British) classification. Each of these two major classes consists of subclasses which subsequently categorize the specific disease entities, the most common of which are shown in Fig. 2. Myeloid neoplasms (Fig. 2, left side) consist of three major subclasses: chronic myeloproliferative disorders, ▶ myelodysplastic syndromes, and acute myeloblastic leukemias, but also include more rare myeloid disorders with both proliferative and dysplastic features and disorders of histiocytes, mast (▶ Mastocytosis) cells, and dendritic cells. Lymphoid neoplasms consist of two major subclasses, precursor and mature neoplasms, with specific disease entities of each subclass defined according to the involved cell type, that is, B-, T-, or NK-cells (Fig. 2, right side). Neoplasms of B-cell origin are exceedingly more common than disorders arising in T- and

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Hematological Malignancies, Leukemias, and Lymphomas

Haematopoiesis CD4-positive T-lymphocyte Platelets

Megakaryocyte Megakaryoblast Thymocyte

Erythrocytes

Erythroblast

Myeloid progenitor cell Granulocytes

CD8-positive T-lymphocyte

Promyelocyte

Myeloblast

Pluripotent progenitor Cell

Lymphoid progenitor cell

B Lymphoblast

CD5-positive B-lymphocyte

CD5-negative B-lymphocyte

Myelomonocytic progenitor cell Plasma cell

Monoblast

Monocyte

NK-cell

Dendritic cell Macrophage

Hematological Malignancies, Leukemias, and Lymphomas, Fig. 1 Hematopoiesis

NK-cells (▶ Malignant Lymphoma: Hallmarks and Concepts). Major Myeloid Subclasses Within the WHO Classification Chronic Myeloproliferative Disorders (MPDs) are disorders of effective hematopoiesis initially presenting with increased levels of one or more of the circulating myeloid cell types: erythrocytes, platelets, and granulocytes. On the basis of specific genetic events they can be subdivided into Philadelphia chromosome positive disorders, carrying the t(9;22) (▶ BCR-ABL1) hallmark of ▶ Chronic Myeloid Leukemia (CML), and Philadelphia chromosome negative MPDs (Philadelphia chromosome negative myeloproliferative disorders), described as essential thrombocytemia, polycytemia vera, and chronic idiopathic myelofibrosis in the WHO classification. The Philadelphia chromosome negative MPDs are better classified according to the presence or absence of activating mutations in the JAK2 protein kinase (JAK2

mutations). Also recognized within the subclass of chronic MPDs are rare disorders as chronic neutropilic leukemia, chronic eosinophilic leukemia, and unclassifiable myeloproliferative diseases. The “domain” of MPDs is larger than the peripheral blood, since all of these disorders have the potential to transform into acute leukemia (▶ Blast crisis), including leukemias with a lymphoid phenotype. Furthermore, a small but distinct class of myeloproliferative diseases also show dysplastic features. Therefore, the WHO classification has defined a class of myelodysplastic/myeloproliferative diseases, shown in the lower left corner of Fig. 2. This class includes chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, and juvenile myelomonocytic leukemia. The ▶ myelodysplastic syndromes (MDS) are diseases of ineffective hematopoiesis, resulting in decreased levels of one or more of the circulating myeloid cell types. As the myeloproliferative diseases, the myelodysplastic syndromes can also transform but only to acute myeloid

Hematological Malignancies, Leukemias, and Lymphomas

2011

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Hematological Malignancies, Leukemias, and Lymphomas, Fig. 2 Leukemic diseases

leukemias, suggesting that the “domains” of these diseases may be somewhat smaller than that of myeloproliferative disorders. The class includes refractory anemia (with or without ringed sideroblasts), refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts, 5q- syndrome (MDS: 5q-syndrome), and unclassifiable myelodysplastic syndromes. The acute myeloid leukemias (AML) (Acute myeloblastic leukemia) can be divided into subclasses (as shown in red in Fig. 2): AML with recurrent cytogenetic translocations (Chromosomal aberrations including ▶ acute promyelocytic leukemia, AML with multilineage dysplasia (with or without preceeding myelodysplastic syndrome), therapy-related AML and myelodysplastic syndromes, and AML not otherwise categorized. Conversion from myelodysplastic syndrome to overt AML is defined by a blast count exceeding 20% of the

cells in the bone marrow. Finally, acute biphenotypic leukemias are included among myeloid neoplasms in the WHO classification.

Major Lymphoid Subclasses Within the WHO Classification The lymphoid precursor neoplasms are the acute lymphoblastic leukemias (ALL) of either B- or T-lineage origin, but also include the presentation of these diseases as lymphoblastic lymphomas. The Mature T and NK-cell neoplasms include mycosis fungoides (and the leukemic variant of this lymphoma; ▶ Sezary syndrome), ▶ anaplastic large T-cell lymphoma, angioimmunblastic T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified (NOS), and a number of more rare diseases. NK-cell neoplasms are rare, but include both lymphoma and leukemia subtypes (Fig. 2, bottom right).

2012

The mature B-cell neoplasms are historically and based on histology divided into ▶ Hodgkin disease and non-Hodgkin lymphoma (NHL). ▶ Hodgkin disease is further subclassified as either nodular lymphocyte predominant Hodgkin lymphoma or classical Hodgkin lymphoma with the four specific subtypes: nodular sclerosis, lymphocyte-rich, mixed cellularity, and lymphocyte depletion. The non-Hodgkin lymphomas are subclassified according to morphology, immune phenotype, rearrangement of the immunoglobulin genes, and recurrent chromosomal aberrations. ▶ Chronic lymphocytic leukemia/small lymphocytic leukemia and ▶ mantle cell lymphoma are characterized by obligatory expression of CD5 in the cell membrane. The most common NHL is ▶ diffuse large B-cell lymphoma, which is not a single uniform entity as demonstrated by gene expression profiling and molecular genetics (▶ BCL6 Translocations in B-Cell Tumors), but may contain several related disease entities with variable clinical course. The most common small cell lymphoma is ▶ follicular lymphoma, characterized by the t(14;18) recurrent translocation resulting in rearrangement of ▶ BCL2. Several other small cell lymphoproliferative syndromes, often referred to as indolent lymphomas, exists including extranodal ▶ marginal zone B-cell lymphoma of ▶ MALT (mucosa associated lymphoid tissue) type, splenic marginal zone lymphoma, and lymphoplasmacytic lymphoma (with the clinical syndrome of Waldenström macroglobulinemia). ▶ Burkitt lymphoma, a very aggressive disease characterized by the rearrangement of the ▶ MYC oncogene, exists with a number of rare conditions and variants. The non-Hodgkin lymphomas have a very heterogeneous clinical course and biology, from highly aggressive but potentially curable diseases such as diffuse large cell lymphoma and Burkitt lymphoma to indolent but incurable diseases as CLL and follicular lymphoma. Even these seemingly indolent diseases may over time acquire additional molecular changes, resulting in transformation to large cell lymphomas (Richters syndrome). The plasma cell neoplasms are dominated by ▶ multiple myeloma and related variants as

Hematological Malignancies, Leukemias, and Lymphomas

monoclonal gammopathy of unknown significance to localized plasmacytoma or disseminated plasma cell leukemia. Diagnosis The complexity of the hematopoietic system (Fig. 1) is reflected by the existence of a wide spectrum of distinct tumors, with numerous variants (Figs. 1 and 2). Optimal treatment of a given condition is dependent on exact classification of the disease. It is not sufficient to rely solely on morphology, cytochemistry, and immunophenotype, since some specific conditions, which require specific treatments, can only be identified based on cytogenetics (Chromosomal aberrations) or even more sensitive molecular methods as ▶ fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), or DNA-sequencing of specific genes. The classification of hematological malignancies described here is based on these features, but also recognizes the clinical course of specific entities, such as the establishment of MDS- or therapy-related AML’s as diseases with particularly poor prognoses compared to AML’s with recurrent cytogenetic translocations. The continued elucidation of molecular mechanisms underlying hematological malignancies and the application of new technologies, such as ▶ microarray (cDNA) technology differential display or ▶ microRNA analysis, in this research will establish new entities and create a need for further revisions of the dynamic classification scheme in the near future. Treatment Biological risk prediction and targeted therapy, using antibodies alone, or in combination with chemotherapy or as intelligent probes carrying a radionucelotide to the very center of the tumor (▶ Radioimmunotherapy), are now being applied for the treatment of hematopoietic neoplasms. The indications for ▶ immunotherapy using allogeneic bone marrow transplantation are steadily expanding. A multitude of new designed drugs are under systematic development (▶ Drug design).

Hematological Malignancies, Leukemias, and Lymphomas

Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Acute Megakaryoblastic Leukemia ▶ Acute Myeloid Leukemia ▶ Acute Promyelocytic Leukemia ▶ Adoptive Immunotherapy ▶ Allogeneic Cell Therapy ▶ Anaplastic Large Cell Lymphoma ▶ B-Cell Tumors ▶ BCL2 ▶ BCL6 Translocations in B-Cell Tumors ▶ BCR-ABL1 ▶ Benzene and Leukemia ▶ Blast Crisis ▶ Burkitt Lymphoma ▶ Chromosomal Translocations ▶ Chronic Lymphocytic Leukemia ▶ Chronic Myeloid Leukemia ▶ Dendritic Cells ▶ Diffuse Large B-Cell Lymphoma ▶ Drug Design ▶ Follicular Lymphoma ▶ Imatinib ▶ Immunotherapy ▶ MALT Lymphoma ▶ Malignant Lymphoma: Hallmarks Concepts ▶ Mantle Cell Lymphoma ▶ Marginal Zone B-Cell Lymphoma ▶ Mastocytosis ▶ Microarray (cDNA) Technology ▶ MicroRNA ▶ Minimal Residual Disease ▶ Multiple Myeloma ▶ Myelodysplastic Syndromes ▶ MYC Oncogene ▶ Natural Killer Cell Activation ▶ Radioimmunotherapy ▶ Sezary Syndrome ▶ STI-571 ▶ V(D)J Recombination

2013 leukemias: a report of the French-American-British Cooperative Group. Ann Intern Med 103:620–625 Harris NL, Jaffe ES, Stein H et al (1994) A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84:1361–1392 Harris NL, Jaffe ES, Diebold J et al (1999) World health organization of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting – Airlie House, Virginia, November 1997. J Clin Oncol 17:3835–3849 World Health Organisation classification of neoplastic diseases of the hematopoietic and lymphoid tissues. In: Sobin LH (ed) World Health Organisation international histological classification of tumors. Springer, Berlin/ Heidelberg/New York

See Also

and

References Bennet JM, Catovsky D, Daniel MT et al (1985) Proposed revised criteria for the classification of acute myeloid

(2012) Allogeneic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 138. doi:10.1007/978-3-642-16483-5_194 (2012) Bone marrow. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 445. doi:10.1007/978-3-642-16483-5_680 (2012) CD5 B cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 702. doi:10.1007/978-3-642-16483-5_916 (2012) Chromosomal aberrations. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 838. doi:10.1007/978-3-642-16483-5_1138 (2012) FISH. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1415–1416. doi:10.1007/978-3-642-16483-5_2197 (2012) Indolent lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1846. doi:10.1007/978-3-642-16483-5_3038 (2012) JAK2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1923. doi:10.1007/978-3-642-16483-5_3171 (2012) Non-Hodgkin lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4110 (2012) PCR. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2803. doi:10.1007/978-3-642-16483-5_4417 (2012) Philadelphia chromosome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2864. doi:10.1007/978-3-642-164835_4520 (2012) Pluripotent. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2930. doi:10.1007/978-3-642-16483-5_4638 (2012) Translocation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5942 (2012) Waldenstrom macroglobulinemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3941. doi:10.1007/978-3-642-164835_6227

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2014

Hemicentin, him4, FIBL6, HMCN1, FBLN-6 ▶ Fibulins

Henle–Koch Postulates ▶ Koch’s Postulates

Hensin (Rabbit) ▶ Deleted in Malignant Brain Tumors 1

Heparanase Israel Vlodavsky Anatomy and Cell Biology, Technion Israel Institute of Technology, Cancer and Vascular Biology Research Center, Haifa, Israel

Definition Heparanase is a mammalian enzyme (endo-bglucuronidase) degrading heparan sulfate (HS), a ubiquitous strongly anionic linear polysaccharide associated with the cell surface and ▶ extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzyme cleaves glycosidic bonds in HS with a hydrolase mechanism and is thus distinct from bacterial eliminases, called heparinase and heparitinase. The heparanase enzyme facilitates cell migration and egress from blood vessels and hence plays a role in tumor ▶ metastasis, ▶ angiogenesis, ▶ inflammation, and autoimmunity. Heparanase activity has been identified in a variety of human tumors (i.e., melanoma; carcinoma of the breast, liver, colon, prostate, and pancreas; myeloid leukemia) and certain normal cells (i.e.,

Hemicentin, him4, FIBL6, HMCN1, FBLN-6

cytotrophoblasts, platelets, neutrophils, activated T lymphocytes). HS chains (Mr  30,000) are cleaved by heparanase at only a few sites, resulting in HS fragments of still appreciable size (Mr  5,000). Thus, the enzyme recognizes a particular and quite rare HS structure. A 2-Osulfate group on hexuronic acid residue located two monosaccharide units away from the cleavage site appears essential for substrate recognition by heparanase (Fig. 1). Heparanase activity (pH optimum in solution 6.0) has been demonstrated in both lysosomal and endosomal cellular compartments and in the cell membrane. In human neutrophils the enzyme is localized in tertiary granules.

Characteristics Heparan Sulfate Proteoglycans (HSPGs) The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) glycosaminoglycan (GAG) chains are covalently O-linked. The HS chains are typically composed of repeating hexuronic (L-iduronic or D-glucuronic acid) and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (Fig. 1). The HS chains generally consist of clusters of sulfated disaccharide units separated by low- or non-sulfated regions. Studies of the involvement of extracellular matrix (ECM) molecules in cell attachment, growth, and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth, and tissue repair. The HS chains, unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules (i.e., heparin-binding growth factors, chemokines, lipoproteins, enzymes) (Fig. 1) cling to the cell surface and ECM and thereby exert localized cellular effects. Moreover, transmembrane (syndecan) and membrane-anchored (glypican) HSPGs have a co-receptor role in which the proteoglycan, in concert with other cell surface molecules, comprises a functional receptor complex that binds the ligand and mediates its action.

Heparanase

2015

Heparanase cleavage site

1

2

3

O

HO

COO– OH

OH

O O

OH

4

COO–

H2COSO–3

5

H2COX

O HNAc

O O

OH

7

H2COSO–3

O OH

6

OSO–3

O

HNSO–3

COO– OH

O

O O

OSO–3

OH

8

COO–

H2COSO–3 O 3H

O O

O

OH

HNSO–3

OH

C OH H

OX

bFGF HNSO–3 ATIII/Thr LPL

Heparanase cleavage

HS

Perlican FGFR

Heparanase, Fig. 1 Scheme of heparan sulfate and the heparanase cleavage site (top) and of a basement membrane HSPG (perlecan) (bottom). Cleavage is associated with release of HS-bound growth factors and enzymes

Molecular Properties Cloning and expression of a single human heparanase cDNA sequence was achieved using amino acid sequences derived from heparanase enzymes purified from human platelets, placenta, hepatoma cells, and transformed embryonic fibroblasts. The heparanase cDNA contains an open reading frame of 1629 bp, which encodes for a 61.2 kD latent polypeptide of 543 amino acids (Fig. 2). This proenzyme is posttranslationally cleaved by cathepsin-L into 8 and 50 kDa subunits that non-covalently associate to form the active heparanase (Fig. 2). Heterodimer formation is essential for heparanase activity. Site-directed mutagenesis revealed that similar to other glycosyl hydrolases, heparanase has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu225 and a nucleophile at Glu343 (Fig. 2). The sequence also contains a putative N-terminal signal peptide sequence (Met1 to Ala35) and a candidate

transmembrane region (Pro515 to Ile534; Fig. 2). Alignment of the human, mouse, and rat heparanase amino acid sequences, corresponding to the 50 kD human mature enzyme (Lys158 to Ile543), demonstrated 80.0%, 79.7%, and 92.7% identity between the human and mouse, human and rat, and mouse and rat heparanases, respectively. A 58–60% homology was found between these enzymes and the chicken heparanase. The fact that highly homologous cDNA sequences were derived from different species and types of normal and malignant cells is consistent with the notion that one dominant endoglucuronidase is expressed by all mammalian cells. Thus, unlike the large number of proteases that can solubilize polypeptides in the ECM, it appears that only one heparanase is used by cells to degrade the heparan sulfate side chains of HSPGs. The genomic locus that encodes heparanase spans 40 kb. It is composed of 12 exons separated by 11 introns and is localized on human chromosome 4q21.3.

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Heparanase Heparanase gene: chromosome 4q21.3

1 kb

Intron Genomic gene size:∼40 kb Heparanase protein

s.p.

Propeptide

Met1 Ala35

Pre-proheparanase

Gln157

+ Gln36 s.p. Signal peptide Propeptide

Heparanase (50/8)

Gln109 Lys158

50 kDa active heparanase

IIe543

Predicted active site Glycosylation site

Heparanase, Fig. 2 Scheme of the human heparanase gene and protein

Preferential Expression in Human Tumors Expression of the heparanase mRNA and protein in normal tissues is restricted to the placenta, activated immune cells, platelets, and keratinocytes. Heparanase is preferentially expressed at early stages of carcinoma progression. Immunohistochemistry, in situ hybridization, RT-PCR, and real-time PCR analyses revealed that heparanase is upregulated in essentially all human tumors examined. These include carcinomas of the colon, thyroid, liver, pancreas, lung, bladder, cervix, breast, stomach, prostate, head and neck, salivary gland, and nasopharynx, as well as leukemia, lymphoma, and multiple myeloma. Patients exhibiting high levels of heparanase mRNA and/or protein had a significantly shorter postoperative survival time than patients whose tumors contained relatively low levels of heparanase. There is also a correlation between heparanase expression levels and tumor vascularity in cancer patients, indicating a significant role in tumor angiogenesis.

Involvement in Tumor Metastasis A critical event in the process of cancer invasion and metastasis is degradation of various ECM constituents including collagen, laminin, fibronectin, and HSPGs. The ability of HSPGs to interact with various ECM macromolecules and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of HS may therefore result in disassembly of the subendothelial ECM and hence play an important role in fundamental biological phenomena involving cell migration and ranging from pregnancy, morphogenesis, and development to inflammation, angiogenesis, and cancer metastasis. Expression of HS-degrading heparanase correlates with the metastatic potential of tumor cells. Moreover, elevated levels of heparanase were detected in sera of metastatic tumor-bearing animals and cancer patients, in tumor specimens, and in the urine of patients with aggressive metastatic disease. A direct role of heparanase in tumor metastasis was

Heparanase

provided by the increased lung, liver, and bone colonization of melanoma, lymphoma, myeloma, and prostate carcinoma following transfection and overexpression of the heparanase gene. A marked decrease in the metastatic potential of cells expressing high levels of endogenous heparanase was noted in response to administration of antiheparanase siRNA, or neutralizing monoclonal antibodies, indicating that heparanase is causally involved in cancer progression. Involvement in Tumor Angiogenesis Angiogenesis represents a coordinated multicellular process that requires the functional activity of a wide variety of molecules including growth factors, ECM components, adhesion receptors, and matrix-degrading enzymes. HSPGs and HSPG-degrading enzymes have been implicated in a number of angiogenesis-related cellular events including cell invasion, migration, adhesion, differentiation, and proliferation. The heparin affinity of fibroblast growth factors (FGFs) and other heparin-binding growth factors appears to be the basis for their storage in basement membrane (BM) and ECM, where they are bound to HS and can be released in an active form by HS-degrading enzymes. The released angiogenic factors may then stimulate endothelial cell proliferation and migration associated with neovascularization. An important early step in the angiogenic cascade is degradation of the subendothelial capillary BM by proliferating endothelial cells and formation of vascular sprouts. Heparanase, degrading the polysaccharide scaffold of BM, markedly contributes to the invasive ability of endothelial cells and their migration through the ECM toward the angiogenic stimulus. Apart from direct involvement in BM invasion by endothelial cells, the heparanase enzyme elicits an indirect angiogenic response by releasing HS-bound angiogenic growth factors from ECM and BM (Fig. 1) and by generating HS degradation fragments that stabilize and promote the mitogenic and angiogenic activity of heparin-binding growth factors (i.e., bFGF, ▶ VEGF). In fact, recombinant mammalian heparanase, as well as heparanase secreted by

2017

platelets, tumor cells, and inflammatory cells, releases FGF as a complex with HS fragments. This yields a highly active form of FGF that readily interacts with high-affinity receptors on the surface of endothelial cells and thus elicits an angiogenic response. The molecular size of the HS chain required for optimal stimulation of FGF receptor binding, dimerization, and signaling is similar to that of HS fragments released by heparanase. A profound angiogenic response is elicited in vivo by lymphoma cells overexpressing the heparanase gene and embedded in a reconstituted BM (Matrigel). It appears that cooperative interaction between heparanases from tumor, inflammation, and endothelial sources plays an important role in the angiogenic cascade. Apart from the well-studied catalytic feature of the enzyme, heparanase was noted to exert biological functions apparently independent of its enzymatic activity. Nonenzymatic functions of heparanase include enhanced cell adhesion and induction of p38 and Akt phosphorylation. Moreover, enzymatically active and inactive heparanase were noted to induce VEGF expression, thus providing, among other mechanisms, a molecular basis for the potent angiogenic capacity of heparanase. The anticancerous potential of heparanase inhibitors is, therefore, not restricted solely to suppression of the invasive metastatic phenotype, but also to suppression of tumor angiogenesis and growth. Clinical Relevance Heparanase-inhibiting molecules (e.g., non-anticoagulant species of heparin, polysulfated polysaccharides, polyanionic molecules) markedly reduce (>90%) the incidence of lung colonization induced by various tumor cells, in correlation with their anti-heparanase activity. The occurrence of a single heparanase species and its ability to promote both tumor angiogenesis and metastasis, the most critical steps in tumor progression, make it a promising target for cancer therapy. The heparanase-inhibiting pentasaccharide, phosphomannopentaose sulfate (PI-88), as well as other heparin mimetics (i.e., Roneparstat, Necuparanib, PG545) are being tested in clinical

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2018

trials for various cancers. The heparanase crystal structure has been resolved paving the way for rational design of heparanase-inhibiting small molecules. Notably, because heparanase plays important roles in other diseases (e.g., arthritis, colitis, sepsis, diabetes, diabetic nephropathy) these compounds may exhibit wide-ranging applicability to public health.

References Ilan N, Elkin M, Vlodavsky I (2006) Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38:2018–2039 McKenzie EA (2007) Heparanase: a target for drug discovery in cancer and inflammation. Br J Pharmacol 151:1–14 Parish CR, Freeman C, Hulett MD (2001) Heparanase: a key enzyme involved in cell invasion. Biochim Biophys Acta 1471:M99–M108 Pisano C, Vlodavsky I, Ilan N, Zunino F (2014) The potential of heparanase as a therapeutic target in cancer. Biochem Pharmacol 89:12–19 Sanderson RD, Yang Y, Suva LJ, Kelly T (2004) Heparan sulfate proteoglycans and heparanase – partners in osteolytic tumor growth and metastasis. Matrix Biol 23:341–352 Vlodavsky I, Ilan N, Naggi A, Casu B (2007) Heparanase: structure, biological functions, and inhibition by heparin-derived mimetics of heparan sulfate. Curr Pharm Des 13:2057–2073 Weissmann M, Arvatz G, Horowitz N, Feld S, Naroditsky I, Zhang Y, Ng M, Hammond E, Nevo E, Vlodavsky I, N. Ilan N (2016) Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis. Proc Natl Acad Sci USA 113:704–709 Wu L, Viola CM, Brzozowski AM, Davies GJ (2015) Structural characterization of human heparanase reveals insights into substrate recognition. Nat Struct Mol Biol 22:1016–1022

Heparanase Inhibitors Jian Ding State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China

Definition Heparanase inhibitors are a group of compounds inhibiting/decreasing heparanase enzymatic

Heparanase Inhibitors

activity, which abolish degradation of heparanase on heparan sulfate (HS) of extracellular matrix (ECM) and thus subsequential cascade.

Characteristics ▶ Heparanase, a single-functional HS-degrading endoglycosidase, is a mammalian endo-bD-glucuronidase cleaving HS chains at a limited number of sites. The enzyme is synthesized as a latent 65-kDa precursor that undergoes subsequent proteolytic cleavage, yielding 8-kDa and 50-kDa subunits. These subunits heterodimerize to form a highly active enzyme. The enzyme is thought to participate in the cleavage of HS chains from HS-containing proteoglycans (HSPGs), leading to ECM remodeling that facilitates the cell invasiveness and ▶ angiogenesis associated with cancer metastasis. The development of heparanase inhibitors for the treatment of highly malignant tumors is therefore of considerable interest. In view of its enzymatic characteristics, much effort has devoted in drug development targeting the substrate as mimetics based on the highaffinity interaction between the enzyme and its substrate. These include heparin, chemical derivatives of heparin, nonanticoagulant heparin, and other polyanionic molecules such as sulfated phosphomannopentaose (PI-88), oligomannurarate sulfate (JG3), laminarin sulfate, and suramin. Others targeting heparanase itself include small molecules, peptides, and neutralizing antibodies. In addition, effort also has been made on several fronts to inhibit heparanase gene expression. Inhibitors Targeting the Heparanase Substrate Heparin, Chemically Modified Derivative and Nonanticoagulant Heparin

As an analog of the natural substrate of heparanase, heparin is commonly considered to be a potent inhibitor of heparanase. This activity is attributed, in part, to the high-affinity heparin–enzyme interaction and the limited

Heparanase Inhibitors

degradation of heparin. Heparin thus serves as an alternative enzyme substrate. Early reports showed that heparin and some of its derivatives, as well as other sulfated polysaccharides inhibiting tumor cell heparanase, also inhibited experimental metastasis in animal models. Other related compounds lacking heparanaseinhibiting activity failed to exert an antimetastatic effect. No significant differences in tumor inhibition were found between the currently used unfractionated heparins and low-molecularweight heparins (LMWH). Regardless of the mode of action, heparin and LMWH were reported to exert a beneficial effect in cancer patients. The use of heparin as an antimetastatic agent is, however, limited due to its potent anticoagulant activity. This drawback has stimulated research on the potential use of modified, nonanticoagulant species of heparin and HS in cancer therapy. Glycol-split N-acetyl heparin, which shows dramatically increased heparanase-inhibiting activity compared with unmodified heparin, is a valuable compound. Notably, glycol splitting causes substantial loss of the anticoagulant activity of heparin due to the complete removal of heparin affinity for antithrombin. The main reason for such an effect is the cleavage of C-2–C-3 bonds of the GlcA residue of the pentasaccharidic sequence, which is essential for binding to antithrombin. Encouragingly, glycol-split N-acetyl heparins also exhibit marked decreases in the ability to release basic fibroblast growth factor (bFGF) from the ECM. Mitogenic activity in the ECM is thus inhibited. Work on heparanase inhibition by glycol-split heparin is mostly recent. The data from this nonanticoagulant heparin species applying on animals gave us clues that it is possible to separate the antimetastatic and anticoagulant activities of heparin. We thus predict that nonanticoagulant heparin species may have clinical potential, because they can be given in doses higher than those used for heparin, thereby exploiting fully the antimetastatic component of heparin species. Together, the combination of potent heparanase inhibition, the lack of anticoagulant activity, and the low release of ECM-bound bFGF promises

2019

that N-acetylated glycol-split heparins will be highly effective and specific antiangiogenic and antimetastatic agents. Sulfated Phosphomannopentaose

Heparanase enzyme activity also can be efficiently inhibited by shorter but more extensively sulfated oligosaccharides. The use of such small molecules may overcome intractable side effects caused by administration of larger inhibitors. Researchers have therefore sought small saccharides potently inhibiting heparanase activity but without side effects. The first such molecule, PI-88, showed good safety and tolerability in Phase I trials, and the Food and Drug Administration has now cleared PI-88 for a Phase III clinical trial. PI-88, a highly sulfated phosphosulfomannan (Fig. 1), is a hemisynthesized product obtained by sulfation of a saccharide from the yeast Pichia holstii. PI-88 is a mixture of chemically sulfated oligosaccharides, ranging from disaccharides to hexasaccharides, with the majority (60%) being pentasaccharides. PI-88 exerts its biological effects by blocking the enzymatic activity of heparanase and by interfering with the action of HS-binding angiogenic growth factors such as aFGF, bFGF, and VEGF. In an animal model, PI-88 was shown to inhibit the primary tumor growth of the highly invasive rat mammary adenocarcinoma 13762 MAT by 50%, to inhibit metastasis to the draining popliteal lymph node by 40%, and to reduce the vascularity of tumors by 30%. All these effects are highly significant. Acute hematogenous metastasis assays also demonstrated that PI-88 was a potent (>90%) inhibitor of blood-borne metastasis. The data from a completed Phase II clinical trial showed that PI-88 was especially well suited to postoperative use. In particular, PI-88 was effective in postoperative liver cancer patients in whom tumor burdens were low. PI-88 is one of a new class of multi-targeted cancer drugs that have an antiangiogenesis effect. A particular concern with PI-88 systemic treatment is drug interference with a relatively broad range of protein–HS interactions. An interpretation of drug specificity is thus difficult.

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2020

Heparanase Inhibitors

OPO3Na2 OR O

RO

OPO3H2 OR O

RO RO O

OR

n O O

RO RO

R=SO3Na or H, n=4

OR

Heparanase Inhibitors, Fig. 1 Phosphomannopentaose (PI-88)

Oligomannurarate Sulfate (JG3)

The success of PI-88 inspired a surge in the development of additional oligosaccharide-based innovative heparanase inhibitors, using substrates mimicking GAG as prototypes. Oligomannurarate sulfate, a new, hemisynthesized, structurally novel, sulfated oligosaccharide derived from marine oligomannurarate blocks (Fig. 2), showed promising potential substrate-based heparanase inhibitor. JG3 is a highly sulfated oligosaccharide with molecules ranging in size from tetrasaccharides to decasaccharides. JG3 significantly inhibited tumor angiogenesis and metastasis both in vitro and in vivo. As a nondegradable substrate mimetic, JG3 specifically binds to the KKDC and QPLK domains of heparanase, with high affinity, and significantly inhibited heparanase activity (the IC50 was 6.55 ng/ml). The JG3–heparanase interaction was competitively inhibited by LMWH but not by other GAGs. In addition, JG3 abolished heparanase-driven invasion, inhibited the release of HS-sequestered bFGF from the ECM, and repressed subsequent angiogenesis.

Moreover, JG3 inactivated bFGF-induced FGFR and ERK1/2 phosphorylation and blocked bFGFtriggered angiogenic events by directly binding to bFGF. Thus, JG3 appears to inhibit both major heparanase activities by simultaneously acting as both a substrate mimetic and a competitive inhibitor of heparan sulfate. In vivo, the B16F10 ▶ melanoma lung metastasis model, orthotopic xenografts of human breast carcinoma MDA-MB-435, and ▶ hepatocellular carcinoma BEL-7402 in athymic mice have been used to evaluate the effects of JG3 on tumor growth, angiogenesis, and metastasis. JG3 dramatically decreased the numbers of B16F10 metastases, with an inhibition rate of 82.2%. Also, JG3 suppressed MDA-MB-435 tumor growth by 37.6%, abated tumor metastasis by 88.3%, and inhibited tumor-related angiogenesis. In addition, JG3 inhibited hepatocellular carcinoma BEL-7402 primary tumor growth by 44.2%. Encouragingly, unlike other polyanionic compounds, JG3 showed very low toxicity, probably due to its weak anticoagulant activity (13-fold

Heparanase Inhibitors

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NaOOC

O O

OR-

O

COONa

R2O ORl

R2O

n

OH

NaOOC

R1 =SO3Na. R2 =SO3Na or H, n = 3-9

Heparanase Inhibitors, Fig. 2 Oligomannurarate sulfate (JG3)

SO3Na

SO3Na NaO3S

SO3Na

SO3Na HN

O

NH

O

O

SO3Na

O H N

H N

N H

N H

R

R

R=Me

Heparanase Inhibitors, Fig. 3 Suramin

less than that of heparin and nearly threefold less than that of PI-88). These data all suggest that JG3 is potentially valuable as an anticancer agent. Suramin

Suramin (Fig. 3) is a polysulfonated naphthyl urea, inhibiting heparanase with an IC50 of 48 mM. Suramin inhibited B16 melanoma cell invasion (IC50 = 10 mM) through reconstituted basement membrane, but had no effects on melanoma cell growth. Suramin has not been widely used because it has significant toxic effects in humans, including neurotoxicity, renal toxicity, adrenal insufficiency, and anticoagulant-mediated blood dyscrasias. This old medical terminology originally referred to the imbalance of the four humors or blood, black bile, yellow bile, or water/phlegm. The incorrect balance of these four humors was thought to lead to pathological conditions or disease. Blood dyscrasias simply refer to disorders of any cellular elements within

the blood. This term is essentially the same as hematologic disorders. In efforts to avoid these side effects, analogs of suramin have been synthesized and are undergoing evaluation. Compounds NF 227, NF 145, and NF 171 are three such analogs, all of which possess heparanase inhibitory activities more potent than that of suramin (the IC50 values were 20–30 mM). These compounds effectively inhibited heparanase-mediated angiogenesis in an animal model. Noncarbohydrate Substrate Mimetic Polymers

Synthetic, linear, noncarbohydrate polyanionic polymers have also been studied as heparin mimetics. The materials were initially tested for inhibition of heparanase-mediated degradation of HS in the subendothelial ECM. One series of polymers, synthesized by polymerization of phenol-based monomers, showed high inhibitory effects on heparanase activity. Of these, RG-13577, polymerized from 4-hydroxyphenoxy

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2022 Heparanase Inhibitors, Fig. 4 2-[4-propylamino5-[5-(4-chloro)phenylbenzoxazol-2-yl]phenyl]2,3-dihydro-1, 3-dioxo-1Hisoindole-5-carboxylic acid

Heparanase Inhibitors

O HOOC N

NH

O

O N

Cl

monomers, has a molecular weight of 5,800 and showed almost complete inhibition of heparanase activity at 2.5 mg/ml. Another group of polymers, derived from N-acryl amino acid monomers bearing charged functional groups, range from 3,000 to 6,000 Da in size and also showed potent inhibition of heparanase. Polymers with hydroxyl groups, when sulfonated, showed 70–80% inhibition of heparanase activity even at 1 mg/ml. This activity is comparable to that of heparin. Inhibitors Targeting Heparanase Small Molecules

Besides the efficacy of heparanase substrate mimetics, the development of small-molecularweight compounds directly inhibiting heparanase activity is still required. Several heparanase inhibitors have been discovered among microbial metabolites or have been obtained by organic synthesis. This group of compounds does not contain any sulfate moieties, and the group members have clearly defined chemical structures.

Compounds showing high potency against heparanase in vitro and in animal models would be developed as therapeutic leads. A class of inhibitors, exemplified by 1H-isoindole-5-carboxylic acid, was reported. The structure activity relationship study of this class compound led to 2-[4-propylamino-5-[5(4-chloro)phenyl-benzoxazol-2-yl]phenyl]-2,3dihydro-1,3-dioxo-1H-isoindole-5-carboxylic acid (Fig. 4), which displayed potent heparanase inhibitory activity (IC50 = 0.2 mM) and a much improved antiangiogenic effect (IC50 = 1 mM) when compared to other derivates. Other valuable small molecules are furanyl1,3-thiazol-2-yl-acetic acids and derivatives developed as potent heparanase and angiogenesis inhibitors. Although they showed promising in vitro potency against heparanase, this series of compounds suffered drawbacks such as synthetic difficulties and poor DMPK properties. On its structure basis, the changed chemical structure (benzoxazol-5-yl-acetic acids) showed much more activity and potency on DMPK. The most

Heparanase Inhibitors

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F N

O

COOH N H

Br

O

Heparanase Inhibitors, Fig. 5 Benzoxazol-5-yl-acetic acids

H N

O N

R

R N H

N H

N H

N

R=di-Me

Heparanase Inhibitors, Fig. 6 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea

powerful inhibitor in this class (Fig. 5) showed antiheparanase activity with an IC50 of 0.4 mM and antiangiogenesis with an IC50 of 1 mM. More importantly, the material attained a plasma concentration ca. tenfold above its IC50 value for heparanase inhibition. Novel 1-[4-H-benzoimidazol-2-yl]-phenyl]-3[4-(1H-benzoimidazol- 2-yl)-phenyl]-ureas were described as potent inhibitors of heparanase. Among these compounds, 1,3-bis-[4-(1Hbenzoimidazol-2-yl)-phenyl]-urea (Fig. 6) had high heparanase inhibitory activity (IC50 = 0.075 mM) in vitro and was effective in a B16 experimental metastasis model. Two other types of small inhibitory molecules are those in the RK-682 series and the KI-105 series. Compound RK-682 was isolated from a Streptomyces species and showed antiheparanase activity (IC50 = 17 mM). The derivative 4-Bn-RK-682 showed better selectivity for heparanase than did RK-682. Compound KI-105 was synthesized to mimic the anionic groups of HS using nitro and benzoic acid moieties. Peptides Competing with the Heparin/HS-Binding Domains of Heparanase

It has been reported that a peptide with the KKDC motif of the 50 kDa heparanase subunit region

(Lys158–Asn171) physically associated with both heparin and HS and inhibited heparanase enzymatic activity in a dose-dependent manner. Antiheparanase Antibodies

Very limited work has been performed in this area. In one study, a rabbit antibody against recombinant heparanase demonstrated dose-dependent inhibition of heparanase activity. The antibody inhibited ovarian carcinoma cell invasion at a concentration of 0.67 mM.

Conclusions and Perspectives As the predominant enzyme in tumor cells, heparanase represents an attractive target for the development of novel anticancer agents. Most research on heparanase inhibitors remains, however, at the in vitro or early preclinical stages. Currently, all heparanase inhibitors raise specificity concerns, because they may cross-react with other biological molecules including heparinbinding proteins or even tyrosine kinases. The phenotypes of animals with heparanase gene knockouts should provide great insights into the roles of heparanase in the complex in vivo environment. Studies on enzyme kinetics will help to

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obtain complete information on heparanase biochemistry. The determination of heparanase crystal structure will tremendously benefit rational drug design, leading to the development of more specific and more potent inhibitors. Also, researchers continue to focus on further defining heparanase substrate specificity, exploring both catalytic and noncatalytic enzyme activities. This work, in combination with enzyme crystal structure determination, allows us to hope that a more “rational” approach to the development of effective and highly specific heparanase inhibitors will soon be possible. We predict that future data will further reinforce the idea that heparanase represents a tractable and highly attractive target for novel types of anticancer agents.

Heparin-Affin Regulatory Peptide

Heparin-Binding Growth Factors ▶ Fibroblast Growth Factors

Heparin-Binding Growth-Associated Molecule ▶ Pleiotrophin

Heparin-Binding Neurite-Promoting Factor ▶ Pleiotrophin

References Ilan N, Elkin M, Voldavsky I (2006) Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38:2018–2039 Miao HQ, Liu H, Navarro E et al (2006) Development of heparanase inhibitors for anti-cancer therapy. Curr Med Chem 13:2101–2111 Zhao HJ, Liu HY, Chen Y et al (2006) Oligomannurarate sulfate, a novel heparanase inhibitor simultaneously targeting basic fibroblast growth factor, combats tumor angiogenesis and metastasis. Cancer Res 66:8779–8787

Heparin-Binding Neurotropic Factor ▶ Pleiotrophin

Hepatic Carcinoma ▶ Hepatocellular Carcinoma

Heparin-Affin Regulatory Peptide ▶ Pleiotrophin

Hepatic Epithelioid Hemangioendothelioma

Heparin-Binding Brain Mitogen

Hamidreza Fonouni and Arianeb Mehrabi Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany

▶ Pleiotrophin

Synonyms

Heparin-Binding Growth Factor 8 ▶ Pleiotrophin

Sclerosing angiogenic tumor; Sclerosing endothelial tumor; Sclerosing epithelioid angiosarcoma; Sclerosing interstitial vascular sarcoma

Hepatic Epithelioid Hemangioendothelioma

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Hepatic Epithelioid Hemangioendothelioma, Table 1 Comparison of epithelioid hemangioendothelioma in different sites Organ Soft tissue

Age 2nd–9th Dec

Gender mf

Angiocentricity 50% arise from vessel

Mortality (%) 13

mf

Distribution Solitary, rarely multifocal 50% multifocal

Bone Lung

1st–8th Dec; peak 2nd/3rd Dec Median 40y

?

?

f>m

>50% multifocal

65

f>m

>50% multifocal

Intravascular; spread common Intravascular; spread common

Liver

2nd–9th Dec

Definition Epithelioid hemangioendothelioma (EH) is a neoplasm of vascular origin involving soft tissue and visceral organs such as liver, spleen, bone, brain, meninges, breast, heart, head and neck, soft tissue, stomach, and lymph nodes (Table 1). The majority of primary epithelioid hemangioendothelioma arises from soft tissue, bone, lung, and liver. The worst outcome is seen in the lung and hepatic tumors. There is a difference in age and gender distribution of EH in various sites (Mehrabi et al. 2006).

Characteristics The term EH was first recognized as soft tissue vascular tumors of endothelial origin with clinical course between benign hemangioma and angiosarcoma. The incidence of EH is less than 0.1 per 100,000 population. Hepatic EH (HEH) affects more commonly adult females and is characterized by an epithelioid or histiocytoid morphology and a growth pattern with evidence of endothelial histogenesis. Two different types of HEH have been described so far: the nodular type in the early stage of HEH, and the diffuse type that reflects the advanced stage of the disease with coalescence of the lesions which are associated with hepatic vascular invasion (Fig. 1a). The clinical manifestation of HEH is heterogeneous varying from cases with no symptoms to hepatic failure. In the symptomatic patients, the most common clinical manifestations include right upper quadrant pain, hepatomegaly, and

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weight loss. Weakness, anorexia, epigastric mass, Aurora A, nausea/vomiting, jaundice, and fatigue are the other common presenting manifestations. The majority of cases have a multifocal tumor involving both liver lobes. The right lobe is affected more than the left lobe in both multifocal and unifocal presentations. Lung, regional lymph nodes, peritoneum, bone, spleen, and diaphragm are the most common sites of extrahepatic involvement at the time of diagnosis. Pleura, mediastinum, retroperitoneum, myocardium, pericardium, brain, cervical lymph nodes, common bile duct, pancreas, and uterus are the other reported sites with HEH ▶ Metastases. Etiology The definitive etiology of HEH is unclear. Some possible etiologic factors include oral contraceptives, vinyl chloride, ▶ asbestosis, thorotrast, major trauma to the liver, viral hepatitis, primary biliary cirrhosis, and alcohol consume. In contrast to many other types of primary liver tumors, HEH does not arise upon a background of chronic liver disease. Diagnosis Due to the rarity of the tumor, only strong suspicion can help to perform necessary evaluations and establish an appropriate diagnosis. Laboratory. The majority of the cases have abnormal liver laboratory parameters such as elevated alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transpeptidase (GGT), and bilirubin. Tumor markers are mostly within normal range and only suitable to rule out other primary or metastatic tumors.

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Hepatic Epithelioid Hemangioendothelioma

Hepatic Epithelioid Hemangioendothelioma, Fig. 1 Hepatic epithelioid hemangioendothelioma. (a) Macroscopy: Multifocal growth pattern of nodules and coalescence of them to form confluent masses specifically in the periphery with extension to and retraction of liver capsule. (b) Hematoxylin–eosin stain: The tumor cells

form intracytoplasmic lumina containing erythrocytes. (c) Tissue stain for factor VIII-related antigen: In addition to the intracytoplasmic lumens, the pleomorphic cells of tumor are immunoreactive for factor VIII related Ag in this tissue section

Imaging. Imaging studies include ultrasonography, computed tomography (CT) scan, magnetic resonance imaging (MRI), scintigraphy, and angiography. On ultrasonography, the most common finding is hypoechoic lesions. On CT scan, low-density pattern is the most common abnormal feature. Moreover, the majority of cases show contrast enhancement in CT images. Two major manifestations of HEH in CT images are multiple, nodular lesions, and/or large masses which can be the result of coalescence of smaller nodules. The nodular type is relatively nonspecific, whereas the diffuse type is very suggestive. On MRI, HEH is usually hypointense on T1-weighted and hyperintense on T2-weighted images (T1- and T2-weighted images) (hypointense, hyperintense).

One of the characteristic features of the HEH is the target shape of the lesions which could be due to the presence of a central sclerotic zone and a peripheral region of cellular proliferation (Fig. 2). On scintigraphic imaging, low uptake is the major finding, which can be useful for the follow-up of patients under therapy. Angiographic findings are nonspecific ranging from hypo- to hyperperfusion. It is noteworthy that imaging studies can only lead to a strong suspicion regarding the presence of HEH and its pattern. Histopathology. Definitive diagnosis requires a histopathological examination. Often, a laparoscopic wedge or core biopsy is sufficient to depict architectural features of the HEH such as the intravascular characteristics. The diagnosis is

Hepatic Epithelioid Hemangioendothelioma

Hepatic Epithelioid Hemangioendothelioma, Fig. 2 MRI sections of HEH T2-weighted hyperintense lesions and central necrosis showing a target appearance

mostly confirmed with immunohistochemical (▶ Immunohistochemistry) evidence of endothelial differentiation (Fig. 1b, c). Differential Diagnosis. More than two thirds of HEH cases may be initially misdiagnosed. The most common misdiagnoses are ▶ cholangiocarcinoma, angiosarcoma, ▶ hepatocellular carcinoma (HCC), metastatic carcinoma (▶ Metastasis), and sclerosing hemangioma. Some important features for differential diagnosis include the infiltrative growth pattern with preservation of the hepatic acinar (Hepatic acinus) landmarks such as portal areas, the characteristic vascular invasion with tufting of portal vein branches and terminal hepatic venules, the identification of epithelioid tumor cells especially with intracytoplasmic lumina, and the delay of staining for epithelial differentiation markers. Therapy There is no generally accepted strategy for the treatment of HEH due to its heterogeneous dignity and variable clinical outcome. Theoretically, liver resection is the first choice for curative treatment of HEH, but in the majority of the cases an oncological resection is impossible due to multicentricity of the lesions or anatomical difficulties. Liver transplantation is generally the most common treatment modality. According to the Pychlmayr classification of hepatic malignancies, HEH is placed among the favorable indications

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for liver transplantation. Life expectancy of the patients with HEH is potentially good. Long-term disease-free survival after liver transplantation is reported in patients with disseminated disease at the time of diagnosis; conversely, some patients with disease confined to the liver developed rapid recurrence and metastases following liver transplantation. The experience with other therapeutic modalities such as systemic or regional chemotherapy, transarterial chemoembolization (▶ Chemotherapy), and radiotherapy (▶ Chemoradiotherapy) is limited and variable; therefore, generally these treatments are of limited value especially as the first line therapy. Considering only follow-up without any treatment as a management option is controversial. There are some cases with long-term survival or even complete spontaneous tumor regression without receiving any treatment, but until now it is impossible to reliably identify HEH patients with a nonaggressive tumor and to consider them for a “wait and see” strategy. The mode of hepatic involvement and the presence or absence of extrahepatic involvement are the main factors in the decision of the treatment modality. When less than one lobe is involved and there is no extrahepatic involvement, liver resection could be done as the first choice in treatment of HEH. The therapeutic strategy in the presence of extrahepatic involvement is especially controversial. When extrahepatic involvement exists, independent of performing liver resection or not, adjuvant chemotherapy (▶ Adjuvant chemoendocrine therapy) may be considered. In the case of massive involvement of the liver, liver transplantation is the best therapeutic choice. Extrahepatic involvement by itself does not exclude liver transplantation. Although chemotherapy in this situation is questionable, it may control the growth of the extrahepatic tumor. It is noteworthy that the clinical course of HEH is variable, ranging from a favorable disease with prolonged survival, even without therapy, to a rapidly progressive disease with a grave outcome. Therefore, the decision of the treatment strategy has to be tailored for each case, and the individual rate of progression, severity of signs, and symptoms, and response to other treatment modalities

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may be important determinants for decision making. Clinical Outcome Three main causes for tumor recurrence and treatment failure after liver transplantation are error in the pretransplant evaluation, enhanced tumor growth under immunosuppressive therapy, and lack of effective anticancer therapy following surgery. The 5-year survival in different reports varies from 45% to 70%. Generally, surgical therapies such as liver resection or transplantation have the best survival rates and chemoradiotherapy or no treatment leads to the worst clinical outcome. The presence of tumor necrosis may be associated with poor outcome, while typical indicators of biologic aggression such as nuclear atypia, capsule penetration, and number of mitosis are found to be unrelated to clinical outcome. The unpredictable natural course and prognosis of HEH makes it difficult to give a correlation between the morphological grading or clinical staging and outcome.

Cross-References ▶ Adjuvant Chemoendocrine Therapy ▶ Asbestos ▶ Chemoradiotherapy ▶ Chemotherapy ▶ Cholangiocarcinoma ▶ Hepatocellular Carcinoma ▶ Immunohistochemistry ▶ Metastasis ▶ Metastatic Colonization

References Ishak KG, Sesterhenn IA, Goodman ZD et al (1984) Epithelioid hemangioendothelioma of the liver: a clinicopathologic and follow-up study of 32 cases. Hum Pathol 15:839–852 Lauffer JM, Zimmermann A, Krahenbuhl L et al (1996) Epithelioid hemangioendothelioma of the liver. A rare hepatic tumor. Cancer 78:2318–2327 Makhlouf HR, Ishak KG, Goodman ZD (1999) Epithelioid hemangioendothelioma of the liver: a clinicopathologic study of 137 cases. Cancer 85:562–582

Hepatic Epithelioid Hemangioendothelioma Mehrabi A, Kashfi A, Fonouni H et al (2006) Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy. Cancer 107(9):2108–2121

See Also (2012) Common Bile Duct. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 960. doi:10.1007/978-3-642-16483-5_1277 (2012) Epithelioid Hemangioendothelioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1296. doi:10.1007/978-3-642-16483-5_1964 (2012) Hemangioendothelioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1640. doi:10.1007/978-3-642-16483-5_2612 (2012) Hepatic Acinus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1656. doi:10.1007/978-3-642-16483-5_2650 (2012) Hypoechoic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1791. doi:10.1007/978-3-642-16483-5_2920 (2012) Jaundice. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1926. doi:10.1007/978-3-642-16483-5_3174 (2012) Low Density Lipoprotein. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2076. doi:10.1007/978-3-642-16483-5_3417 (2012) Mediastinum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2199. doi:10.1007/978-3-642-16483-5_3597 (2012) Mitosis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2342. doi:10.1007/978-3-642-16483-5_3774 (2012) Nuclear Atypia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2567. doi:10.1007/978-3-642-16483-5_4147 (2012) Peritoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2819. doi:10.1007/978-3-642-16483-5_4467 (2012) Primary Biliary Cirrhosis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2984. doi:10.1007/978-3-642-16483-5_4730 (2012) Radiotherapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3158. doi:10.1007/978-3-642-16483-5_4926 (2012) Retroperitoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3292. doi:10.1007/978-3-642-16483-5_5078 (2012) Scintigraphy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3341. doi:10.1007/978-3-642-16483-5_5179 (2012) T1- and T2-Weighted Images. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3601. doi:10.1007/978-3-642-16483-5_6235 (2012) Transarterial Chemoembolization. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3748. doi:10.1007/978-3-642-16483-5_5896 (2012) Tumor Markers. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3796. doi:10.1007/978-3-642-16483-5_6036

Hepatic Ethanol Metabolism

Hepatic Ethanol Metabolism Iain H. McKillop1 and Laura W. Schrum2 1 Department of General Surgery, Carolinas Medical Center, Charlotte, NC, USA 2 Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC, USA

Definition The majority of ethanol metabolism occurs in the liver following the ingestion of alcoholic beverages. The enzymatic reactions involved in ethanol metabolism can in turn lead to a variety of deleterious effects on cells both within the liver and at the systemic level which has led to chronic ethanol consumption being identified as a major risk factor in the development of liver cancer and a significant risk factor for the development of non-hepatic tumors.

Characteristics The liver demonstrates a “dual circulation” vasculature in which oxygenated blood is delivered via the hepatic artery and deoxygenated blood, containing substances that have been absorbed from the gastrointestinal tract, via the hepatic portal vein. Following ingestion ethanol is rapidly absorbed and enters the hepatic portal vein where it is delivered to the functional subunits of the liver termed the hepatic lobules. As blood flows through the hepatic vasculature (sinusoids), specialized epithelial cells, termed hepatocytes, that line the vasculature process materials absorbed in the GI tract to maintain normal physiological homeostasis. In addition to performing essential physiological functions such as protein, lipid, and carbohydrate metabolism, bile production, and regulation of vitamin A storage, the liver also plays a central role in drug and hormone metabolism. Following ethanol consumption metabolism occurs in the hepatocyte via three main enzymatic pathways: alcohol dehydrogenase (ADH),

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cytochrome p450 2E1 (CYP2E1), and catalase. The metabolism of ethanol is a two-step process: the first involves the conversion of ethanol to acetaldehyde (via ADH, CYP2E1, or catalase), and the second requires the conversion of acetaldehyde to acetate by the aldehyde dehydrogenase (ALDH) enzyme (Fig. 1). In both instances, these reactions lead to the production of the reduced form of nicotinamide dinucleotide (NADH). These metabolic pathways for ethanol directly contribute to many of the deleterious effects of ethanol intake on normal hepatic and systemic physiology. While the metabolism of acetaldehyde by ALDH is an efficient metabolic pathway, acetaldehyde is a highly reactive species that, if allowed to accumulate, can cause significant protein damage and formation of ▶ adducts to DNA and ▶ DNA damage. In addition to the damaging effects of acetaldehyde/NADH, once formed, it must be recycled to the oxidized NAD+ form by the electron transport chain in the mitochondria of hepatocytes. This process requires increased oxygen demand and can in turn lead to the production of reactive oxygen species (ROS) and increase cellular ▶ oxidative stress that can cause ▶ oxidative DNA damage and protein/lipid peroxidation. In the instance of moderate ethanol consumption, the ADH–ALDH system is sufficient to metabolize ethanol. However, following chronic, excessive ethanol intake, the CYP2E1 enzyme becomes induced leading to increased acetaldehyde/NADH production and further elevating ROS synthesis and hepatic oxidative stress (Fig. 1). Although ethanol metabolism is localized primarily in the hepatocytes, the nonparenchymal cells of the liver including hepatic stellate cells (HSCs) and ▶ Kupffer cells (KCs) have also been shown to express ethanol-metabolizing genes. Human HSCs express both ADH and ALDH, and CYP2E1 expression has been observed in both HSCs and KCs, therefore further contributing to the detrimental effects of ethanol metabolism in the liver. Depending on the level and period of ethanol consumption, normal hepatic function becomes increasingly compromised with complications ranging from moderate steatosis (fatty liver) to acute alcoholic hepatitis and eventually the development of fibrosis and cirrhosis. In

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Hepatic Ethanol Metabolism Protein & DNA adducts

Ethanol + (CH3CH2OH) + NAD

ADH CYP2E1 catalase

Acetaldehyde (CH3CH=O)

+ NADH + H+

Acetate + NADH + H+ (CH3CH=O)

CO2+ H2O

e– transport chain

ALDH

Ros

Peroxidation (LIPID, PROTEIN, DNA)

Hepatic Ethanol Metabolism, Fig. 1 Hepatic ethanol metabolism as it pertains to potential mechanisms of liver damage. Ethanol is metabolized by a two-step process to

acetaldehyde that, in turn, is metabolized by acetaldehyde dehydrogenase (ALDH) to acetate

addition, epidemiological data indicates that chronic ethanol consumption acts synergistically to accelerate the progression of HCC in patients exposed to other common risk factors including exposure to ▶ aflatoxins and hepatitis virusassociated HCC. The damage caused to the liver through acetaldehyde adduct formation and/or ROS generation/oxidative stress is thought to be a major risk factor for ethanol-related liver cancer development. Greater than 80% of all primary tumors diagnosed in the liver arise as a result of hepatocyte cell transformation (▶ hepatocellular carcinoma, HCC). Unlike other common malignancies, HCC occurs most commonly in the absence of familial patterns and largely within the realms of known risk factors. Chronic ethanol consumption, metabolism, and the cellular and genetic damage associated with these events thus represent a significant risk factor for hepatic damage, cell transformation, and the development of HCC. In addition to the direct effects of ethanol metabolism on the hepatocyte integrity, such as impaired microtubule polymerization, several other direct and indirect factors can combine to

augment the effects of ethanol consumption in the liver and the body as a whole. Glutathione (GSH) is an antioxidant important in reducing the oxidative stress caused by the synthesis and release of ROS. In the mitochondria, GSH is the only source of hydrogen peroxide metabolism and becomes significantly depleted following chronic ethanol ingestion due, at least in part, to decreased GSH transport into the mitochondria. While these detrimental effects on hepatocyte function directly increase the probability of cell transformation, increasing evidence suggests that the sequential nature of tumor formation and/or other systemic effects may play an increasing role in the effects of ethanol during the development and progression of HCC. Ethanol abuse accounts for the majority of liver fibrosis and cirrhosis cases in the western world; however, even though the clinical progression is well described, the ▶ molecular pathology is less well understood. Current models propose that alcoholic liver disease is initiated by an inflammatory response due to the activation of KCs as well as the infiltration of leukocytes including macrophages, neutrophils, and lymphocytes. The

Hepatic Ethanol Metabolism

activation of these inflammatory cells has been shown to be due to elevated gut-derived endotoxin plasma levels. Ethanol alters gut permeability to macromolecules, decreases gut motility, and increases growth of Gram-negative bacteria in the intestinal microflora which ultimately leads to the introduction of endotoxin into the portal microcirculation. The activation and recruitment of inflammatory cells lead to the production of several profibrogenic cytokines. These cytokines likely induce and perpetuate the activation of HSCs leading to increased deposition of extracellular matrix (ECM) like type I collagen. The HSCs themselves can also exacerbate the inflammatory response through the secretion of chemoattractants and adhesion molecules necessary for leukocyte adhesion and infiltration. The presence and activation of KCs, other infiltrating inflammatory cells and damaged hepatocytes, lead to increased ROS resulting in the activation of quiescent HSCs. The quiescent HSCs reside in the perisinusoidal space of Disse, and one of their primary functions in the healthy liver is the storage and homeostasis of vitamin A, namely, retinol, retinal, and retinoic acid. Upon a fibrogenic stimulus, including ethanol, the HSC transdifferentiates from a quiescent, vitamin A storing cell to that of an activated myofibroblast-like cell which proliferates, migrates, to the site of injury, and is responsible for the excessive accumulation of ECM. This continued deposition of ECM leads to scarring of the liver and ultimately liver dysfunction. Reduced levels of serum and hepatic vitamin A have been reported in persons with alcoholic liver disease (ALD). Ethanol exposure to HSCs inhibits retinoic acid production and intracellular retinol levels. Possible mechanisms which interfere with retinoid metabolism in the cell may include reduced vitamin A uptake and enhanced degradation of vitamin A. In addition to oxidative damage incurred directly by the hepatocyte, liver damage also affects cell membrane integrity due to ethanol metabolism via a nonoxidative pathway leading to the generation of fatty acid ethyl esters (FAEE). Fatty acid ethyl esters accumulate in the cell plasma membrane as well as in the mitochondrial membrane leading to organelle dysfunction.

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Accumulation of FAEE, particularly linolenic acid ethyl ester (LAEE), has been reported to activate signaling pathways important in regulating collagen expression which may further contribute to ethanol-induced fibrosis and subsequently HCC progression. In addition to the effects of ethanol and ethanol metabolism on hepatocytes and other cell populations, other factors must also be considered as to how ethanol can affect tumorigenesis. For example, the high incidence of cigarette smoking in ethanol-dependent patients, regional differences in dietary intake, the type of beverage consumed, and the socioeconomic status and relative balance of diet in ethanol-dependent patients can all affect the incidence and rate of hepatic tumor development and progression. Similarly, the induction of CYP2E1 (following chronic ethanol intake) has also been demonstrated to play a significant role in procarcinogen and ▶ carcinogen metabolism, many of which are present in cigarette smoke and alcoholic beverages. Since oxidative stress has been linked to the development of ALD, the use of antioxidants as possible therapeutic strategies has been explored. The addition of antioxidants such as vitamin E, superoxide dismutase, GSH precursors such as S-adenosyl-L-methionine (SAMe) and the green tea extract ()-epigallocatechin-3-gallate (EGCG), has been shown to prevent or ameliorate ethanol-induced liver injury in a variety of animal models of ALD and HCC. However, the use of antioxidants should be approached with caution due to the possible toxic properties of antioxidants under certain conditions. Similarly, the use of “over the counter” antioxidants raises the possibility of drug–drug interactions and altered endogenous and exogenous agent metabolism caused by antioxidant intake, many of which are currently poorly studied and reported.

References McKillop IH, Schrum LW (2005) Alcohol and liver cancer. Alcohol 35:195–203 McKillop IH, Moran DM, Jin X et al (2006) Molecular pathogenesis of hepatocellular carcinoma. J Surg Res 136:125–135

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Hepatic-Fixed Macrophages

Definition

Hepatic-Fixed Macrophages ▶ Kupffer Cells

Hepatitis B Virus Synonyms HBV

Definition A member of the Hepadnaviridae family (hepatotropic DNA viruses). The HBV genome is a partially relaxed double-stranded circular DNA of 3,200 base pairs and encodes four partly overlapping open reading frames (ORFs). Thirty to fifty percent of persons who acquire the infection before the age of 5 years develop chronic HBV infection. HBV is a high-risk factor for ▶ hepatocellular carcinoma.

Cross-References ▶ Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention ▶ Hepatocellular Carcinoma Molecular Biology

Hepatitis B Virus x AntigenAssociated Hepatocellular Carcinoma Mark A. Feitelson Department of Biology, Temple University, Philadelphia, PA, USA

Keywords ▶ HCC; ▶ Hepatocellular carcinoma; ▶ Liver cancer

The term hepatitis B x antigen, or HBxAg, refers to the gene product of hepatitis B virus (HBV) that contributes importantly to virus replication, the pathogenesis of chronic liver disease (CLD), and the development of HCC. CLD is the process that involves the immunologically mediated damage or destruction of various cell types within the liver. CLD encompasses hepatitis (▶ inflammation of the liver), fibrosis (scarring, which involves the accumulation of connective tissue in the place of liver tissue), and cirrhosis (extensive scarring in which connective tissues completely surrounds islands of remaining liver tissue).

Characteristics There are an estimated 350 million people worldwide who are chronically infected with HBV and often replicate virus for many years or decades. These people are at high risk for the development of chronic hepatitis, which may progress onto cirrhosis (end stage liver disease) and then HCC. Although the pathogenesis of infection is immune mediated, this is characterized most often by responses that trigger hepatocellular damage and destruction but do not clear virus. An important characteristic of chronic liver disease (CLD) is that liver cell regeneration provides opportunities for integration of virus DNA into the replication forks of cellular DNA. It turns out that the region encoding HBxAg is the most frequently integrated region of HBV DNA into the host genome. This region often encodes HBxAg, which is a trans-activating protein that alters the expression of cellular genes in many chromosomes throughout the host genome. This is quite an accomplishment for a small protein of only 17 kDa and is achieved by constitutively activating a number of signal transduction pathways in the cytoplasm (such as JAK/STAT (▶ signal transducers and activators of transcription in oncogenesis), NF-kB, ▶ AP-1, AP-2, ras, src, PI3K/Akt (▶ PI3K signaling; Akt signal transduction pathways in oncogenesis), and

Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma

b-catenin (▶ Wnt signaling), among others) and by binding to transcription factors in the nucleus (such as Oct-1, CREB, ATF-2, b-zip, TBP, and other basal transcription factors) or sequestering them in the cytoplasm (e.g., p53). HBxAg also alters gene expression at the posttranscriptional level by blocking the activity of the ▶ proteasome, which normally degrades proteins, and by altering the integrity of translation. In addition, HBx indirectly promotes the accumulation of mutations by inhibiting multiple DNA repair pathways. Some of these pathways have been shown to be operative in the mechanism (s) whereby HBxAg promotes cell growth in culture dishes and anchorage independent growth in soft agar (a property often associated with cancer cells). The findings that HBxAg is capable of conferring tumorigenicity upon nonmalignant liver cells and that the sustained overexpression of HBxAg in transgenic mice gives rise to HCC further underscore the centrality of HBxAg expression to the development of HCC. It is likely that inadequate immune responses permit the persistence of infected hepatocytes during chronic infection. Although HBV is sensitive to interferon, the sequestration of HBV replication complexes within immature core particles during the virus life cycle largely prevents their being sensed by endosomal toll-like receptors, resulting in little or no induction of interferon. However, there is increasing evidence that HBxAg inhibits a number of apoptotic pathways (e.g., Fas and tumor necrosis factor alpha (TNFa), both of which trigger programmed cell death) during chronic infection, thereby promoting the persistence of infected cells during CLD. HBxAg also promotes fibrogenesis, in that it promotes the expression of extracellular matrix proteins such as ▶ fibronectin, promotes the cross-linking of collagen, and potentiates TGFb1 signaling, in part, by inhibiting expression of the TGFb1-binding partner, a-2-macroglobulin. In this context, it is not surprising to find a strong direct correlation between intrahepatic HBxAg expression and CLD. This relationship would protect virus infected cells from immune destruction, but these same features also promote carcinogenesis, since tumor cells are also resistant to apoptosis

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and demonstrate constitutively active signaling pathways that had been previously altered in preneoplastic tissue by HBxAg. There are few natural effectors of HBxAg that are known to be responsible for these profound biological changes that ultimately result in cancer. HBxAg appears to stimulate ▶ insulin-like growth factor 2 (IGF2), which promotes hepatocellular growth, activates wild-type b-catenin (and its effector, c-myc), trans-activates several unique “oncogenes,” and promotes ▶ angiogenesis. HBxAg also binds to and inactivates the tumor suppressors, p53 and PTEN; downregulates expression of the natural cell cycle inhibitor, ▶ p21; and promotes phosphorylation (inactivation) of the Rb tumor suppressor protein (retinoblastoma protein), suggesting the molecular basis upon which HBxAg stimulates unchecked cell growth. HBxAg also binds to and inactivates a novel senescence factor, p55sen, suggesting that HBxAg contributes to carcinogenesis, in part, by overcoming cellular senescence. The observation that HBxAg also downregulates ▶ E-cadherin expression, the latter of which is involved in cell adhesion, provides at least part of the explanation as to how HBxAg stimulates cell motility (metastasis) in carcinogenesis. Studies have also shown that HBx up-regulates Hippo signaling, which promotes cell growth, activates Hedgehog and Notch signaling, which impact upon cell fate, and promotes the appearance of stem cell like characteristics. It is thought that the latter plays a central role in the development of cancer. ▶ Oxidative stress is a common feature of CLD, resulting in the activation of HBxAg in CLD and HCC. Overexpression of HBV envelope-associated polypeptides, cytokine signaling, and cell-mediated immunity against HBV-infected cells all promote the development of oxidative stress. This was associated with decreased intrahepatic levels of superoxide dismutase (Cu/Zn), which normally protects cells from ▶ reactive oxygen species. These conditions stimulate HBxAg, which compromises transcription-coupled DNA repair and ▶ nucleotide excision repair, resulting in increased chromosomal alterations (genetic instability) and

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micronuclei formation (where metaphase plates separate prior to complete DNA replication). In addition, HBxAg is associated with the outer mitochondrial membrane, where it binds to the voltage-dependent anion channel (VDAC3), resulting in decreased mitochondrial membrane potential and the further development of oxidative stress. In this way, oxidative stress augments HBxAg function in propagating chronic infection and in stepwise hepatocarcinogenesis.

References Arbuthnot P, Capovilla A, Kew M (2000) Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways. J Gastroenterol Hepatol 15:357–368 Breuhahn K, Longerich T, Schirmacher P (2006) Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 25:3787–3800 Feitelson MA (2004) Molecular and genetic determinants of primary liver malignancy. In: Khatri VP, Schneider PD (eds) Surgical clinics of North America, vol 84, Liver surgery: modern concepts and techniques. WB Saunders, Philadelphia, pp 339–354 Jin YM, Yun C, Park C et al (2001) Expression of hepatitis B virus X protein is closely correlated with the high periportal inflammatory activity of liver diseases. J Viral Hepat 8:322–330 Waris G, Siddiqui A (2003) Regulatory mechanisms of viral hepatitis B and C. J Biosci 28:311–321

Hepatitis C Virus Wolfgang H. Caselmann Medizinische Klinik und Poliklinik I, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany

Synonyms HCV

Definition Hepatitis C virus (HCV) belongs to the Flaviviridae family (Hepacivirus genus) and is a

Hepatitis C Virus

pathogenic human RNA virus, which causes chronic liver disease and hepatocellular carcinoma (▶ Hepatocellular Carcinoma, Molecular Biology) (HCC).

Characteristics The size of this enveloped virus is 60 nm. Its nucleocapsid contains a single-stranded RNA genome of 9.6 kb genome (Fig. 1) of plus(+) strand polarity that carries a single open reading frame. At least six major genotypes (1–6) with up to three subtypes (a to c) exist, which differ not only in their nucleic acid sequence but also in their pathophysiological properties. All structural and nonstructural viral proteins are processed from a polyprotein precursor of 3010–3033 amino acids in the cytoplasm or endoplasmic reticulum of the infected cell. Untranslated Regions The 50 - and 30 -untranslated regions (UTR) are highly conserved within the different genotypes. The 341–344 nucleotides 50 -UTR (Fig. 1) forms extensive stem-loop structures, which are important in translation initiation. It also harbors an internal ribosomal entry site. The 30 -UTR is composed of a poorly conserved variable region (28–42 nucleotides), a variable polypyrimidine stretch, and conserved 98 nucleotides at the 30 end (x region). Both UTRs seem to be crucial for regulation of HCV translation and possibly also for controlling HCV replication and are, therefore, candidate targets for experimental antiviral strategies. Structural Proteins The core protein C (21 kD) polymerizes to an icosahedral capsid and binds RNA to form the nucleocapsid. The envelope proteins E1 (31 kD) and E2 (70 kD) form heterodimers whose formation is mediated by the chaperon calnexin. These dimers are embedded in a host-derived lipid bilayer. Within the E2 sequence there are two hot spots of mutations (hypervariable regions, HVR1 and HVR2). Mutations occurring during HCV infections are due to a selection driven by

Hepatitis C Virus

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Hepatitis C Virus, Fig. 1 Genomic organization and designation of HCV RNA and amino acid positions and function of HCV proteins

the host immune system and account for the regular existence of a variety of quasispecies. The E2 protein is produced as two precursors, E2/NS2 and E2/p7, that differ in their C-terminus. The hydrophobic p7 is probably important for membrane anchoring of E2. The function of p7 or E2/p7 during the HCV life cycle is not known. All structural protein processing is performed by cellular signal peptidases. Nonstructural Proteins The nonstructural protein NS2 (23 kD) is released from the polyprotein precursor both by host signal peptidases at the C-terminus of p7 and by an NS2/3 proteinase at the NS2-NS3 junction. NS3 (68 kD) has three different functions: it is part of the NS2/3 proteinase, its N-terminal third contains a serine proteinase, and at the C-terminus an RNA-dependent NTPase/helicase was discovered. Together with NS4A (6 kD) as a stable complexed cofactor, the N-terminal NS3 serine proteinase catalyzes the cleavage between NS3 and NS4A, followed by cleavage between

NS5A-NS5B, NS4A-NS4B, and NS4B-NS5A. The NS3 helicase can unwind double-stranded (ds)RNA, dsDNA, and RNA/DNA heteroduplex molecules. For this purpose any NTP or dNTP is used as source of energy. NS4B is a 26 kD membrane-associated protein of unknown function. NS5A (56–58 kD) represents two cytoplasmic proteins, p56 and p58, which are both phosporylated at serine residues. This process may be essential for the viral replication cycle, but the biological functions of these proteins are not understood. There is some evidence that NS5A mediates interferon-alpha resistance of HCV. The NS5B (65 kD) protein is responsible for HCV RNA replication; it represents an RNA-dependent RNA polymerase that is not found in humans and may therefore be another attractive target for antiviral strategies. The RNA minus() strand is transcribed in the host cytoplasm into a plus(+) strand RNA. This serves as a template to produce new minus() strands for packaging into the envelope. Both steps are accomplished by the NS5B protein.

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Cellular and Molecular Regulation Although the HCV genome does not harbor acutely transforming oncogenes, the HCV core and the NS3 gene are candidate genes whose products may mediate malignant hepatocyte transformation. Oncogene complementation assays using HCV core and H-ras or c-MYC oncogenes showed that HCV core cooperates with these oncogenes and transforms primary rat embryo fibroblasts to the tumorigenic phenotype. Focus formation, soft agar growth, and tumor development was also shown in nude mice using rat-1 fibroblasts. Also in this model a loss of contact inhibition, morphological changes, and anchorage- and serumindependent growth occurred when HCV core and v-H-ras were cooperatively expressed. The most striking evidence of an oncogenic potential of HCV comes from an HCV core-transgenic mouse model, in which hepatic tumors arose in up to 30% of animals of two different strains. Interestingly, all tumors occurred in male animals. However, other mice transgenic for HCV core or C-terminally truncated (amino acid 384–715) HCV core failed to develop histological or biochemical signs of liver disease. The postulated underlying molecular mechanisms of HCV core-induced hepatocyte transformation are manifold. They comprise sequestration of LZIP in the cytoplasm leading to a loss of CRE-dependent transcription and regulation of cell proliferation and subsequently to morphological transformation of NIH 3T3 cells. Other mechanisms may be interference with ▶ apoptosis or transactivation of cellular c-fos, c-myc, p53, or b-interferon promoters. Stable expression of the 50 -portion of the NS3 gene was able to induce focus formation and soft agar growth in NIH 3T3 cells. It was demonstrated that internal cleavage of the NS3 protein occurs at cleavage sites FCH(1395)//S(1396)KK and IPT(1428)//S(1429) GD within the HCV RNA helicase domain in presence of NS4A. These findings were confirmed in two different isolates of HCVof genotype 1b. The 50 -portion of NS3 was more oncogenic than the full-length NS3 protein. Clinical Relevance An estimated 2% of the world’s population is chronically infected with HCV. It accounts for

Hepatitis C Virus

20% of acute and 70% of chronic hepatitis cases. HCV is mainly transmitted by blood. Since screening and treatment of blood products for HCV has been routinely performed, posttransfusion hepatitis has become extremely rare. Intravenous drug addiction is today a major way of HCV transmission. Acute hepatitis is often clinically inapparent, and in chronic hepatitis symptoms occur mainly in later stages of disease. A high chronicity rate of up to 75% of acute infection and its silent course account for the pathogenic potential of this virus, which includes extrahepatic manifestations. Twenty percent of chronically infected patients develop liver cirrhosis, and 2–5% per year will progress to hepatocellular carcinoma (HCC). Therapy for HCV infection comprises the combined use of 3–6 MU of a-interferon administered subcutaneously three times a week and 800–1200 mg of the nucleoside analog ribavirin po daily for 6–12 months dependent on the genotype and viral load. Roughly 40–45% of previously untreated or relapse patients benefit from the treatment, i.e., eliminate HCV and normalize liver enzymes. In addition, this treatment is capable of improving histological signs of liver damage. Its effect on HCC prevention cannot clearly be judged at present. Therapeutic innovations comprise pegylated interferons that allow single weekly dosing due to slow effector release from subcutaneous depots and a decrease of systemic side effects. Sustained response rates up to 70% can be reached. A synthetic consensus interferon-alpha showed promising effects in monotherapeutic use in a preliminary trial and is presently being evaluated in combination with ribavirin. Major future achievements are expected from the development of inhibitors of HCV protease, helicase, or RNA-dependent RNA polymerase, which may improve effectivity of antiviral treatment similarly to the situation in HIV infection but are not yet available for clinical use. Therapeutic nucleic acids such as antisense oligodeoxynucleotides, RNAs, or ribozymes may be another experimental concept for the treatment of HCV infection in the future.

Hepatoblastoma

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Cross-References

Characteristics

▶ Apoptosis ▶ Hepatocellular Carcinoma Molecular Biology ▶ MYC Oncogene ▶ RAS Genes

Hepatoblastomas are the most frequent malignant liver tumors of childhood. Their annual incidence is 0.5–1 cases per million children under 15 years of age in the Western countries. The affected children are most frequently between 6 months and 3 years old. Hepatoblastoma has also been detected in utero by prenatal ultrasound examination. Since liver tumors lack early clinical symptoms, hepatoblastoma patients often present with locally extended tumors at diagnosis. However, distant metastases usually occur very late in the disease progression. The patients frequently have highly elevated ▶ a-fetoprotein levels. In these cases this ▶ oncofetal antigen can be useful as a sensitive diagnostic marker and also as a marker for the monitoring of treatment response.

References Moriya K, Fujie H, Fukuda K et al (1998) The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 4:1065–1067 Wedemeyer H, Caselmann WH, Manns MP (1998) Combination therapy of chronic hepatitis C – an important step but not the final goal. J Hepatol 29:1010–1014

See Also (2012) Hepatitis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1663. doi:10.1007/978-3-642-16483-5_2658 (2012) Liver cirrhosis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2067. doi:10.1007/978-3-642-16483-5_3396 (2012) Pegylated interferons. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2805. doi:10.1007/978-3-642-16483-5_4434 (2012) Quasispecies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3132. doi:10.1007/978-3-642-16483-5_4885 (2012) Transactivation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3747. doi:10.1007/978-3-642-16483-5_5893

Hepatoblastoma Torsten Pietsch1 and Dietrich von Schweinitz2 1 Institut für Neuropathologie, Kinderchirurgie, Universitätskliniken Bonn, Bonn, Germany 2 Klinikum der Universität München, Kinderchirurgische Klinik im Dr. von Haunerschen Kinderspital, München, Germany

Definition Hepatoblastoma is a childhood malignant embryonal liver tumor consisting of immature epithelial cells with or without additional mesenchymal component.

Pathological Classification Hepatoblastomas always consist of immature epithelial liver cells resembling fetal or embryonal liver cells. Approximately one third of the cases contain additional mesenchymal components and are then termed “mixed” hepatoblastomas. According to Weinberg and Finegold (1983), the epithelial component is further classified into well-differentiated “fetal,” less-differentiated “embryonal,” and undifferentiated “small cell anaplastic” categories. The latter is rare, as are “macrotrabecular” or “teratoid” variants. Small cell anaplastic and macrotrabecular variants indicate a bad prognosis. A histopathological hallmark of hepatoblastomas, in particular of the fetal differentiated cases, is the occurrence of hematopoietic foci mainly consisting of erythropoietic or thrombopoietic progenitor cells mimicking fetal hematopoiesis in the liver (Fig. 1). Staging In the last 30 years, the treatment modalities and outcome of hepatoblastoma patients have significantly improved. It turned out that hepatoblastomas are responsive to chemotherapy. Today, most hepatoblastoma patients are enrolled in multicenter studies and receive a stage- and riskadapted multimodal therapy. Different staging systems are used including the American and

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Hepatoblastoma, Fig. 1 Histopathology of an epithelial hepatoblastoma showing foci of hematopoietic cells

German four-graded postoperative staging system, the Japanese TNM classification, and the European SIOP PreTreatment Extent of Disease (PRETEXT) grouping system. The patients are stratified into standard- and high-risk patients, the latter often presenting with extended, multifocal, or metastatic disease. Therapy and Outcome In current protocols, most patients receive a neoadjuvant chemotherapy with ▶ cisplatin and doxorubicin, which in some studies is combined with ifosfamide, 5-fluorouracil, or carboplatin. The combination carboplatin and etoposide has also been effective. The overall survival after chemotherapy and resection is 70–80%. Unfortunately, approximately one quarter of the patients still die from the disease, so that predictive factors are important to recognize early these high-risk patients and to offer them an intensified therapy. High-risk patients have been treated with prolonged preoperative chemotherapy (SIOP, USA) or with mega-therapy (Germany). Predictive Factors Several histological, clinical, as well as serological factors have been evaluated for their predictive value. In particular, the extension of the tumor in the liver, multifocality, and vascular invasion and the presence of metastases have been of predictive importance in most studies. The decline of alphafetoprotein levels under chemotherapy predicts the clinical response. In contrast, the impact of distinct histological features such as mixed versus

Hepatoblastoma

epithelial or fetal versus embryonal differentiation and chromosome ploidy of the tumors is still under discussion, and the data is controversial. The etiology of hepatoblastomas is unknown. Environmental factors do not seem to play a major role in the pathogenesis of hepatoblastomas. Preterm infants may have an elevated risk for the development of hepatoblastomas. Although most hepatoblastomas occur sporadically, familial cases have been described. The incidence is highly elevated in families with ▶ adenomatous polyposis coli (FAP [▶ APC gene in familial adenomatous polyposis]) and ▶ BeckwithWiedemann syndrome. Genetic and Molecular Characteristics Cytogenetic analyses have revealed several recurrent numerical aberrations as well as structural alterations. Most frequently trisomies of chromosomes 1, 2, 8, and 20 have been described as well as a recurrent translocation t(1;4)(q12;q34) in 15% of hepatoblastomas. Approximately 50% of the cases show gain of material of chromosome 2q. A few cases have an ▶ amplification of material from chromosomal band 2q24, indicating the location of a hepatoblastoma-related oncogene. ▶ Microsatellite analyses have uncovered frequent allelic losses of chromosomal regions of chromosome arms 1p and 11p. The losses of chromosomal region 11p15.5 are always of maternal origin indicating the existence of one or more imprinted hepatoblastoma-associated gene (s) expressed from the maternal allele. Studies on the mutational status of the ▶ TP53 gene resulted in conflicting data. Whereas TP53 mutations were described in a small Japanese tumor collection, these were absent in other studies. Similarly, somatic missense mutations were described in one study but not confirmed by others. The activation of the ▶ WNT signaling pathway by activating mutations in the b-catenin gene is a frequent event in hepatoblastomas. Approximately 50% of the hepatoblastomas save point mutations or deletions of exon 3 encoding the protein degradation targeting site of the b-catenin protein, resulting in a destruction of N-terminal phosphorylation sites that are necessary for protein degradation. This leads to the

Hepatocellular Carcinoma

accumulation of a mutated protein, which transfers oncogenetic signals to the nucleus and increases transcription of specific target genes of the TCF/LEF family of transcriptions factors such as ▶ cyclin D1 and c-myc. Similar mutations have been described in other tumor entities including colorectal cancer. However, hepatoblastoma represents the malignant tumor with the highest incidence for b-catenin mutations. Cellular Characteristics Hepatoblastoma cells resemble liver progenitor cells during embryonic and fetal development suggesting that hepatoblastomas are derived from such progenitors. Hepatoblastoma cells are still dependent on growth factors and use specific growth factor signaling systems. The important fetal mitogen, insulin-like growth factor II, has been demonstrated to be highly overexpressed in hepatoblastomas. The encoding IGF2 gene maps to chromosome 11p15.5, a region frequently altered in hepatoblastomas and related embryonal tumors. They also produce several hematopoietic ▶ cytokines such as interleukin 1, ▶ stem cell factor, ▶ erythropoietin, and thrombopoietin that induce hematopoietic foci in hepatoblastomas. Hepatoblastoma cells overexpress the receptor ▶ Met for hepatocyte growth factor (HGF, ▶ scatter factor) and proliferate in response to HGF in vitro. Concentrations needed for this effect are usually found in the serum of patients after liver surgery. This may explain why hepatoblastomas often show rapid regrowth after partial resection. Therefore it is now common sense that the tumors should undergo primary resection, only when they can be removed without residual tumor cells.

2039 Von Schweinitz D, Hecker H, Schmidt-von-Arndt G et al (1997a) Prognostic factors and staging systems in childhood hepatoblastoma. Int J Cancer 74:593–599 Von Schweinitz D, Byrd DJ, Hecker H et al (1997b) Efficiency and toxicity of ifosfamide, cisplatin and doxorubicin in the treatment of childhood hepatoblastoma. Study Committee of the Cooperative Paediatric Liver Tumour Study HB89 of the German Society for Paediatric Oncology and Haematology. Eur J Cancer 33:1243–1249 Weinberg AG, Finegold MJ (1983) Primary hepatic tumors of childhood. Hum Pathol 14:512–537

Hepatocarcinoma ▶ Hepatocellular Carcinoma

Hepatocellular Carcinoma Toshihiko Mizuta Department of Internal Medicine, Imari Arita Kyoritsu Hospital, Saga, Japan

Synonyms HCC; Hepatic carcinoma; Hepatocarcinoma; Liver cancer; Liver cell carcinoma

Definition Hepatocellular carcinoma (HCC) is a primary cancer that arises from hepatocytes, the major cell type of the liver. Most cases of HCC are secondary to either hepatitis virus (usually type B or C) infection or cirrhosis.

References Brown J, Perilongo G, Shafford E et al (2000) Pretreatment prognostic factors for children with hepatoblastoma – results from the International Society of Paediatric Oncology (SIOP) study SIOPEL 1. Eur J Cancer 36:1418–1425 Mann JR, Kasthuri N, Raafat F et al (1990) Malignant hepatic tumours in children: incidence, clinical features and aetiology. Paediatr Perinat Epidemiol 4:276–289

Characteristics Epidemiology HCC is the fifth most common cancer in men and the eighth most common cancer in women worldwide. An estimated more than half a million new cases are diagnosed annually, while there are

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major geographical differences in incidence. The annual incidence rates in Eastern Asia and sub-Saharan Africa exceed 15/10,000 inhabitants, while the figures are intermediate (between 5/10,000 and 15/10,000) in the Mediterranean Basin and Southern Europe and very low (50% chance of surviving 3 years, even without treatment. In contrast, a patient with multiple tumors involving both lobes of the liver with decompensated cirrhosis is unlikely to survive more than 6 months, even with treatment. AFP levels have been shown to be prognostically important, with the median survival of AFP-negative patients significantly

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Hepatocellular Carcinoma, Table 1 Definitions of the CLIP score Variables ChildTurcottePugh stage Tumor morphology

AFP (ng/ml) Portal vein thrombosis

Score 0

1

2

A

B

C

Unilobular and extension 50% 50% –

No

Yes



longer than that of AFP-positive patients. Other prognostic variables include performance status (measure of general well-being), liver functions, and the presence or absence of cirrhosis and its severity in relation to the Child-Pugh classification. Prognostic Staging System A clinical staging system provides guidance for patient assessment and appropriate therapy. It is useful for the decision to treat certain patients aggressively while avoiding the overtreatment of other patients who would not tolerate therapy or whose life expectancy rules out any chance of success. Clinical staging is also an essential tool for comparison between groups in therapeutic trials and between different studies. The current classifications most commonly used for HCC are the Okuda staging system, the Child-Pugh staging system, tumor node metastasis (TNM) staging, and Cancer of the Liver Italian Program (CLIP) score (Table 1). Among these, the CLIP score is currently the most commonly used integrated staging score, for both tumor and liver disease stages. The Japan integrated staging (JIS) system, a new system that is based on a combination of the Child-Pugh system and the Liver Cancer Study Group of Japan (LCSGJ) system – the LCSGJ system is also concordant with TNM classification for HCC by the International HepatoPancreato-Biliary Association and the

Hepatocellular Carcinoma, Table 2 Japan integrated staging (JIS) scoring system Variables Child-Pugh grade TNM stage by LCSGJ

Score 0 1 A B I II

2 C III

3 IV

International Union Against Cancer (UICC) – has been proposed in Japan (Table 2). The stratification ability of the JIS scoring system is much better than that of the CLIP scoring system. The JIS scoring system also performed better than the CLIP scoring system in selecting the best prognostic patient group. Definition of TNM stage by the Liver Cancer Study Group of Japan (LCSGJ) T factor Fulfilling three factors Fulfilling two factors Fulfilling one factor Fulfilling zero factors T1 N0 M0 T2 N0 M0 T3 N0 M0 T4 N0 M0 or any T N1 M0 Any T N0-1 M1

I. Single

II. 20%, and chromosome arms 6q, 9p, 13q, 16p, and 17p LOH have been linked to inactivation of the tumor suppressor insulin-like growth factor 2 receptor (IGF2R), p16, retinoblastoma (RB1), axin 1, and p53. The genetic changes involved in hepatocarcinogenesis can be divided into at least five pathways: the p53 pathway, which is involved in the response to DNA damage or genomic instability; the p16/p27/RB1 pathway, which is involved in cell-cycle control; the transforming growth factorb (TGF-b) pathway, which is involved in growth inhibition and apoptosis of hepatocytes; the

G0

p16

p27

Wnt/b-catenin/APC pathway, which is involved in intercellular interactions; and the E-cadherin/ integrin and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways, which are involved in cancer cell migration and metastasis. Among the different signaling pathways involved in hepatocarcinogenesis, deregulation of the cell-cycle machinery is thought to be closely involved in the progression of HCC. Cell-cycle progression in mammalian cells is mainly governed by cyclin A, cyclin D, and cyclin E, along with cyclin-dependent kinase 2 (CDK2) and CDK4. The kinase activities of these proteins are negatively regulated by the CDK inhibitors (CDKIs) p16 and p27. RB1, which is phosphorylated by CDKs and activates the E2F family of transcription factors, is mutated in 15% of HCCs. The cyclin D1 gene, which is involved in the G1 progression and G1/S transition phases of the cell cycle, has been shown to be amplified in 10–20% of HCCs. By contrast, both p16 and p27 are frequently inactivated in HCCs. P16 is inactivated in 50% of HCCs, mainly due to the de novo methylation of the DNA-promoter region. Somatic mutation of the p16 gene has also been described in some cases of HCC. P16 DNA methylation was identified in preneoplastic liver tissues of individuals with chronic hepatitis virus infections. P27 is a

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member of the KIP family of CDKIs, and its expression is reduced in 40–60% of HCCs. Decreased p27 expression is closely associated with poor prognoses of individuals with HCCs, indicating that it is an adverse prognostic factor. In some cases of HCC, increased cell proliferation has been noted despite relatively high levels of p27 expression, suggesting the inactivation of p27 by sequestration into cyclin D1–CDK4containing complexes. Although many genes appear to be altered in HCC, the frequencies of individual mutations are low, and the patterns of genetic alteration differ among patients. Connections between different pathways might thus be plausible mechanisms underlying hepatocarcinogenesis.

Advances in the use of imaging tools, such as CT scanning with contrast medium and magnetic resonance imaging (MRI), have enabled the precise detection of HCCs. Accuracy levels have reached >90% using enhanced CT and MRI techniques, and the current limit of detection for HCCs is 10 mm diameter. HCCS can develop from small nodules into large nodules when given access to an arterial blood supply rather than a portal blood supply. Small HCCs with poor arterial blood supplies are difficult to distinguish from adenomatous hyperplasia, large regenerative nodules, or angiomyolipoma. Direct tumor biopsy guided by ultrasound with a fine needle is therefore useful for making a final diagnosis in such cases.

Diagnosis

Treatment

Serum alpha-fetoprotein (AFP) and des–gcarboxy prothrombin, which is also known as protein induced by vitamin K antagonist (PIVKA II), are useful serological markers of HCC. Although AFP exists in normal human tissues (both fetal and adult), serum levels are sensitive to the presence of HCC. The overall diagnostic accuracy of AFP is 80%. By contrast, PIVKA II is only present in HCC tissues, so its specificity is relatively high, and its overall diagnostic accuracy is 55%. The simultaneous measurement of AFP and PIVKA II could therefore be useful for the detection of HCC. However, as elevated AFP levels can be the result of liver cirrhosis, it is important to determine whether they are caused by active liver cirrhosis or by HCC that has arisen from liver cirrhosis. The elevation of serum AFP reflected as regeneration of hepatocytes. The levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19-9) are also often elevated in the serum of patients with HCC. However, the specificities of these markers are lower than those of AFP and PIVKA II. Total imaging is important for detecting small HCCs. The majority of HCCs arise from chronic hepatitis and liver cirrhosis; periodical checking using ultrasound scanning and computed tomography (CT) is therefore crucial to detect the early stages of HCCs.

There are several treatment modalities for HCC. Surgical treatment is divided into two categories: hepatic resection and liver transplantation. The former is based upon the hepatic functional reserve, tumor location, and tumor numbers, and includes many procedures ranging from partial resection to extended right or left hepatic lobectomy. The latter is based on the tumor size and tumor numbers alone. The Milan criteria (that is, the presence of one nodule 95%) among all RAS proteins in mammals. The aa region 85–165 is also well conserved (70–80%), in contrast to the 166–186 aa region, called the hypervariable region (HVR) that precedes the final CAAX motif (aa 186–189) (Fig. 1b). While the HVR is divergent among HRAS, KRAS, and NRAS, it is highly conserved in mammals for each gene. On the other hand, the CAAX motif is unique for each RAS isoform. To exert their biological functions, p21ras proteins must be attached to cellular membranes. Therefore, upon synthesis in the cytosol, the inactive precursor p21ras proteins must first undergo a series of posttranslational modifications, before attaining full biological activity. The first step increases hydrophobicity at the C-terminus through farnesylation of Cys186 in the CAAX box. As a result, p21ras is targeted to the surface of the endoplasmic reticulum (ER). Following, a proteolytic cleavage of the –AAX motif and a carboxy-methylation of the resulting C-terminal cysteine residue take place. The third step requires palmitoylation of Cys residues in the HVR region adjacent to Cys186 of the CAAX box (Fig. 1b). This sequel of processing provides the signals that enable p21ras proteins to traffic via the ER-Golgi secretory pathway to their final plasma membrane locations. For p21Hras, the destinations in the plasma membrane include cholesterol-rich

HRAS

microdomains (lipid rafts and caveolae) as well as bulk-disordered plasma membrane. Trafficking between these domains is dynamic and depends upon the activation state. Of note, p21Hras can be palmitoylated at both Cys181 and Cys184, but the two residues have different significance for intracellular trafficking. Palmitoylated Cys181 is linked with cell surface localization, while Cys184 restricts p21Hras to the Golgi apparatus. Overall, the specific subcellular distribution is generated by a constitutive de-/ reacylation cycle that operates on palmitoylated p21Hras, driving its rapid exchange between the plasma membrane and the Golgi apparatus. Nevertheless, location on the ER can also occur, but transiently, as well as on recycling endosomes derived from plasma membrane. This continuous de-/reacylation cycle prevents p21Hras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. Other, noncanonical subcellular localizations have also been reported. The p19Hras-IDX isoform that lacks the C-terminus HVR region has been found in the cytoplasm and the nucleus. p21Hras has also been observed in the nucleus. Biochemical Properties of p21Hras Protein The p21Hras protein exhibits GTPase activity and can bind GDP as well as GTP. In normal cells, p21Hras functions as a molecular switch being in an inactive state when bound with GDP and active upon GTP binding. Cycling between these two conditions is controlled by different classes of regulatory proteins. These factors regulate the rates of GDP release and GTP hydrolysis and are summarized as follows: 1. GTPase-activating proteins (GAPs) that increase the rate of GTP hydrolysis 2. Guanine nucleotide release proteins (GNRPs, also called guanine nucleotide exchange factors – GEFs) that catalyze the release of bound GDP 3. Guanine nucleotide dissociation inhibitors (GDIs) that inhibit the replacement of GDP by GTP and may also inhibit the action of GAPs

HRAS

Conformational changes mostly in the two motile regions switch I and switch II are responsible for the functional interactions with the negative (GAP) and positive (GEF) cellular regulators. Functional Properties of p21Hras Protein The p21Hras protein participates in many signal transduction pathways. In normal cells, these pathways control various cell aspects, like proliferation, differentiation, survival, inflammation, and cell death. Nevertheless, the specific outcome is tissue/cell type or embryonic origin dependent, as described in the next section. Many of the effector pathways are shared by the products of all three RAS genes (HRAS, KRAS, or NRAS), but each isoform displays different qualitative/quantitative abilities to activate a specific pathway that is also cell context dependent. This complexity in RAS signaling is further increased due to the presence of isoforms for these effectors as well as by their subcellular localization. The initially studied and best-known effector pathway activated by p21Hras is the RAF/MEK/ ERK one. It represents the prototype of the MAP kinase pathways. It is also one of the most essentials in cells responding to mitogenic stimuli through tyrosine kinase receptors. Activation of this pathway leads to a wide range of cellular responses, including growth, differentiation, inflammation, and apoptosis. Of note, p21HrasRAF interactions are transient in comparison to p21Kras-RAF, which are more stable. Moreover, only the ER-associated p21Hras can activate the RAF1-ERK signaling. The second best-studied RAS effector pathway is that of phosphoinositide 3-kinases (PI3Ks). The pathways involving PI3Ks play important roles in RAS-mediated cell survival and proliferation. When activated, PI3K converts phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3). In turn, PIP3 binds the pleckstrin homology (PH) domain of Akt/PKB, stimulating its kinase activity. This results in phosphorylation of a series of other proteins that affect cell growth, cell cycle entry, and cell survival. PI3K also activates Rac, which is involved in cytoskeleton

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reorganization. Additionally, it can suppress the RAF/MEK/ERK one. The PI3Ks-mediated pathways are more robustly activated by p21Hras in contrast to p21Kras. Another representative, RAS effector pathway, involves Ral-GEF proteins. Ral-GEF members (RalGDS, RGL, RGL2, and RGL3) link RAS proteins to activation of the RalA and RalB small GTPases. The biological function of these proteins is not yet fully understood. An additional ability of p21Hras is to induce activation of NF-kB. Similar ability possesses p21Kras but not p21Nras. An increasing number of molecules that specifically interact with RAS have been described, including Tiam1, p120GAP, NF1, MEKK1, Rin1, AF-6, PKC-z, Nore1, Canoe, and others. Finally, it has been shown that cyclopentenone 15-deoxy-D12,14-prostaglandin J2 can activate p21Hras, but not p21Kras or p21Nras. In a different approach, HRAS null fibroblasts were shown to exhibit a specific transcriptomic profile in comparison to that of NRAS. Specifically, its absence affects mid-G1 progression, relating lack of HRAS to impairment of transcriptional programs driving processes of cell growth and proliferation. Tissue Expression of p21Hras Protein Although all three RAS genes are ubiquitously expressed in mammalian tissues, the quantitative ratios among them vary widely. Expression of each RAS isoform is dependent on the cell lineage, tissue, and developmental stage. During mouse development, expression of HRAS is stable from day 10 up to day 19 of gestation. In contrast, NRAS and KRAS expression levels are highest at day 10 and decrease toward the end of gestation. Analyses per various organs showed that during the postnatal development, HRAS transcript expression displayed a slight increase in the gut, kidney, and testis, with levels in brain being the highest up to the adulthood. In adult mice, the levels of HRAS transcripts are high in the brain, muscle, and skin, while the lowest are in the liver, spleen, thymus, and ovary. On the other hand, KRAS and NRAS exhibit different patterns of expression.

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The high levels of HRAS in the brain and muscle (skeletal and heart) of adult animals, where cell division is minimal, support the notion that this gene is involved in maintaining cell differentiation in these organs. Generation and analysis of single or multiple RAS gene knockout animals revealed that only KRAS4B (a KRAS isoform with alternative exon IV spliced in) is necessary and sufficient for the development of mice to the adult stage. HRAS, KRAS4A, or NRAS null animals show normal growth rates and are viable and fertile, with no obvious phenotypic abnormalities. Also double HRAS/NRAS knockouts are viable, exhibiting normal growth. Interestingly, lower than expected, according to Mendelian ratios, numbers of adult, double knockout animals were obtained. HRAS and Cancer Point mutations represent the most frequent mechanism of oncogenic activation of the RAS genes. Mutations detected in tumors involve codons 12, 13, 59, and 61. They affect the interaction of RAS with guanine nucleotide. Specifically, their presence inhibits GTP hydrolysis, either by diminishing GTPase activity or, for codon 59, by modulating the rate of guanine nucleotide exchange. In the HRAS gene, they are frequently detected at condon 12 (54%), followed by codon 61 (34.5%) and codon 13 (9%). In about 30% of all human tumors, RAS genes carry point mutations. They predominantly occur in the KRAS gene (25–30%). Comparatively, their frequency in the NRAS and HRAS genes is lower (8% and 3%, respectively). In human tumors, HRAS gene point mutations have been reported (http://sanger.ac.uk/genetics/CGP/cos mic/) in bladder, stomach, testis, thyroid, breast, cervix, skin, lung, and prostate carcinomas, as well as soft tissue sarcomas (malignant fibrous histiocytoma-pleomorphic sarcoma), and certain types of lymphomas (Burkitt and Hodgkin). Nevertheless, its frequency of activation in these types of malignancies is not as high as that reported for KRAS in other types of tumors. Overall, the three RAS isoforms, when oncogenic activated, display a distinct transforming

HRAS

potential that is dependent upon cellular/tissue, species, and embryological origin. For example, in a bone marrow transduction/transplantation model, the three RAS genes have the potential to induce myeloid leukemia in mice, but with different terms of potency and resulting disease phenotype. In other cellular settings, oncogenic activated HRAS induces adipocytic or neural differentiation. Also, activated HRAS has been shown to induce differentiation in tissues of endodermal origin, providing a possible explanation for the absence of mutations in this gene in tumors derived from this embryonic layer. This is in contrast with its ability to promote tumorigenic transformation in tissues of the bladder and salivary glands, as these two tissue types arise from the transitional zone, a region where the endoderm and the ectoderm meet. Other reported genetic events affecting HRAS locus involve the frequent association of hypomethylation at CCGG sites, in the 30 region surrounding the VTR, with loss of heterozygosity in non-small cell lung cancer. HRAS and Familial Syndromes Costello syndrome (CS; MIM 214080) is an autosomal dominant congenital disorder. The characteristics displayed by the CS patients involve craniofacial features, failure to thrive, developmental delay, mental retardation, cardiac and skeletal abnormalities, and a predisposition to develop neoplasia, both benign and malignant. Together with the Noonan syndrome, neurofibromatosis type 1 (NF1) syndrome, Legius syndrome, Leopard syndrome, the cardio-facio-cutaneous (CFC) syndrome, hereditary gingival fibromatosis (HGF) type 1 syndrome, autoimmune lymphoproliferative syndrome (ALPS), and capillary malformation-arteriovenous malformation (CM-AVM) are complex developmental disorders related to RAS/MAPK pathway germline mutations. The known mutations in HRAS gene related to CS are p.G12S, p.G12A, p.G12V, p.G12C, p. G12E, p.G13D, p.G13C, p.K117R, and p.A146T. Apart from the p.G12V mutation, which is also prevalent in tumors, the other mutations are more frequent in CS.

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene

HRAS and Other Pathological Conditions Altered RAS signaling has been reported to be involved in the development of several pathological conditions. Specifically, HRAS pathway activation has been associated with nonobese diabetes and diabetic retinopathy, hyperinsulinemia, glomerulonephritis, and certain types of X-linked mental retardations.

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HRT ▶ Hormone Replacement Therapy

HSD18 ▶ Methylation-Controlled J Protein

References Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y (2008) The RAS/MAPK Syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat 29:992–1006 Arozarena I, Calvo F, Crespo P (2011) Ras, an actor on many stages: posttranslational modifications, localization, and site-specified events. Genes Cancer 2:182–194 Castellano E, Santos E (2011) Functional specificity of ras isoforms: so similar but so different. Genes Cancer 2:216–231 Fernandez-Medarde A, Santos E (2011) Ras in cancer and developmental diseases. Genes Cancer 2:344–358 Kotsinas A, Gorgoulis VG, Zacharatos P, Mariatos G, Kokotas S, Liloglou T, Ikonomopoulos J, Zoumpourlis V, Kyroudi A, Field JK, Asimacopoulos PJ, Kittas C (2001) Additional characterization of a hexanucleotide polymorphic site in the first intron of human H-ras gene: comparative study of its alterations in non-small cell lung carcinomas and sporadic invasive breast carcinomas. Cancer Genet Cytogenet 126:147–154

HRAS1 ▶ HRAS

HRAS1DX ▶ HRAS

HSF ▶ Interleukin-6

HSF-III ▶ Leukemia Inhibitory Factor

hSNF5 ▶ HSNF5/INI1/SMARCB1 Tumor Suppressor Gene

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene Franck Bourdeaut1 and Paul Fréneaux2 1 Département de pédiatrie, INSERM 830, Biologie et génétique des tumeurs, Institut Curie, Paris, France 2 Département de Pathologie, Institut Curie, Paris, France

Synonyms

HRPC ▶ Hormone-Refractory Prostate Cancer

BAF47; BRG- and BRM-associated factor, 47 kDa; hSNF5; Human non-sucrose fermenting 5; SMARCB1; SWI/SNF-related, matrix-

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HSNF5/INI1/SMARCB1 Tumor Suppressor Gene

associated, actin-dependent regulator chromatin subfamily b, member 1

of

Definition hSNF5/INI1 is a member of the highly conserved SWI/SNF complex, involved in the ATP-dependent ▶ chromatin remodeling. hSNF5/INI1 acts as a ▶ tumor suppressor gene.

Characteristics The Chromatin Remodeling and the SWI/SNF Complex In the nucleus of eukaryotic cells, DNA is wrapped around histones, constituting a dense histone–DNA complex referred to as nucleosomes. The closed conformation of DNA in nucleosomes precludes the binding of transcription factors to their target promoters. By regulating the compaction degree of nucleosomes, cells can allow interactions between promoters and transcription factors. This regulation relies on two main mechanisms: (i) stable covalent modifications of histones, including acetylation, methylation, ubiquitination, etc., and (ii) ATP hydrolysis-dependent chromatin remodeling, capable to affect the spatial organization of the chromatin and the stability of nucleosomes in a dynamic way. The SWI/SNF complex, first

described in Saccharomyces cerevisiae, is the prototype of ATP-dependent chromatin-remodeling complex. Approximately 5% of the genome is affected by the SWI/SNF-dependent regulation of the transcription, either by induction or by repression (Fig. 1). The SWI/SNF complexes are conserved throughout the evolution. In humans, chromatography has led to identify two fractions of SWI/SNF complexes, PBAF or SWI/SNF-B, characterized by the presence of polybromo protein, and BAF or SWI/SNF-A, which may be the actual homolog of yeast SWI/SNF. The BAF complexes are variably composed of approximately ten subunits, in a cell-type-specific manner. However, all BAF complexes contain one of the two ySnf2 human homologs, hBRM or hBRG1 (Fig. 2). Either of them brings the ATPase activity of the complex. In vitro, hBRG1 and hBRM are sufficient to induce a chromatin remodeling. Nevertheless, the addition of other members of the core complex increases the efficiency of the remodeling. Furthermore, the integrity of the entire complex seems to be indispensable in vivo. Among the BAF proteins, at least three are ubiquitously expressed, demonstrating a critical role in the function of the complex: BAF155, BAF170, and BAF47. Altogether, these indispensable subunits constitute the core complex of SWI/SNF. The latter, BAF47, maintains the stability of the whole complex in yeasts but might not play such a role in mammals. Since Regulation of transcription

HMT

ATP

ADP+Pi

HDMT M SWI/SNF

TF

Ac

TF

HAT

HDAC

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene, Fig. 1 Chromatin remodeling and regulation of transcription. The chromatin is wrapped around histones. Acetyl or methyl radicals are added or removed by histone acetyltransferases (HAT) or histone deacetylases (HDAC) and histone methyltransferases (HMT) and histone

demethylases (HDMT), respectively. ATP hydrolysis (ATP = ADP + Pi) also participates to a direct chromatin remodeling through the ATPase activity of remodeling complexes such as SWI/SNF, regulating the accessibility of the chromatin to transcription factors (TF)

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exon 2 produces two different transcripts of 1155 and 1128 nucleotides. hSNF5 protein has strong homologies with other SWI/SNF proteins in various species (yeast Snf5, Drosophila dSNR1, Caenorhabditis elegans CeSnf5). The homology domain encompasses 193 amino acids and two highly conserved repeated sequences, Rpt1 and Rpt2 (Fig. 3b). This repeated domain brings a nuclear export signal, consistent with the subcellular localization of the protein. Rpt1 directly interacts with several partners, such as HIV IN, c-MYC, hBRM, or ALL1. A coiled-coil domain at the C-terminal extremity plays a role in the binding to interaction partners.

its interaction with the HIV1 integrase (IN) was its first role identified, it has been called “INI1.” hSNF5/INI1 Gene and Protein Structures In human, hSNF5/INI1 gene is located in 22q11.2. It is composed of nine exons and spreads over  50 kb (Fig. 3a). An alternative splicing in

SNF5/ INI1

BA F 15 5

F BA 0 17 (SNF2) BRM/BRG1

Studies of Snf5 inactivation in cell and mouse models have brought numerous insights on Snf5 biological functions. First, there are some strong evidences of Snf5 being involved in cell survival. Indeed, complete inactivation of Snf5 leads to early embryonic lethality and decreases cell survival in vitro. In MEFs, loss of Snf5 also impairs the cell cycle progression and, consistently, alters the expression of various Rb-E2f-responsive genes. Furthermore, the defect of Snf5 enhances sensitivity to DNA damage and subsequent

ADP + Pi

ATP

H

Biological Roles of hSNF5/INI1

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene, Fig. 2 The SWI/SNF complex. SWI/SNF complexes are composed of up to 12 proteins. Alternatively hBRM or hBRG1 carries the ATPase activity. The other proteins of the core complex are BAF155, BAF170, and BAF47

a 1−31

1

32−78

2

78−121

3

121−167 167−211

4

211−265

266−329

329−373

373−386

6

7

8

9

5

Exons

50 kb

b

Amino acids

DNA SNF5 homology domain

184

377 Rpt 1

Rpt 2

Coiled coil −C

N− 1

c-MYC HIV IN BRM ALL1

47 kDa

385



HSNF5/INI1/SMARCB1 Tumor Suppressor Gene, Fig. 3 hSNF5/INI1 gene and protein structure. Interacting proteins are indicated in front of the Rpt2 domain. (a)

Structure of the gene: 9 exons encompassing 50 kb. (b) Structure of the protein with the highly conserved homology domain

2132 HSNF5/INI1/SMARCB1 Tumor Suppressor Gene, Fig. 4 hSNF5/INI1 and the G1–S transition. Arrows in green and red indicate, respectively, a positive and negative regulation. Dot lines indicate not fully consensual links. SWI/SNF has a physical interaction with RB and directly coregulates E2F1 transcription. hSNF5/INI1 on its own represses the G1 to S transition. This effect can be due either to CCND1 downregulation or to p16/ INK4a overexpression. hSNF5/INI1 acts upstream RB

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene

SNF5 E2F

RB SWI/SNF CCND1

Phosphorylation

P16/ink4A

P RB

E2F CDK4/6

G1

apoptosis through a p53-dependent manner. These paradoxical roles in survival and apoptosis might be time, cell-type, and stress dependent. However, the most relevant observation is that conditional inactivation of Snf5 leads to the development of a tumor in 100% of the mice. Moreover, the short median delay of 11 weeks before tumor development is rarely observed with the inactivation of a single gene and indicates a tremendously potent tumor suppressor role for SNF5. To which extent the tumor suppressor function of SNF5 depends on SWI/SNF remains unclear. However, reexpression of hSNF5/INI1 in rhabdoid cells has demonstrated its critical role in the control of the G1–S transition. This effect could rely either on a repressive effect upon cyclin D1 expression or on a strong induction of p16/INK4a. Interestingly, a functional RB protein is required for INI1-dependent cycle control, whereas INI1 is dispensable for normal RB-mediated growth arrest (Fig. 4). Hence, INI1 definitely acts upstream RB. However, the control of the G1–S transition may not fully explain the antioncogenic role of hSNF5/INI. Indeed, missense mutations do not affect the replication checkpoint but might rather lead to polyploidy by promoting chromosomal instability and compromising the mitotic

S

checkpoint. Furthermore, there has been evidence for the involvement of hSNF5/INI1 in dynamic regulation of the cytoskeleton. The restoration of the hSNF5/INI1 gene in rhabdoid cells alters the expression of Rho-family genes, likely to modulate the cytoskeleton, and obviously modifies the organization of actin stress fibers. hSNF5/INI1 might therefore play a role in cellular adhesion and migration properties. Finally, taking into consideration the critical role of chromatin remodeling in the activation of cell-type-specific genetic programs, the role of hSNF5/INI1 in driving cell differentiation has also been investigated. INI1-dependent differentiating effect has been observed in hepatocytes. Conversely, reexpression of hSNF5/INI1 in rhabdoid cell lines drives distinct differentiation phenotypes according to the cell anatomic origin. A critical impairment of INI1-dependent differentiation programs may account for the highly undifferentiated phenotype of hSNF5/INI1deficient tumors. Genetic Alterations of hSNF5/INI1 in Human Malignancies Deletions encompassing the hSNF5/INI1 locus are encountered in many tumor types, including ▶ rhabdoid tumors (RTs), proximal epithelioid sarcomas (PES), meningiomas, and

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schwannomas. Biological effects of hemizygous INI1 deletions are questionable, since loss of one allele results only in a 15–20% reduction in total INI1 mRNA levels due to transcriptional compensation by the remaining allele. Hence, “two-hit” events only are likely to drive transformation. Strikingly, biallelic inactivation is encountered in about 80% of rhabdoid tumors. Karyotypic analyses in RTs usually show no or few other alterations than deletions or translocations of 22q11. Altogether, this indicates a very strong association between hSNF5/INI1 extinction and RTs. Both homozygous deletions and hemizygous deletions with point mutations are encountered. In RT cell lines, mitotic recombinations of chromosome 22q and nondisjunction/duplication lead to partial or complete uniparental disomy. This mechanism may account for most homozygous changes and result in the total or partial loss of one chromosome associated with the duplication of the remaining chromosome carrying the deleted or mutated allele. The association of a point mutation with a whole gene deletion is less frequently encountered but seems to be preponderant in ▶ brain tumors. Point mutations, consisting in nucleotide replacement, deletion, or insertion and leading to truncating nonsense codons, are spread all over the nine exons with

no obvious hotspot. Missense mutations are much rarely reported. Studies using immunohistochemistry with anti-SMARCB1 antibody have confirmed the total loss of protein expression in a high proportion of RTs as a direct consequence of the genetic alterations. Whether hSNF5/INI1 inactivation is specific for RT or could be involved in miscellaneous other tumors remains not fully consensual (see next section). Congenital multifocal rhabdoid tumors and familial cases strongly supported the existence of constitutional mutations in a predisposing syndrome. Indeed, about 40 germline mutations have been reported so far, associated to the development of a tumor during early childhood in almost all patients. Hence, the penetrance is very high, though not full. Nucleotides or whole gene deletions, point mutations, and splice site changes have all been observed at the constitutional level.

HSNF5/INI1/SMARCB1 Tumor Suppressor Gene, Fig. 5 Pathological aspects of hSNF5/INI-deficient ▶ rhabdoid tumor. (a) “Rhabdoid” cells: discohesive polygonal or round cells with abundant cytoplasm, juxtanuclear globular eosinophilic cytoplasmic inclusion,

and large nucleus with one prominent nucleolus. (b) Negative staining of rhabdoid cells with anti-hSNF5/INI1 antibody, resulting from a complete loss of the protein and the biallelic inactivation of hSNF5/INI1 gene. Positive staining is retained in normal stromal cells

Pathological and Clinical Aspects of hSNF5/ INI-Deficient Tumors RTs were initially described as highly aggressive variants of Wilms tumors occurring in infants (▶ Nephroblastoma). “Rhabdoid” cells were defined by key morphologic histological and immunophenotypical features: polygonal or

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round shape with abundant cytoplasm, juxtanuclear globular eosinophilic cytoplasmic inclusion, large nucleus with one prominent nucleolus (Fig. 5a), coexpression of vimentin, and epithelial markers such as epithelial membrane antigen and/or cytokeratins. Such features were then observed in other pediatric malignancies, in the brain (atypical teratoid/rhabdoid tumors) (brain tumors) and miscellaneous soft tissues. Presently, the cell origin of RTs remains unknown. The actual incidence of RTs may still be underestimated since those rare tumors are frequently misdiagnosed. However, immunohistochemistry with a monoclonal anti-hSNF5/INI1 antibody is very sensitive and highly specific for the detection of hSNF5/INI1 loss of function (Fig. 5b) and should now facilitate the diagnosis of most RTs. hSNF5/INI1 biallelic genetic inactivation has also been reported in other types of ▶ childhood cancers such as central PNET and choroid plexus carcinomas and in a small subset of undifferentiated malignant tumors without evidence of “rhabdoid” morphological features. In adults interestingly, in which RTs are thought to be much rarer, hSNF5/INI1 complete defect has also been observed in some “proximal type” of epithelioid sarcomas (proximal-type epithelioid sarcomas) and, marginally, in composite tumors with “rhabdoid” component. hSNF5/INI1 deficiency might therefore account for a wider spectrum of tumors. Nevertheless, it still remains unclear whether these different malignancies should be considered as phenotypical variants of a same biological entity. In accordance with this hypothesis, they usually share with typical RTs a highly aggressive clinical behavior.

diagnostic tools, based on molecular screening and specific anti-hSNF5/INI1 immunohistochemistry. Better knowledge on hSNF5/INI1 roles in oncogenesis should lead to more efficient targeted therapies and improve the dramatically poor prognosis of hSNF5/INI1-deficient tumors.

Conclusion There are increasing evidences of chromatin remodeling being a critical process in oncogenesis. hSNF5/INI1 offers a convincing example as a tremendously potent tumor suppressor gene. In humans, hSNF5/INI1 inactivation is strongly associated with rhabdoid tumors, but may not be as restricted to these children malignancies as previously thought. Identification of hSNF5/INI1 involvement in human cancers has given helpful

Definition

References Imbalanzo AN, Jones SN (2005) Snf5 tumor suppressor couples chromatin remodelling, checkpoint control, and chromosomal stability. Cancer Cell 7:294–295 Robert CW, Leroux MM, Fleming MD et al (2002) Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer Cell 2:415–425 Roberts CW, Orskin SH (2004) The SWI/SNF complexchromatin and cancer. Nat Rev Cancer 4:133–142 Sevenet N, Lellouch-Tubiana A, Schofield D et al (1999) Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations. Hum Mol Genet 8:2359–2368 Versteeg I, Sevenet N, Lange J et al (1998) Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394:203–206

Hsp90 Philip J. Tofilon Radiation Oncology Branch, National Cancer Institute, Bethesda, MD, USA

Synonyms Heat-shock protein 90

Hsp90, the 90-kDa heat-shock protein, is a ▶ molecular chaperone that regulates the degradation, folding, and/or transport of a diverse set of critical cellular regulatory proteins. Most Hsp90 The entry “Hsp90” appears under the copyright SpringerVerlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

Hsp90

clients, i.e., those proteins that require its “chaperoning” activity for appropriate function, participate in some aspect of signal transduction including a wide variety of protein kinases, hormone receptors, and transcription factors. Moreover, Hsp90 can stabilize mutated proteins allowing them to maintain normal function despite genetic abnormalities. This ability to buffer genetic changes and serve as a capacitor of phenotypic variation has implicated Hsp90 in evolutionary and oncogenic processes.

Characteristics Hsp90 is an ATP-dependent molecular chaperone and is one of the most highly expressed proteins in eukaryotic cells. There are two major isoforms of Hsp90: the major form Hsp90a and the minor form Hsp90b. The two hsp90 genes differ primarily in their noncoding and regulatory regions. Both the a and b proteins consist of four basic structural domains. The N-terminal domain is the site of ATP binding and is essential for its chaperone function. Inhibitory agents, at least those developed to date, specifically target this site preventing ATP binding and consequently Hsp90 function. The N-terminal domain is connected to the middle domain by a small highly charged linker region that is thought to be the site for co-chaperone binding. The larger middle domain contains the site for client protein binding, and the C-terminal domain provides the dimerization site, which is essential for Hsp90 activity. The Hsp90 proteins function as homodimers of a/a and b/b. Whereas there does appear to be functional differences between the isoforms with b being associated with development, there is also considerable overlap. Although the majority of Hsp90 is located in the cytoplasm, this molecular chaperone can also be found in the nucleus, albeit at considerably lower levels, where it has been associated with the regulation of gene expression and the ▶ DNA damage response to radiation. In addition, Hsp90 is located on the cell surface where it plays a role in antigen processing and the immune response. Finally, studies have shown that Hsp90 can be

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secreted into the extracellular space. Although the specific function has not been clearly defined, in this location Hsp90 has been suggested to play a role in blood clotting, cell migration, and tumor metastatic processes. Mechanism Hsp90 chaperone function is mediated by an ATP-dependent cycling between two conformations, which regulates its interactions with specific co-chaperones and cofactors and drives the loading and off-loading of client proteins. The Hsp90 dimer typically exists in a multi-protein chaperone complex that includes the co-chaperones Hsp70 (70-kDa heat-shock protein) and Hsp40 (40-kDa heat-shock protein), the adaptor protein HOP (hsp co-chaperone organizing protein), and the cofactor p23. In addition, there are specific immunophilins and other co-chaperones that regulate substrate binding. For example, the co-chaperone Cdc37 is involved in Hsp90mediated stabilization of protein kinases. A client protein initially binds to the Hsp70/ Hsp40 complex, which links via HOP to an ADP-bound Hsp90. Exchanging ADP with ATP alters Hsp90 confirmation such that HOP and Hsp70/Hsp40 are released and p23 and other co-chaperones such as Cdc37 are recruited to the complex. In the ATP-bound confirmation and associated with these co-chaperones, Hsp90 folds and stabilizes a client protein, maintaining it in a responsive conformation. Although the specific processes have not been completely defined, in the absence of the appropriate stimulus or ligand binding to the client protein, Hsp90 through its ATPase activity cycles back to its ADP-bound conformation, recruiting the initial set of co-chaperones, which ultimately leads to client protein degradation. Considerable insight into the mechanism of Hsp90 chaperone activity has been generated through the use of the inhibitor geldanamycin. This benzoquinone ansamycin binds to the nucleotide binding site of Hsp90, resulting in a conformation that resembles the ADP-bound conformation. The inability of Hsp90 to cycle to its ATP-bound conformation then maintains the chaperone complex in a state that favors client protein degradation. Studies to

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date indicate that in cells treated with geldanamycin or one of its analogs, the half lives of the Hsp90 client proteins are uniformly and significantly reduced. Clinical Aspects As a Single Modality

Hsp90 has received considerable attention as a potential target for cancer therapy. Because of an increased understanding of the mechanisms and molecules that mediate malignant transformation, strategies in cancer therapy have begun to focus on a target-based approach. The putative advantages of this strategy include tumor selectivity sparing normal cells and the availability of markers indicative of tumor susceptibility. Most attempts to apply target-based therapy have entailed the use of agents that target a single molecule. For tumors in which their phenotype is driven by a single oncogenic molecule, such as the targeting of Bcr-Abl by ▶ STI571 in chronic myelogenous leukemia and ▶ HER-2/neu by ▶ Herceptin in certain breast tumors, this approach has demonstrated clinical activity. However, most tumors, especially solid neoplasms, contain multiple genetic abnormalities and are subject to a high degree of genomic instability. The result is that their malignancy/survival is driven by a variety of molecules existing in a number of different survival pathways. Under these circumstances, targeting a single molecule is likely to be of limited consequence. As an alternative to targeting a single molecule, inhibiting Hsp90 provides a multi-target approach to cancer treatment. Hsp90 client proteins include a large number of kinases, receptors, and transcription factors that have been implicated in transformation and maintenance of the malignant phenotype. Examples of such proteins dependent on Hsp90 include ErbB2 (▶ HER-2/neu), Src, Akt, c-Raf-1, cyclin-dependent kinases Cdk4 and Cdk6, HIF1-a, estrogen and androgen receptors, and hTERT. Thus, Hsp90 inhibition provides a means of simultaneously targeting multiple proteins critical to a malignant cell. In addition,

Hsp90

Hsp90 can stabilize mutant proteins (e.g., ▶ p53), allowing for at least some functional activity. Given the level of genomic abnormalities and instability of tumor cells, the ability to buffer against such genetic variation suggests another avenue through which Hsp90 contributes to tumor cell survival. In laboratory studies exposure of tumor cells to Hsp90 inhibitors such as geldanamycin and its analogs 17-allylaminogeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)17-demethoxygeldanamycin (17-DMAG) results in the combinatorial decrease in its client proteins mentioned above as well as additional client proteins, although there is some cell-type specificity. With respect to a potential cancer therapy, inhibition of Hsp90 induces tumor cell death or significantly slows their proliferation in a number of in vitro and in vivo experimental systems, although the critical target or targets (i.e., client proteins) for the most part have not been defined. An additional characteristic that supports the application of Hsp90 inhibitors in cancer treatment is their relative selectivity for tumor cells over normal cells. The molecular basis for this selectivity has not been clearly defined. However, Hsp90 is typically expressed at higher levels in tumor cells than normal cells suggesting that tumors may be more dependent on its chaperoning activity. Regardless of the specific mechanisms involved, the ability of Hsp90 inhibitors to preferentially kill tumor cells over normal has led to the ongoing evaluation of 17AAG and 17DMAG in clinical trials as singlemodality agents. In Combination with Radiotherapy

Hsp90 has also been identified as a determinant of tumor cell radiosensitivity. Among Hsp90 clients are included a number of proteins (e.g., Raf-1, Akt, and ErbB2/Her2/Neu) that have been associated with protection against radiation-induced cell death; a reduction in their individual activities has been shown to result in radiosensitization in some but not all tumor cell lines. Tumor cell radiosensitivity is regulated by a wide variety of signaling molecules with their specific contributions often

Hsp90

determined by cell type. Because Hsp90 inhibitors induce the simultaneous loss of a number of these molecules that can potentially affect radiosensitivity, the use of these agents allows for implementing a multi-target approach to ▶ radiosensitization. The putative advantages of such a multi-target strategy are increases in the degree and probability of radiosensitization. Indeed, inhibitors of Hsp90 such as geldanamycin, 17AAG, 17DMAG, and radicicol have been shown to significantly enhance the radiosensitivity of a number of tumor cell lines derived from a variety of histologies. As for Hsp90 inhibitor treatment alone, the inhibitors have little to no effect on the radiosensitivity of normal cells evaluated in vitro. This lack of normal cell radiosensitization occurs despite a similar reduction in client proteins suggesting that it is not the difference between Hsp90 per se in tumor and normal cells but the actions of its client proteins. Mechanistic studies of the tumor cell radiosensitization induced by 17DMAG have implicated Hsp90 in two components of the DNA damage response – DNA double-strand break repair and ▶ cell cycle checkpoint activation. The inhibition of double-strand break repair could be traced to the loss of ErbB2 (Her2/neu) in 17DMAG-treated cells and the consequent reduction in ErbB1 (EGFR) activity, which leads to a reduction in the ErbB1 interaction with DNA-PKcs and the subsequent attenuation of DNA-PK activation after irradiation. The abrogation of ▶ cell cycle checkpoint activation by 17DMAG was associated with a reduction in radiation-induced activation of ▶ ATM, which was the result of a reduced interaction between NBS1 and ATM. Whereas most studies regarding Hsp90 as a target for cancer treatment have focused on its cytoplasmic activities, these radiation studies indicate that this chaperone has a critical role within the nucleus. The contribution of Hsp90 to double-strand break repair and cell cycle checkpoint activation in tumor cells suggests that its inhibition may also be of benefit in combination with chemotherapeutic agents that kill tumor cells through DNA damage.

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Cross-References ▶ Cell Cycle Checkpoint ▶ DNA Damage Response ▶ HER-2/neu ▶ Herceptin ▶ Molecular Chaperones ▶ Radiosensitization ▶ Raf Kinase ▶ STI-571

References Dote H, Burgan WJ, Camphausen K et al (2006) Inhibition of Hsp90 compromises the DNA damage response to radiation. Cancer 66:9211–9220 Eustace BK, Jay DG (2004) Extracellular roles for the molecular chaperone, hsp90. Cell Cycle 3:1098–1100 Sangster TA, Lindquist S, Queitsch C (2004) Undercover: causes, effects and implications of Hsp90-mediated genetic capacitance. Bioessays 26:348–362 Sharp S, Workman P (2006) Inhibitors of the Hsp90 molecular chaperone: current status. Adv Cancer Res 95:323–348 Sreedhar AS, Kalmar E, Csermely P et al (2004) Hsp90 isoforms: functions, expression and clinical importance. FEBS Lett 562:11–15

See Also (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115X. doi:10.1007/978-3-642-16483-5_163 (2012) CDC37. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 704. doi:10.1007/978-3-642-16483-5_954 (2012) DNA-PK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1147. doi:10.1007/978-3-642-164835_1686 (2012) Geldanamycin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1516– 1517. doi:10.1007/978-3-642-16483-5_2357 (2012) NBS1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2468. doi:10.1007/978-3-642-16483-5_3984 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Radiosensitivity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3154. doi:10.1007/978-3-642-16483-5_4923

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HSV-TK-/Ganciclovir-Mediated Toxicity Donna Shewach Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

Synonyms GDEPT; Gene-directed enzyme prodrug therapy

Definition A form of ▶ suicide gene therapy in which the cDNA for a viral enzyme, the herpes simplex virus thymidine kinase (HSV-TK), is transferred to tumor cells followed by treatment with the antiviral drug ganciclovir (GCV). Expression of HSV-TK enables cells to phosphorylate GCV to a monophosphate derivative. Cellular enzymes convert the monophosphate to GCV triphosphate, which elicits toxicity through incorporation into DNA.

Characteristics HSV-TK-/GCV-mediated killing of tumor cells, and indeed suicide gene therapy in general, has been developed as a mechanism to improve the selectivity of cancer chemotherapy. Since traditional antitumor drugs target rapidly dividing tissues, such as tumor cells, they also can destroy normal dividing host tissue such as cells in the bone marrow, gastrointestinal tract, or hair follicles. Normal tissue toxicity is the major impediment to traditional chemotherapy. With HSV-TK/GCV therapy, a foreign gene (encoding HSV-TK) which can activate a normally innocuous prodrug (GCV) to a toxic product is transferred selectively to the tumor. When the patient is treated with the prodrug, only the tumor cells expressing the foreign gene will be affected (thus the designation of “suicide” gene therapy); since normal host tissues cannot activate the prodrug, they are spared from toxicity.

HSV-TK-/Ganciclovir-Mediated Toxicity

Toxicity to HSV-TK-Expressing Cells GCV is an acyclic analog of 20 -deoxyguanosine that requires phosphorylation for biologic activity (Fig. 1). It was originally discovered as an antiherpesvirus agent, and it is used clinically in the treatment of cytomegalovirus infection. When herpes simplex virus infects human cells, a number of proteins are expressed from the viral genome to facilitate virus replication and spread. One of these proteins, HSV-TK, contrasts with mammalian thymidine kinase in that it can phosphorylate purine as well as pyrimidine nucleoside substrates and their analogs. GCV serves as a substrate for HSV-TK with a Km value of 50 mM, but it is not an efficient substrate for any of the mammalian nucleoside kinases, thus accounting for its selectivity in herpesvirusinfected cells. Following subsequent phosphorylation by mammalian kinases to its triphosphate metabolite, the drug is incorporated into the viral DNA leading to cessation of replication. Based on this mechanism of selectivity, it was proposed that tumor cells genetically engineered to express HSV-TK would be killed when treated with GCV. Proof of this principle was first shown in murine sarcoma cells, and since then numerous reports have demonstrated similar results in many different cell types. At the triphosphate level, GCV can compete with the endogenous 20 -deoxyguanosine 50 -triphosphate (dGTP) for incorporation into DNA by mammalian DNA polymerases. Since GCV has the equivalent of both the 50 and 30 OH

OH N

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HSV-TK-/Ganciclovir-Mediated Toxicity, Fig. 1 Structure of GCV

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GCV

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GCVTP dGTP Bystander cell

HSV-TK-/Ganciclovir-Mediated Toxicity, Fig. 2 Mechanism of cytotoxicity for GCV in HSV-TKexpressing cells and bystander cells. GCV is selectively phosphorylated to the monophosphate in HSV-TKexpressing cells. Further phosphorylation can be accomplished by cellular enzymes. GCV triphosphate competes

with the endogenous dGTP for incorporation into DNA, leading to cell death. GCV at the mono-, di-, or triphosphate level can be transferred directly to bystander (non-HSV-TK-expressing) cells via GJIC channels, resulting in its incorporation into DNA of bystander cells with subsequent cytotoxicity

hydroxyls of deoxyguanosine, it can be incorporated into DNA and allow further extension of the DNA strand (internucleotide addition). In contrast, the structurally related acyclovir lacks the equivalent of a 30 hydroxyl group and thus is an obligate DNA chain terminator. GCV and its metabolites do not interfere with RNA or protein synthesis. The incorporation of GCV monophosphate into DNA is the primary lesion that results in cell death. For some cell types, GCV induces cell death through ▶ apoptosis (Fig. 2). Compared to other substrates for HSV-TK, such as acyclovir, GCV is significantly more toxic and more mutagenic to cells that express HSV-TK. The action of acyclovir is primarily cytostatic, whereas GCV induces cell killing at low, clinically achievable concentrations. Although GCV triphosphate accumulates in cells to a relatively low level of 10–20 mM, this is sufficient to produce several logs of cell death. This high toxicity may be attributable to the avid incorporation and lengthy retention of GCV into DNA. GCV triphosphate and its incorporation into the nascent DNA strand do not produce strong inhibition of DNA synthesis, so cells incorporate high levels of this drug, complete DNA replication, and go on to divide. Daughter cells

become irreversibly blocked when they enter S-phase, suggesting that GCV monophosphate cannot serve as a template for DNA replication. A strong G2/M block has also been observed in some cell types after GCV treatment. The cell cycle position in which cells become blocked may depend on the concentration of GCV. Toxicity to Non-HSV-TK-Expressing Cells (▶ Bystander Effect) With current gene transfer technologies, only a small percentage of tumor cells will express the foreign gene. For this approach to be successful in cancer treatment in patients, there must be a mechanism by which cells that do not express the transgene (bystander cells) can be killed. It was noted early on that when only a fraction of the cell population expressed HSV-TK, treatment with GCV resulted in killing of both the HSV-TKexpressing and HSV-TK-nonexpressing bystander cells. The strong cell killing ability of GCV in HSV-TK-expressing and neighboring bystander cells has resulted in complete regressions of experimental tumors in animals, spurring clinical interest in this approach. In suicide gene therapy, bystander cell killing generally occurs through the transfer of a toxic

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metabolite, produced in the transgene-expressing cell, to bystander cells. The bystander cell killing with HSV-TK/GCV was an unexpected finding since the toxic metabolite, GCV triphosphate, is negatively charged and therefore would not readily pass through cell membranes to kill bystander cells. However, GCV mono-, di-, and triphosphate accumulate in bystander cells when cocultured with HSV-TK-expressing cells and GCV. The primary mechanism that appears to account for transfer of GCV phosphates is gap junctional intercellular communication (GJIC). GJIC allows the direct exchange of small molecules (90%), although its function in the nucleus has not been well characterized. Studies implicate a role in pre-mRNA processing in the nucleus (e.g., RNA splicing and polyadenylation). However, the best characterized functions of HuR occur in the cytoplasm in response to cellular stress. Thus, HuR-related cancer research has primarily focused on HuR-mRNA binding and translocation of HuR to the cytoplasm where it regulates its mRNA cargo in response to stress. Structurally, HuR has three RNA recognition motifs (RRMs 1–3), with 90% amino acid sequence homology with the other three Hu proteins. RRMs 1 and 2 typically recognize RNA sequences that are U or AU rich (AU-rich elements or AREs). HuR most often binds to AREs located in the 3’-untranslated regions (UTR) of transcripts and stabilizes these transcripts. HuR may also bind to sites within the coding sequence of target transcripts or at the 5’-UTR. Unlike the 3’-UTR, AREs at the 5’-UTR are often associated with increased transcript degradation and an internal ribosomal binding element. The RRM 3 region is believed to affect RNA stability through its interactions with the poly-AAA tail of transcripts. This region may also be important for proteinprotein interactions and HuR multimerization. HuR forms an import/export competent ribonucleoprotein complex with mRNA targets and adaptor proteins. In many instances, a hinge region (called HNS or HuR nucleocytoplasmic shuttling sequence), spanning about 30 amino acids and located between RRMs 2 and 3 on the HuR protein, is central to this process. The HuR-associated complex moves as a unit between the nucleus and

HuR

cytoplasm (or vice versa) through the nuclear pore complex. Transportin 2 is an example of an import factor that binds directly to the HuR HNS sequence in the hinge region and facilitates HuR movement back into the nucleus. A parallel import mechanism involves a separate adaptor protein, importin-a1, but is independent of the HNS nuclear localization sequence. Importin-a1 is phosphorylated and activated by AMPK in response to stress and facilitates HuR reuptake into the nucleus. An HNS-independent export mechanism has also been described and involves two phosphoprotein ligands of HuR: pp32 and APRIL. Both adaptor proteins contain leucine-rich nuclear export signals (NES) that interact with the nuclear export receptor, CRM1. HuR nucleocytoplasmic shuttling is usually dependent on Ran (a GTP-binding protein with GTPase activity). The GTP/GDP gradient between the nucleus (high) and cytoplasm (low) determines the directional flow of HuR-associated exportins and importins through the nuclear pore complex. HuR Cancer-Associated Targets (Pro-survival Network) Computational analyses of mRNA sequencing data reveal that roughly 5–8% of coding transcripts have AREs and are potential targets of HuR. This has been validated experimentally through HuR immunoprecipitation-based studies. While high-throughput assays such as ribonucleoprotein immunoprecipitation (RIP-Chip) and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CliP) have identified thousands of potential mRNA targets, dozens of HuR targets have been characterized in greater detail in cancers through focused expression studies of targets (such as RT-qPCR or immunoblot assays). Thorough investigations of HuR targets usually detail HuR binding sites and describe the functional impact of HuR regulation in the setting of a particular stress. An abbreviated list of validated HuR targets highlights a number of key oncogenes and tumor suppressor genes under HuR control and offers empiric evidence of HuR’s pro-survival influence in cancer: cyclin A, cyclin B1, cyclin D1, cyclin E1, FOS, MYC, DCK, DR5, ERBB2, SUMO1, WEE1,

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HuR, Fig. 1 HuR regulates pathways involved in tumorigenesis and drug responsiveness. Light blue depicts the cytoplasm of a cancer cell where HuR is “activated” and regulates tumor proliferation pathways

BRCA1, KRAS, VEGF, MMP9, SNAI1 (SNAIL), VHL, HIF1A, BCL2, SIRT1, TP53, and MAP2K1 (MEK1). It is possible that cancer cells with intact HuR may not need to select for conventional genetic alterations in certain tumorpromoting genes since HuR may already provide a selective pressure of dysregulation. The accompanying figure highlights some pro-survival pathways that are regulated by HuR through its interactions with key genes (Fig. 1). Other gene clusters (i.e., regulons) important in cancer which are regulated by HuR include inflammatory genes (COX2, TNF-a, IL8) and metabolic genes (GLUT1, HIF1A, SLC7A1 (CAT1)). As further evidence of HuR’s importance in regulating cancer genes, 540 cancer genes that are overexpressed in pancreatic cancer relative to normal pancreatic cells were interrogated as potential HuR targets using both bioinformatics and an experimental approach. Somatic mutations and epigenetics accounted for oncogenic transformation in 1% of these genes, while over 11% were validated as HuR targets, suggesting that HuR may be underappreciated as a key regulator of tumor-promoting pathways. HuR-Mediated Response to Stress and Cancer Therapy In basic terms, a primary function of HuR in cancer is to organize an adaptive response to

acute stress. As previously stated, HuR is able to rapidly induce global changes to the proteome by stabilizing target transcripts and facilitating protein translation and therefore serves as a powerful tool used by cancer cells to adapt to the dynamic tumor microenvironment. Upon acute stress, three key steps occur that define the “activated” HuR state: HuR (1) binds to targets, (2) translocates to the cytoplasm, and (3) regulates RNA stability and/or translation of its targets. Several environmental or metabolic stressors have previously been shown to activate HuR in this way, including amino acid deprivation, glucose deprivation, hydrogen peroxide, ultraviolet light, nitric oxide, and heat shock. Counterintuitively, HuR reacts differently to radiation exposure; rather than translocate to the cytoplasm, HuR responds by dissociating from its targets in a CHK2-dependent manner. However, similar to responses to other stressors, transcript dissociation is believed to provide a survival advantage through downstream changes to the cell’s proteome. In light of HuR activation in response to various toxic stimuli, it is not surprising that certain pharmacologic stressors, including some certain chemotherapeutics, engage the HuR network. In some cases, HuR activation leads to drug resistance. For instance, agonism of the TRAIL death receptor DR5 (with an agonist monoclonal

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antibody) is the focus of multiple ongoing trials in cancer. Activation of DR5 increases cytoplasmic HuR in pancreatic cancer cells, which in turn downregulates (or destabilizes) DR5 (an HuR target). This negative feedback loop could account for TRAIL resistance observed in many clinical trials. Indeed, silencing HuR sensitizes cells to DR5 agonism and increases levels of apoptosis. Similarly, tamoxifen treatment induces HuR movement to the cytoplasm, which leads to tamoxifen resistance in breast cancer. As expected, silencing HuR increases sensitivity to tamoxifen, although the HuR targets involved in this process have not been delineated. Like DR5 and tamoxifen, calcineurin inhibitors (such as cyclosporine) activate HuR to the cytoplasm. This phenomenon is proposed to contribute to pro-tumorigenic effects of this drug, possibly through the action of HuR targets such as VEGF. While HuR contributes to drug resistance patterns, HuR can potentially be exploited in instances where activation increases drug sensitivity. Like the aforementioned drugs, gemcitabine induces HuR movement to the cytoplasm, but this action sensitizes cancer cells to gemcitabine treatment through stabilization of the deoxycytidine kinase (dCK) transcript. Under normal conditions, dCK phosphorylates deoxyribonucleosides as part of a nucleoside salvage pathway. HuR’s role in encouraging nucleotide synthesis is consistent with the RBP oncogenic tendency. However, dCK also phosphorylates the gemcitabine prodrug (an anticancer nucleoside) into active metabolites. As a result, cancers with high cytoplasmic expression are particularly responsive to gemcitabine. HuR also stabilizes topoisomerase 2A, which is a target of the commonly used cytotoxic agent, doxorubicin. One may speculate that gemcitabine and doxorubicin would be synergistic in high HuR-expressing tumors; they both induce HuR translocation to the cytoplasm and increase expression of targets required for drug sensitivity. To complicate the matters, while HuR activation paradoxically increases cancer cell sensitivity to gemcitabine, new evidence suggests other HuR targets may include gemcitabine resistance genes.

HuR

HuR, Fig. 2 Pancreatic ductal adenocarcinoma with widespread activation of cytoplasmic HuR

HuR as an Oncogene in Cancer As preclinical experiments suggest, HuR functions primarily as an oncogene in cancer (i.e., a proto-oncogene in the nucleus and an activated oncogene when translocated to the cytoplasm). It stands to reason that cancer cells with a robust HuR pro-survival network are well suited to thrive in the harsh tumor microenvironment relative to neighboring cancer cells and stroma, particularly in the face of acute stress. This concept has been validated in patient tissues, where HuR expression (principally in the cytoplasm) is associated with aggressive cancer biology and worse outcome (Fig. 2). In fact, studies of patient samples in virtually every tumor system ever examined establish HuR as a poor prognostic marker. In breast cancer, elevated cytoplasmic HuR was observed in roughly 1/3 of cases. High expression was associated with high grade, large size, and worse survival in a multivariate model. In a subgroup analysis of lymph node negative breast cancers, HuR stratified patients prognostically, with high expression tied to worse outcome. In pancreatic cancer, elevated cytoplasmic HuR was also observed in 1/3 of patients. Increased expression was associated with a higher T-stage in two independent studies. Interestingly, HuR expression was not associated with worse survival outcomes, in large part because patients receiving standard-of-care gemcitabine likely had more responsive cancers

Hurthle Cell Adenoma and Carcinoma

to adjuvant treatment. In colon cancer, cytoplasmic HuR expression was associated with worse tumor stage and grade, but not survival in a multivariate analysis. Ovarian cancers have among the highest amounts of cytoplasmic HuR expression (~50%), as observed in multiple studies, and increased expression was associated with worse survival and increased grade. In non-small cell lung cancer, cytoplasmic HuR expression was an independent predictor of survival and associated with increased angiogenesis and lymphangiogenesis. Prognostic studies of HuR in multiple tumor systems have evaluated COX2 expression, which is an established HuR target. An association between cytoplasmic HuR expression with increased COX2 expression and worse survival was observed in ovarian cancer, gastric cancer, mesothelioma, prostate cancer, head and neck cancer, and renal cell carcinoma. While HuR was not prognostic in colon cancer, there was a strong association with COX2 expression. Conclusions The RNA-binding protein, HuR, is ubiquitously expressed in normal tissues and exploited by cancers to orchestrate a pro-survival network, particularly in response to acute stress. Future studies will identify key regulated targets under different stressors (e.g., nutrient deprivation, hypoxia, chemotherapy, radiation, etc.). This line of research will provide novel molecular insights into the acute stress response by cancer cells and mechanisms of drug resistance. Based on these studies, novel therapeutic targets under these conditions may be identified.

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Hurthle Cell Adenoma and Carcinoma Ronald A. Ghossein Department of Pathology, Memorial SloanKettering Cancer Center, New York, NY, USA

Synonyms Follicular adenoma, oncocytic variant; Follicular carcinoma, oncocytic variant

Definition Hurthle cell adenomas and carcinomas are, respectively, benign and malignant epithelial tumors arising from the follicular epithelium of the thyroid gland, containing at least 75% of oncocytes and lacking the characteristic nuclear features of papillary thyroid carcinoma (i.e., irregular clear nuclei with grooves, overlapping and pseudo-inclusions). The oncocytes in Hurthle cell adenomas and carcinomas are characterized by an eosinophilic granular cytoplasm on hematoxylin and eosin sections with a vesicular nucleus and a large centrally located nucleolus. The oncocytic nature of the cytoplasm is due in the vast majority of the cases to its high content in mitochondria. Max Askanazy, a German pathologist (1865–1940), was the first to describe the follicular oncocytic cells of the thyroid in 1898 also known as Hurthle cells.

References

Characteristics

Abdelmohsen K, Gorospe M (2010) Posttranscriptional regulation of cancer traits by HuR. Wiley Interdiscip Rev RNA 1(2):214–229 Brody JR, Gonye GE (2011) HuR’s role in gemcitabine efficacy: an exception or opportunity? Wiley Interdiscip Rev RNA 2(3):435–444 Lopez de Silanes I, Lal A, Gorospe M (2005) HuR: posttranscriptional paths to malignancy. RNA Biol 2(1):11–13

Clinical and Histopathologic Characteristics. Hurthle cell carcinomas are a rare tumor accounting for only 3–4% of the 33,550 newly and yearly diagnosed thyroid malignancies in the United States. The median age at diagnosis for Hurthle cell carcinomas is 61 years, and the female to male ratio is 2:1 while it is 8:1 for

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Hurthle cell adenomas. The exact incidence of Hurthle cell adenoma is unknown but probably much higher than the incidence of its malignant counterpart. Most patients with Hurthle cell adenoma and carcinoma present with a painless thyroid mass. Patients with carcinoma tend to present at an older age and usually have larger tumors than adenoma patients. Rarely patients with Hurthle cell carcinoma present with distant metastases usually to lung and bone. Hurthle cell carcinomas should be separated from Hurthle cell adenoma on the basis of presence or absence of invasion of the tumor capsule (capsular invasion) as well as presence or absence of vascular invasion. The latter is defined as invasion of a vessel within or beyond the tumor capsule in the surrounding thyroid or extra-thyroid tissue. Hurthle cell adenoma and carcinoma cannot be separated on the basis of growth pattern whether follicular (i.e., making glands) or solid. The size of the tumor cell and its nuclear-cytoplasmic ratio cannot also be used to diagnose malignancy. Therefore, the differentiation between benign and malignant Hurthle cell tumors is impossible in smears from fine needle aspiration since it is not possible to assess capsular or vascular invasion in cytology specimens. The diagnosis of malignancy can be a very difficult exercise in tissue sections, especially after fine needle aspiration. In addition to causing infarction, the needle biopsy can lead to artifactual irregularities of the tumor capsule, rendering the evaluation of capsular and vascular invasion very difficult. Hurthle cell carcinomas should also be differentiated from the tall cell variant of papillary carcinoma and oncocytic variant of papillary carcinoma. These two entities share with Hurthle cell carcinoma the presence of an oncocytic cytoplasm but differ from it by displaying the nuclear features of papillary carcinoma (i.e., irregular clear nuclei with grooves, pseudo-inclusions, and nucleoli apposed to the nuclear membrane). Hurthle cell carcinomas are divided into minimally invasive and widely invasive tumors. The minimally invasive carcinoma, also termed encapsulated, is totally surrounded by a fibrous capsule and displays capsular invasion and/or foci of vascular invasion. It is unusual for these foci of invasion to be detected grossly. In contrast, widely

Hurthle Cell Adenoma and Carcinoma

invasive Hurthle cell carcinoma is defined by extensive areas of invasion at both the macroscopic and microscopic levels, and the capsule remnant is often difficult to identify. This classification correlates very well with outcome, such that minimally invasive tumors have an overall excellent prognosis, whereas widely invasive tumors have a much poorer outcome with a 55% mortality rate according to one series. However, there are cases of encapsulated minimally invasive follicular carcinoma that recur and metastasize. Identifying these cases at the time of diagnosis is crucial because a minimally invasive tumor will be treated by lobectomy alone followed by observation in some centers. This approach will risk undertreating those few minimally invasive tumors with poor outcome. At variance with the minimalist surgical approach, many surgeons will perform a total thyroidectomy followed by radioactive iodine therapy on any minimally invasive Hurthle cell carcinoma, most likely overtreating a large number of cases. In view of the limitations of the above subclassification, many authors have attempted to revise this classification. Indeed, some researchers will exclude from the minimally invasive category any tumor with vascular invasion, even if microscopic, while we will exclude from the minimally invasive group any tumor with 4 foci of microscopic vascular invasion since the latter has a significant recurrence rate. Within widely invasive Hurthle cell carcinoma, adverse predictors of survival are extrathyroidal extension, nodal metastasis, positive margin, and a solid growth pattern. On multivariate analysis, extrathyroidal extension and nodal metastases are independent predictors of outcome within widely invasive Hurthle cell carcinoma, present in 59 and 21% of cases, respectively. Immunohistochemistry. Hurthle cell tumors are typically positive for the thyroid markers thyroid transcription factor-1 (TTF-1) and thyroglobulin. In carcinomas with a solid growth pattern, thyroglobulin positivity can be very focal. Various cell cycle proteins have been used to differentiate between benign and malignant Hurthle cell tumors and stratify patients with Hurthle cell

Hurthle Cell Adenoma and Carcinoma

carcinoma. Only Ki67 was found to be significantly elevated in widely invasive Hurthle cell carcinoma in comparison to Hurthle cell adenoma and minimally invasive Hurthle cell carcinoma. But this marker is not an independent prognostic marker since it correlates very well with the extent of vascular invasion and the presence of extrathyroid extension. Genetics. Hurthle cell tumor frequently shows chromosomal DNA imbalance. Somatic mutations have been identified in the mitochondrial DNA of Hurthle cell carcinoma. These findings have no clinical use in these tumors. Imaging. It is important to note that the followup imaging of Hurthle cell carcinomas include radioactive iodine scans and positron emission tomography (PET). Hurthle cell adenomas can also be positive by both modalities. This is important to know since PET performed for non-thyroid malignancies (like melanoma) can lead to the discovery of Hurthle cell tumors whether benign or malignant. These are referred to as PET incidentalomas. The presence of PET incidentalomas should lead to a thyroid nodule workup. Treatment. All authors agree that non-minimally invasive Hurthle cell carcinoma should be treated by total thyroidectomy followed by radioactive iodine administration. For minimally invasive carcinomas, there is disagreement between authors. While some recommended full therapy for these patients (similar to widely invasive tumors), we believe minimally invasive tumors should be treated by thyroid lobectomy alone as long as they display no more than two foci of capsular and/or vascular invasion. All authors agree that lobectomy is sufficient for the treatment of Hurthle cell adenoma. Current and Past Controversies Hurthle cell tumors have generated a large number of controversies since their original description. One controversy regards the cell type. While most physicians, especially in North America, go by the term Hurthle cell, it is a misnomer. It is Askanazy and not Hurthle who provided the description of these follicular oncocytic cells. A much more debated issue is whether Hurthle cell carcinoma

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is a distinct tumor entity. Some authors believe this tumor should be separated from conventional non-oncocytic follicular carcinoma. Indeed, compared to non-oncocytic follicular carcinoma, Hurthle cell malignancies generally take up less radioactive iodine and have a higher frequency of extra-thyroid extension, local recurrence, and nodal metastases. Hurthle cell carcinomas are overall more aggressive tumors. Since many of the differences are not evident when patients are stratified by extent of invasion, other authors believe Hurthle cell carcinoma is simply a morphologic variant of follicular carcinoma. This is the official position emanating from the last World Health Organization classification of endocrine tumors. Another controversy arose in the 1970s when some authors suggested that all Hurthle cell tumors are malignant. This view is no longer accepted and all current thyroid pathologists believe it is possible to differentiate benign from malignant Hurthle cell tumors on histological grounds. As mentioned above, there is still controversy as to what constitutes a minimally invasive tumor and there is still a debate regarding the optimal therapy for minimally invasive Hurthle cell carcinomas.

References Chan JK (2000) Tumors of the thyroid and parathyroid glands. Part A. The thyroid gland. In: Fletcher CDM (ed) Diagnostic histopathology of tumors, vol 2, 2nd edn. Churchill-Livingstone, London, pp 986–988 Ghossein RA, Hiltzik DH, Carlson DL et al (2006) Prognostic factors of recurrence in encapsulated Hurthle cell carcinoma of the thyroid gland. A clinicopathologic study of 50 cases. Cancer 106:1669–1676 Rosai J, Caracangui M, DeLellis R (1992) Tumors of the thyroid gland. In: Rosai J, Sobin L (eds) Atlas of tumor pathology (series 3), Fascicle. Armed Forces Institute of Pathology, Washington, DC, pp 161–182 Sobrinho-Simoes M, Asa SL, Kroll TG et al (2004) Follicular carcinoma. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C (eds) Pathology and genetics tumours of endocrine organs. International Agency for Cancer, Lyon, pp 67–72 Stojadinovic A, Ghossein R, Hoos A et al (2001) Hürthle cell carcinoma: a critical histopathological appraisal. J Clin Oncol 19:2616–2625

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Hutchinson–Weber–Peutz Syndrome

Characteristics

Hutchinson–Weber–Peutz Syndrome ▶ Peutz–Jeghers Syndrome

HXK2 ▶ Hexokinase 2

Hyaluronan Receptor ▶ CD44

Hyaluronan Synthases Natalie Thomas1 and Vera Evtimov2 1 Clinical Network Services Pty Ltd, St Albans, UK 2 Monash University, Melbourne, VIC, Australia

Synonyms Hyaluronic acid (HA); Hyaluronic acid synthases (HASs)

Definition Hyaluronan synthases (HASs) are multi-isoform transmembrane proteins which catalyze the synthesis of the high molecular weight carbohydrate, hyaluronan (HA). High expression of hyaluronan synthases in a neoplasm or associated peritumoral stroma is often a poor prognostic indicator in cancer. Hyaluronan is a ubiquitous component of the extracellular matrix where it has various roles in matrix hydration, promotion of cellular growth and proliferation, induction of malignant transformation, and invasion and metastasis. The entry “Hyaluronan Synthases” appears under the copyright Crown Copyright both in the print and the online version of this Encyclopedia.

Hyaluronan Synthases and Hyaluronan Hyaluronan synthases (HAS1, HAS2, HAS3) are integral plasma membrane proteins that utilize UDP-glucuronic acid and UDP-N-acetylglucosamine substrates to alternately add monosaccharide units to the reducing end of a growing chain of hyaluronan (HA). Three isoenzymes (designated HAS1, HAS2, and HAS3) are localized to separate chromosomes and exhibit varying enzymatic properties despite having relatively high amino acid sequence homology. Each isoform, however, is topologically and structurally similar with predicted clusters of transmembrane domains seen at ends of the protein which vary from 1 to 2 in number at the amino terminus and from 4 to 6 at the carboxyl terminus. A large cytosolic region separates the transmembrane domains of the amino and carboxyl termini and contains the active sites and substrate-binding residues to fulfill HA synthetic capacity. All isoforms catalyze the polymerization of UDP-monosaccharides at the internal face of the plasma membrane, where catalysis occurs within the large cytoplasmic domain which constitutes approximately half of the overall protein. The growing HA chain is extruded into the extracellular matrix (ECM) or onto the pericellular surface where it can remain bound to HAS or associate with cellular HA receptors to form a glycocalyx. Expansion of the HA chain into the ECM allows the size constraints that would normally be imposed by cellular dimensions to be overcome and thus permits unrestrained polymer growth. Upon release of HA into the extracellular milieu, it is retained within the matrix via interaction with proteoglycans and receptor proteins (Fig. 1). Hyaluronan (HA) exhibits a diverse range of physiological functions despite its simple structure of repeating disaccharide units. The disaccharide units are linked by glycosidic bonds (in which a single oxygen atom links the two sugar units) into a macromolecule which typically contains 2,000–25,000 disaccharides and ranges in molecular weight (MW) between 105 and 107 Da. It is highly negatively charged due to the prevalence of carboxyl groups contributed from the glucuronic

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a

Extracellular

Catalytic domain

b

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c

H

H O

−O2C

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d

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Cytoplasm Plasma membrane

Hyaluronan Synthases, Fig. 1 (a) The HA synthase is predicted to be a multipass transmembrane protein with a large cytoplasmic domain, which holds key substratebinding and catalytic residues. (b) HA is extruded through the plasma membrane following polymerization of UDP-glucuronic acid and UDP-N-acetylglucosamine. (c) HA is composed of repeating units of glucuronic acid and

N-acetylglucosamine linked by glycosidic bonds. (d) HA chains can associate with HA synthases (red) and cell surface receptors such as CD44 and RHAMM (blue) and interact with other matrix components including proteoglycans (green), forming a structured, malleable, and hydrated pericellular and extracellular matrix

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acid moiety. Within an aqueous environment, HA becomes heavily hydrated primarily due to formation of large solvation cavities which are a result of electrostatic repulsion between intrahyaluronan anionic groups. Hyaluronan adopts a random coil structure in solution where molecules entangle to form a viscous, gel-like meshwork that can contract in response to external pressure but will relax and expand when released as a result of charge repulsion. In this way, a HA-rich ECM is malleable, elastic, and flexible and provides an ideal medium for growth, expansion, and cushioning of tissues. The broad range of physiological activities of HA are typically dependent on MW and concentration as these impact on the physical and mechanical properties, viscosity, hydration, matrix-forming capacity, and interactions between HA and cellular receptors. Therefore, as each HAS isoenzyme differs in catalytic rate and molecular weight of the synthetic product, the differential regulation and control of HAS expression are important determinants in HA function. HAS1 characteristically drives the synthesis of high molecular weight HA (ranging from 2  105 to ~2  106 Da); however, it exhibits a relatively low catalytic rate suggesting a role in maintenance of a basal level of cellular HA. HAS2 produces higher molecular weight HA and is more catalytically active; increased expression of this isoenzyme is observed in many developmental and repair process involving tissue expansion and growth. HAS3 is most active and synthesizes lower molecular weight HA (105–106 Da) that has been associated with providing HA for release into the pericellular matrix or for interaction with cell surface receptors. The molecular weight of native HA can be degraded through cleavage mediated by ▶ hyaluronidase enzymes; Hyal-1 and Hyal-2 hyaluronidases are the predominant forms responsible for HA catabolism in somatic tissues. Hyal-2 is anchored to the plasma membrane and can cleave HA to 20 kDa fragments, enabling internalization for lysosomal degradation into disaccharides in a process mediated by Hyal-1. The concurrent activity of HAS and hyaluronidase enzymes results in significant polydispersity of HA within

Hyaluronan Synthases

the extracellular matrix. While originally thought to be an inert, space-filling macromolecule, the diverse sizes of HA molecules can bind to specific cell surface receptors such as ▶ CD44 and RHAMM (receptor for hyaluronan-mediated motility) to induce transduction of various sizedependent cell signals, thereby highlighting the importance of action of HA synthetic and degradative enzymes. Hyaluronan Synthases and Hyaluronan in Cancer The extracellular environment within and surrounding a tumor mass can have a profound effect on tumorigenesis, progression, and invasion. This is perhaps most evident in metastasis where cell migration and invasion must be mediated by manipulation and breakdown of the extracellular matrix; inevitably, alteration and disruption of the matrix composition are characteristic of cancer initiation and progression. Increased HAS expression and deposition of HA in comparison to that in normal and benign tissue is one such morphological change in matrix composition that is commonly observed in many malignancies. The correlation between high levels of HA and tumor aggressiveness and invasiveness is long established, and in many malignancies, elevated intra-tumoral HA is associated with poor prognosis and low survival rates. Elevated intra-tumoral HA can be resultant of higher expression levels of HAS within the tumor cells and/or through the HA synthesized by stromal cells that surround a tumor. There is a dynamic interplay between stromal and malignant tissue, where paracrine signaling by tumor cells can induce upregulation of expression of HAS in stromal cells. Paracrine stimulation of HAS activity can be attributed to tumor release of growth factors such as TGF-b, FGF, and PDGF or by inflammatory cytokines such as TNF-a. These observations have established a clear link between HAS, HA, and tumorigenesis; the mechanisms by which HA production augments the malignant process have begun to be elucidated by manipulation of HAS enzyme expression and disruption of endogenous interactions that occur

Hyaluronan Synthases

between HA and tumor cells via receptors such as CD44 and RHAMM. Proliferation and Tumor Growth Increases in pericellular HA are observed in proliferating and migrating cells during normal physiological processes such as morphogenesis, growth, and wound healing, so it is not surprising that elevated HA also correlates to pathologies like cancer that are defined by rapid and inappropriate cell division and motility. Structurally, increased synthesis and deposition of HA results in hydration and expansion of the tissue volume creating less dense connective tissue that differs significantly from the architecture of the ECM associated with non-diseased cells and normal tissue. This flexibility and malleability of HA-rich matrices provides a medium that can readily surround rapidly dividing cells concomitantly accommodating changes in shape that occur during mitosis. The HA-rich ECM can also promote proliferation through the facilitation of diffusion of nutrients, growth factors, and cytokines to a tumor and creation of a concentrated reservoir of various growth effectors. Expression of HAS fluctuates within the cell cycle, with maximal activity occurring during mitosis where HA facilitates partial cell rounding and detachment. Interaction of HA and the centrosomally localized RHAMM receptor results in stabilization of the mitotic spindle during mitosis. Cell division is inhibited when the pericellular HA matrix that forms immediately prior to mitosis is disrupted, demonstrating the multifaceted roles that both HA and HAS play in cell cycle regulation and proliferation. Experimentally induced overexpression HAS isoenzymes enhances the growth kinetics of a wide range of tumor cell lines; conversely, disruption of interactions of HA with ▶ CD44 or RHAMM induces cell cycle arrest or apoptosis, suggesting that the role of HA in a tumor is structurally permissive but also has more complex roles in the initiation of cell signaling cascades promoting proliferation. Very high overexpression of HAS isoenzymes and high concentrations of high MW HA can conversely inhibit tumor growth, indicating a tightly

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controlled active regulation of HAS expression during the development of malignancy. Increased expression of hyaluronidase in many malignancies is suggestive of a cooperative balance between HA synthesis and degradation in tumors, where an imbalance in expression may lead to growth inhibition. Controlled regulation of HAS and hyaluronidase expression is evident in induction of angiogenesis. When diffusion of nutrients within a high MW HA-rich matrix is no longer sufficient for growth and proliferation, the HA can be degraded to low MW products which stimulate endothelial cell proliferation, migration, and tumor vascularization. HA also contributes to acquisition of an antiapoptotic phenotype in cancer, thereby promoting tumorigenesis. Normal cell survival requires anchorage to the substratum; cancer cells can avert this requirement, growing independently in a manner dependent on HA-CD44 binding. This interaction can facilitate cell survival by promoting activation of the ▶ PI3K signaling/AKT pathway and stimulating phosphorylation of pro-survival effectors, FAK and BAD. HA-CD44 binding is also required for complex assembly of a number of factors critical for signaling in the ▶ HER-2/neu pathway; when this signaling pathway dysfunctions, there is a direct correlation with malignancy development in a number of solid tumors. RHAMM-HA interaction similarly initiates signaling cascades directing phosphorylation of BAD and appears implicated in aberrant mitosis through disruption of RHAMM binding to microtubules leading to chromosomal misaggregation. Invasion and Metastasis Hyaluronan synthase expression and production of HA can augment the invasion and metastasis of tumor cells in several ways: • By creation of hydrated, expanded matrix pathways which are compatible with cell migration and facilitate tissue penetration. • Promotion of anchorage-independent growth. • Stimulation of cell locomotion and migration mediated by HA interaction with CD44 and RHAMM: CD44 has the capacity to interact

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with cytoskeletal proteins such as actin and ankyrin. The activation of CD44 induced by HA binding can initiate signaling cascades involving ▶ Src kinases and Ras/Rho GTPases that lead to cytoskeletal rearrangement, membrane ruffling, and migration. Cellular locomotion also requires turnover of focal adhesions (cell substratum adhesions); this process appears to require RHAMM-mediated activation of Src kinase and transient phosphorylation of focal adhesion kinase dependent on HA-RHAMM interaction. • Stimulation of expression of ▶ matrix metalloproteinases (MMPs): MMPs are required for proteolytic degradation of the ECM; this occurs when cells invade normal tissue and penetrate through the basement membrane into the surrounding vasculature. HA-CD44 association can promote expression of MMP-9 and MMP-2 and membrane type 1matrix metalloproteinase (MT1-MMP). MT1-MMP additionally mediates the proteolytic cleavage of CD44 from the cell surface and the detachment from underlying HA during cell locomotion. Shedding of CD44 promotes CD44 expression, and the continual receptor cycling allows continued adherence and detachment from the matrix as a tumor cell migrates. • Induction of ▶ epithelial-mesenchymal transition (EMT): Acquisition of an invasive and metastatic phenotype in carcinomas is akin to EMT seen in embryogenesis. Increased HAS expression and production of high molecular weight HA can be sufficient to induce stimulation of ErbB and PI3K signaling/Akt pathways and EMT. Elevated HA production can interfere with formation of intercellular and matrix adhesions increasing cellular autonomy and inducing anchorage-independent growth, MMP expression, and cytoskeletal rearrangement which are typical characteristics of mesenchymal tissue.

Multidrug Resistance Multidrug resistance hinders administration of an effective chemotherapy regime. Acquisition

Hyaluronan Synthases

of resistance is most commonly associated with increased drug efflux by ATP-binding pumps such as MDR (multidrug resistance) proteins. Overproduction of hyaluronan promotes MDR, with upregulation of HA synthesis rendering drug-sensitive cancer cells resistant. Administration of hyaluronidase as an adjunct to chemotherapy regimens increases sensitivity of tumors to therapeutic agents by reducing barriers to drug penetration. Hyaluronan synthesis also stimulates the expression of MDR1 and acquisition of drug resistance via HA-CD44-mediated activation of the ErbB2 signaling complex; this leads to downstream activation of PI3 kinase signaling and stimulation of HAS and MDR1 expression. Cancer Stem Cells A small subset of cells within the heterogeneous tumor mass commonly termed “cancer stem cells” (CSCs) are responsible for treatment resistance and disease recurrence. These characteristics can be attributed to a slow proliferative rate allowing CSCs to evade chemotherapeutics that commonly target rapidly dividing cells and progenitor behavior which endows them with potential to differentiate and recapitulate the heterogeneous population from which they were derived. The exact phenotype of this subpopulation is yet to be fully characterized; the CSC phenotype is likely to vary between cancers; however, CD44 is regarded as a key, conserved biomarker and is routinely used for CSC identification and isolation. The origin of CSCs remains elusive. It has been postulated that they may arise from deregulation of normal cellular pathways or from EMT of either normal or malignant cells. The role of HAS2 in EMT has been characterized (as previously described); there is emerging evidence to suggest that EMT expands the proportion of CSCs in a HA-dependent manner. Furthermore, it appears that the “CSC niche” (the HA-rich extracellular environment) and the maintenance of this subpopulation are intimately linked. Upregulation of HAS expression or depletion of endogenous HA demonstrates an inverse impact on the CSC subpopulation, where an increase in

Hyaluronic Acid Synthases (HASs)

HAS results in the expansion of this subpopulation; conversely, HA depletion reduces the incidence of CSCs. In turn, data provide circumstantial evidence for exploration of the HA metabolic pathway as a target for CSC eradication.

Cross-References ▶ CD44 ▶ Epithelial-to-Mesenchymal Transition ▶ Fibroblast Growth Factors ▶ Focal Adhesion Kinase ▶ HER-2/neu ▶ Hyaluronidase ▶ Matrix Metalloproteinases ▶ PI3K Signaling ▶ Platelet-Derived Growth Factor ▶ Src ▶ Transforming Growth Factor-Beta

References Adamia S, Maxwell CA, Pilarski LM (2005) Hyaluronan and hyaluronan synthases: potential therapeutic targets in cancer. Curr Drug Targets Cardiovasc Haematol Disord 5:3–14 Itano N, Kimata K (2002) Mammalian hyaluronan synthases. IUBMB Life 54:195–199 Stern R (2005) Hyaluronan metabolism: a major paradox in cancer biology. Pathol Biol (Paris) 53:372–382 Toole BP (2004) Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 4:528–539 Visvader JE, Lindeman GJ (2012) Cancer stem cells: current status and evolving complexities. Cell Stem Cell 10:717–728

See Also (2012) Actin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 18–19. doi:10.1007/978-3-642-16483-5_42 (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163 (2012) Extracellular matrix. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067

2165 (2012) Glycocalyx. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1569. doi:10.1007/978-3-642-16483-5_2445 (2012) HA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1625. doi:10.1007/978-3-642-16483-5_2550 (2012) HAS1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1632. doi:10.1007/978-3-642-16483-5_2569 (2012) HAS2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1632. doi:10.1007/978-3-642-16483-5_2570 (2012) HAS3. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1632. doi:10.1007/978-3-642-16483-5_2571 (2012) Hyaluronan. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1767. doi:10.1007/978-3-642-16483-5_2876 (2012) Pericellular matrix. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2814. doi:10.1007/978-3-642-16483-5_4447 (2012) Proteoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3100. doi:10.1007/978-3-642-16483-5_4816 (2012) Resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3263. doi:10.1007/978-3-642-16483-5_5052 (2012) RHAMM. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3301. doi:10.1007/978-3-642-16483-5_5095 (2012) Stroma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3541. doi:10.1007/978-3-642-16483-5_5532 (2012) TGF-b. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3661. doi:10.1007/978-3-642-16483-5_5753 (2012) TNF-a. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3713. doi:10.1007/978-3-642-16483-5_5841 (2012) UDP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3835. doi:10.1007/978-3-642-16483-5_6092

Hyaluronic Acid (HA) ▶ Hyaluronan Synthases

Hyaluronic Acid Synthases (HASs) ▶ Hyaluronan Synthases

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Hyaluronidase Ronny Racine1 and Vinata B. Lokeshwar2 1 Department of Urology, University of Miami – Miller School of Medicine, Miami, FL, USA 2 Department of Biochemistry and Molecular Biology, Medical College of Georgia; Augusta University, Augusta, GA, USA

Definition Hyaluronidases (HAases) are endoglycosidases that degrade hyaluronic acid (HA). HA is a non-sulfated glycosaminoglycan. Exhaustive digestion of HA with HAase generates tetrasaccharides, whereas limited digestion generates HA fragments, some of which induce angiogenesis.

Characteristics Six HAase genes are present in the human genome and these occur in two linked triplates. HYAL-1, HYAL-2, and HYAL-3 genes are clustered in the chromosome 3p21.3 locus, whereas HYAL-4, HYAL-P1, and PH20 (which encodes testicular HAase) reside in the chromosome 7q31.3 locus (Stern and Jedrzejas 2006). Based on their pH activity profiles, HAases are divided into two categories. HYAL-1, HYAL-2, and HYAL-3 are considered acidic HAases because they are active at acidic pH. For example, HYAL-1 has a pH optimum around 4.0–4.2, and the enzyme is inactive above pH 5.0. It is normally expressed in serum and urine. On the contrary, PH20 or the testicular HAase is a neutral HAase as it is active at pH 7.0 (pH activity profile 3.0–9.0) and is involved in ovum fertilization. Among the six mammalian HAases, HYAL-1 is the major tumor-derived HAase and is Modified version of Lokeshwar VB, Selzer MG (2012) Hyaluronidase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 1772–1775. doi:10.1007/978-3-642-16483-5_2881

Hyaluronidase

expressed by a variety of tumor cells. HYAL-1 was initially purified from the urine of patients with high-grade bladder cancer and has since been shown to be expressed in tumor cells, tumor tissues, urine, and saliva from cancer patients. HAase Expression as a Cancer Biomarker Both HAase activity and the expression of primarily HYAL-1 gene have been examined as biomarkers for cancer detection and predicting prognosis. Measurement of HAase activity by an ELISA-like assay (referred commonly as the HAase test) has shown high sensitivity and specificity to detect bladder cancer, in particular highgrade bladder cancer. Recent work has also focused on targeting hyaluronidase activity for tumor imaging. HA-coated nanoparticles have been used to target fluorescent dyes to tumor sites due to the increased levels of HA receptors present on tumor cells (McAtee et al. 2014). HA has also been used to encapsulate imaging fluorophores where tumor-associated HYAL activity releases the dye from the HA gel, increasing fluorescence intensity in tumor tissue. Further work with HA and fluorescent-labeling reagents has produced autoquenched fluorescent HA and Forster resonance energy transfer pair-conjugated HA, which can detect low levels of HAase activity in urine samples. HAase levels are also elevated in the saliva of patients with head and neck tumors and in the urine of children with Wilms tumor. In several studies, elevated HYAL-1 mRNA expression, analyzed by quantitative reverse transcription PCR, and/or protein expression, detected by immunohistochemistry in tumor specimens, has been shown to be independent prognostic predictors of disease recurrence, progression, metastasis, and treatment response in bladder, breast, prostate, and other carcinomas (Lokeshwar et al. 2005, 2014; McAtee et al. 2014). Contrarily, reduced HYAL-1 expression detected by immunohistochemistry appears to be associated with the progression of endometrial carcinomas. Reduced HAase activity is found in colorectal tumors; however, other studies have reported increased HYAL-1 and HYAL-2 expression in colorectal cancer and its correlation with advanced disease. Although the expression of

Hyaluronidase

other HAases are less well studied, decreased HYAL-2 gene methylation has been shown to be associated with increased risk for breast cancer (Yang et al. 2015). It is noteworthy that HAase family members such as HYAL-1 and HYAL-3 are alternatively spliced. The splice variants are functionally inactive and have tumor-suppressing effects. For example, HYAL-1 variant 1 is missing a 30 amino acid region corresponding to exon 2. Expression of this variant induces the formation of a HYAL-1 and HYAL-1-v1 complex possessing diminished hyaluronidase activity. In vivo expression of this variant slows tumor growth and causes bladder cancer tumors to become necrotic and infiltrated by neutrophils (Lokeshwar et al. 2006). Therefore, HYAL-1 protein expression detected by immunohistochemistry may not necessarily translate into high HAase activity, and different tumors may express different splice variants. Regulation of HAase Activity Only the promoter regions of HYAL-1 and HYAL-2 have been cloned. HYAL-1 promoter activity is regulated by methylation and the binding of transcription factors AP-2, EGR-1, SP1, and NF-kB (Lokeshwar et al. 2008). Alternative mRNA splicing is another mechanism by which HAase activity is regulated. For example, in some tumor cells, internal splicing within the 50 untranslated region in exon 1 correlates with HYAL-1 protein levels and HAase activity. HYAL-1 protein is not detected in tumor cells which express a HYAL-1 transcript that retains the 50 untranslated region; however, it is unclear how and why the 50 untranslated region in the HYAL-1 mRNA prevents translation. Several alternatively spliced variants of HYAL-1 and HYAL-3, which map within respective coding regions, have been reported. All of these variants are enzymatically inactive (Lokeshwar et al. 2002). In addition to alternative splicing, the expression of HAase family members may be regulated by DNA methylation. For example, HYAL-1 expression has been shown to be epigenetically regulated by methylation at C(-71) and C (-59), which map in the minimal promoter region; both Cs are part of the SP1/Egr-1-binding sites.

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Similarly, as described above, HYAL-2 expression also appears to be regulated by DNA methylation (Yang et al. 2015). HAase Functions in Cancer In tumor tissues HA is contributed by both tumor stroma and tumor cells and induces intracellular signaling by binding to HA receptors. Most studies have been focused on HYAL-1, which is exclusively expressed by tumor cells. By degrading HA, HAase/HYAL-1 generates small HA fragments, some of which (~10–25 disaccharide units) are angiogenic. In experimental tumor model systems, studies have shown that HYAL-1, either alone or in co-expression with HA-synthase genes, promotes tumor cell proliferation, motility, and invasion or in xenografts enhances tumor growth, metastasis, and angiogenesis. Contrarily, knockdown of these genes inhibits tumor cell functions (Lokeshwar et al. 2014; McAtee et al. 2014). In the case of HYAL-1, promotion of tumor cell function is dose dependent. At levels detected in clinical specimens, HYAL-1 promotes tumor growth, invasion/metastasis, and angiogenesis; however, overexpression of HYAL-1 at levels exceeding those expressed in tumor tissues induces apoptosis and inhibits tumor formation. Therefore, while a limited degradation of the pericellular HA matrix generates angiogenic HA fragments, which induce intracellular signaling, complete degradation of the HA matrix, as a result of experimental overexpression of HYAL-1, will inhibit tumor growth and progression. HAase-Induced Signaling in Cancer Cells Studies on HA-mediated signaling usually do not distinguish between HA and HA fragments present in the pericellular matrix. Smaller angiogenic disaccharide fragments, generated by the tumorassociated HA-HYAL-1 system, have been detected in the urine of patients with bladder cancer and Wilms tumor, in the saliva of patients with head and neck cancer, and in the bladder and prostate tumor tissues (Lokeshwar et al. 2014; McAtee et al. 2014). In contrast to the angiogenic HA fragments, HA oligosaccharides consisting of 2–3 disaccharide units have been shown to have

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antitumor activity, presumably because they inhibit HA-induced signaling. Interaction between pericellular HA/angiogenic fragments and HA receptors induces multiple intracellular pathways. For example, interaction of HA and angiogenic HA fragments with HA receptors (i.e., CD44 and/or RHAMM) promotes cell survival, cancer stemness, motility, and invasion by activating growth factor receptor signaling (e.g., ErbB2, c-Met), PI-3 K/Akt and Erk pathways, small GTPase proteins (i.e., RhoA and Rac1), Ras, NFkB and src signaling, cytoskeleton reorganization, etc. These signaling pathways cause the expression of matrix metalloproteinases, COX-2, inflammatory cytokines, and vascular endothelial growth factor (VEGF). It has also been shown that in a feedback loop, induction of PI-3 K/Akt pathway increases CD44 and RHAMM expression, thus ensuring continued cancer-promoting signaling by the tumorassociated HA-HAase system. HAase for Cancer Therapy HAase is potentially useful for cancer therapy in two seemingly opposite ways (Lokeshwar et al. 2005, 2014). First is the use of HAase to breakdown extracellular matrix, therefore improving the penetrance of chemotherapeutic agents in tumors, and second is the targeting of tumor-derived HAase to control tumor growth and progression. Testicular HAase has been added in cancer chemotherapy regimens to improve drug penetration. Tumor cells growing in three-dimensional multicellular masses, such as spheroids in vitro and solid tumors in vivo, acquire resistance to chemotherapeutic drugs (i.e., multicellular resistance). But this acquired chemoresistance can be abolished by the addition of testicular HAase. In clinical studies, testicular HAase has been used to enhance the efficacy of vinblastine in the treatment of malignant melanoma and Kaposi sarcoma, boron neutron therapy of glioma, intravesical mitomycin treatment for bladder cancer, and chemotherapy involving cisplatin and vindesine in the treatment of head and neck squamous cell carcinoma. Provenzano et al. have shown that intravenous injection of PH20 in a murine model of pancreatic ductal

Hyaluronidase

adenocarcinoma prior to gemcitabine also resulted in a doubling of overall survival. This strategy can be applied to any tumor with high concentrations of HA in the tumor stroma (McAtee et al. 2014). High levels of HA, which is highly hydrophilic, in the tumor stroma increase interstitial pressure, preventing the influx of chemotherapeutics. It is noteworthy that the HAase concentrations (1  105–2  105 IU) used in these clinical studies far exceed the amount of HAase present in tumor tissues, and therefore, it is unlikely that at these concentrations the infused HAase will act as a tumor promoter. Since HAase activity and HYAL-1 appear to be a critical determinant of cancer growth, metastasis, and angiogenesis, targeting them could potentially be an effective strategy for cancer therapy. Many synthetic and naturally occurring compounds have been tested as HAase inhibitors (Isoyama et al. 2006). Polymers of sulfated hyaluronic acid (sHA) with varying degrees of sulfation are effective inhibitors of HYAL-1 activity. sHA polymers inhibit HYAL-1 activity by a mixed inhibition mechanism (i.e., competitive + uncompetitive). Since sHA is a natural compound and is derived from HA, its antitumor activity has been examined against prostate cancer models (Benitez et al. 2011). sHA with 2.75 of sulfation was found to inhibit prostate cancer cell growth through induction of apoptosis. sHA inhibited HA signaling, with inhibition of PI-3 K/Akt pathway as the major HA signaling target. sHA also displays antitumor activity due to inhibition of angiogenesis and induction of tumor cell apoptosis. Tumor inhibitory effects of sHA are accompanied by lack of any serum or organ toxicity, suggesting that targeting of tumorderived HAase could potentially be an attractive strategy for cancer therapy. Summary HAase appears to be an important molecular determinant of tumor growth, infiltration, and angiogenesis. At concentrations that are present in tumor tissues, HAase acts as a tumor promoter. HAases, either alone or together with HA, are potentially accurate diagnostic and prognostic indicators for cancer detection and tumor

Hydrogen Peroxide

metastasis. We are only beginning to understand the complex role that this enzyme plays in cancer. Nonetheless, it is already proving to be a useful target for developing novel cancer therapeutics and diagnostics.

Cross-References ▶ Cancer ▶ Chemotherapy ▶ Tumor Suppression

References Benitez A, Yates TJ, Lopez LE, Cerwinka WH, Bakkar A, Lokeshwar VB (2011) Targeting hyaluronidase for cancer therapy: antitumor activity of sulfated hyaluronic acid in prostate cancer cells. Cancer Res 71(12):4085–4095. doi:10.1158/0008-5472.CAN-10-4610 Isoyama T, Thwaites D, Selzer MG, Carey RI, Barbucci R, Lokeshwar VB (2006) Differential selectivity of hyaluronidase inhibitors toward acidic and basic hyaluronidases. Glycobiology 16(1):11–21. doi:10.1093/ glycob/cwj036 Lokeshwar VB, Schroeder GL, Carey RI, Soloway MS, Iida N (2002) Regulation of hyaluronidase activity by alternative mRNA splicing. J Biol Chem 277(37):33654–33663. doi:10.1074/jbc.M203821200 Lokeshwar VB, Cerwinka WH, Isoyama T, Lokeshwar BL (2005) HYAL1 hyaluronidase in prostate cancer: a tumor promoter and suppressor. Cancer Res 65(17):7782–7789. doi:10.1158/0008-5472.CAN-051022 Lokeshwar VB, Estrella V, Lopez L, Kramer M, Gomez P, Soloway MS, Lokeshwar BL (2006) HYAL1-v1, an alternatively spliced variant of HYAL1 hyaluronidase: a negative regulator of bladder cancer. Cancer Res 66(23):11219–11227. doi:10.1158/0008-5472.CAN06-1121 Lokeshwar VB, Gomez P, Kramer M, Knapp J, McCornack MA, Lopez LE, Fregien N, Dhir N, Scherer S, Klumpp DJ, Manoharan M, Soloway MS, Lokeshwar BL (2008) Epigenetic regulation of HYAL1 hyaluronidase expression. identification of HYAL-1 promoter. J Biol Chem 283(43):29215–29227. doi:10.1074/jbc.M801101200 Lokeshwar VB, Mirza S, Jordan A (2014) Targeting hyaluronic acid family for cancer chemoprevention and therapy. Adv Cancer Res 123:35–65. doi:10.1016/B978-0-12-800092-2.00002-2 McAtee CO, Barycki JJ, Simpson MA (2014) Emerging roles for hyaluronidase in cancer metastasis and therapy. Adv Cancer Res 123:1–34. doi:10.1016/B978-012-800092-2.00001-0

2169 Stern R, Jedrzejas MJ (2006) Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 106(3):818–839. doi:10.1021/cr050247k Yang R, Pfutze K, Zucknick M, Sutter C, Wappenschmidt B, Marme F, Qu B, Cuk K, Engel C, Schott S, Schneeweiss A, Brenner H, Claus R, Plass C, Bugert P, Hoth M, Sohn C, Schmutzler R, Bartram CR, Burwinkel B (2015) DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood. Int J Cancer 136(8):1845–1855. doi:10.1002/ijc.29205

See Also (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 408–409. doi:10.1007/978-3-642-16483-5_6601 (2012) Targeted therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3610. doi:10.1007/978-3-642-16483-5_5677

Hybrid Genes ▶ Fusion Genes

Hybrid Positron Emission Tomography/Computed Tomography ▶ Positron Emission Tomography

Hydrogen Dioxide ▶ Hydrogen Peroxide

Hydrogen Peroxide Miguel Lopez-Lazaro Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain

Synonyms Dihydrogen dioxide; Hydrogen dioxide

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Hydrogen Peroxide 4e– + 4H+ 2H2O

oxphos

e– O2

e– + 2H+ O2• –

e–

H2O2

ATP

e– OH– + OH+

2H– 2OH–

2H2O

2e– + 2H+

Hydrogen Peroxide, Fig. 1 Aerobic cells generally use O2 to generate energy (ATP) through a process called oxidative phosphorylation (OXPHOS), but they can also

use O2 to generate reactive oxygen species such as hydrogen peroxide (H2O2)

Definition

cytochrome oxidases, NADPH oxidases, cyclooxygenases, cytochromes P450, xanthine oxidase, etc.). ROS can be eliminated by endogenous antioxidant systems. For instance, glutathione and thioredoxin systems decrease the cellular levels of H2O2 by catalyzing a reaction in which H2O2 is reduced to H2O. Likewise, the enzyme catalase eliminates H2O2 by transforming two molecules of H2O2 into two molecules of H2O and one molecule of O2. Antioxidant agents can reduce the cellular levels of ROS by preventing their generation or by favoring their elimination. For instance, some polyphenols (e.g., ▶ flavonoids) can prevent the generation of H2O2 by scavenging O2 •, and selenium compounds can favor the elimination of H2O2 by providing selenium atoms, which are essential components of the H2O2 detoxifying enzyme glutathione peroxidase. Prooxidant agents, on the contrary, can increase the cellular levels of ROS by increasing their generation or by reducing their elimination. For instance, arsenic can increase ROS generation by activating the enzyme NADPH oxidase, and buthionine sulfoximine (an inhibitor of g-glutamylcysteine synthetase, the rate-limiting enzyme of glutathione synthesis) can reduce ROS elimination by decreasing glutathionemediated H2O2 decomposition. The controlled generation of ROS plays an important role in the physiological control of cell function. For instance, cells under hypoxic conditions generate H2O2 and use it to activate hypoxia-inducible factor-1 (HIF-1) (hypoxia and tumor physiology), a transcription factor that codes for many proteins that help cells adapt to low oxygen levels. An uncontrolled or excessive cellular production of H2O2, however, can produce carcinogenic effects and cell death (Fig. 2).

Hydrogen peroxide (H2O2) is a ▶ reactive oxygen species (ROS) generated from molecular oxygen (O2). Although the controlled cellular production of H2O2 plays an important physiological role, high cellular levels of H2O2 can produce carcinogenic effects and induce cell death.

Characteristics H2O2 is a pale blue liquid first isolated in 1818 by Louis Jacques Thénard. H2O2 has industrial and domestic uses (e.g., paper bleaching, chemical synthesis, laundry detergents, antiseptic for wound cleaning, etc.), and it is manufactured today through an autoxidation reaction using O2 from the air. Cells of aerobic organisms also generate H2O2 from O2. Most of the energy (ATP) that aerobic cells need to live is obtained through a process called oxidative phosphorylation (OXPHOS). In this process, ATP generation is coupled with a reaction in which O2 is reduced [reduction/oxidation] to water (H2O) by a mitochondrial protein complex called cytochrome oxidase. In this reaction, four electrons and four protons are added to O2 to form two molecules of H2O. But when a molecule of O2 gains only one electron to form superoxide anion (O2 •), this highly reactive oxygen species tends to gain three more electrons and four protons to form H2O; this process involves several reactions and results in the production of other ROS such as H2O2 (Fig. 1). ROS are generated in multiple compartments within the cell (e.g., mitochondria, cytosol, plasma membrane, peroxisomes, endoplasmic reticulum, etc.) and by numerous enzymes (e.g.,

Hydrogen Peroxide

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Hydrogen Peroxide, Fig. 2 While the controlled generation of H2O2 has an important physiological role, a sustained increase in the cellular levels of H2O2 can produce carcinogenic effects, and an excessive increase in the levels of H2O2 can induce cell death. Antioxidant agents can reduce the cellular levels of H2O2 and prevent the

carcinogenic effects induced by H2O2; these agents may therefore exert a cancer-preventive activity. Prooxidant agents can increase the cellular levels of H2O2 and may induce carcinogenic effects. A sufficient increase in the cellular levels of H2O2 induced by prooxidant agents may trigger cell death and be therapeutically useful

Carcinogenic Effects Cancer cells from different tissues have been observed to produce high amounts of H2O2. High cellular levels of H2O2 have been associated with DNA alterations, including DNA damage, mutations, and genetic instability. For instance, transition metals such as iron or copper can react with H2O2 to produce hydroxyl radical (OH•) via the Fenton reaction; OH• is a highly reactive species known to produce ▶ DNA oxidation damage. The production of DNA alterations by H2O2 may play an important role in ▶ carcinogenesis, as cancer is considered to be a genetic disease caused by DNA alterations. Cell proliferation, ▶ apoptosis resistance, ▶ angiogenesis, ▶ invasion, and ▶ metastasis are key events of the carcinogenesis process. It is well known that, in order for cancer to develop, tumor cells must proliferate. Accumulating data have shown that H2O2 stimulates cell proliferation; in fact, the cell proliferation induced by different stimuli can be decreased by H2O2-detoxifying enzymes (e.g., catalase). Under physiological conditions, cells with irreparable damages usually commit suicide by triggering a programmed process called apoptosis. Since tumor cells have important damages in many of their components,

the formation of a cancer requires that tumor cells develop apoptosis resistance. Interestingly, it has been found that non-cytotoxic concentrations of H2O2 can produce apoptosis resistance in cancer cells. Angiogenesis – the generation of new blood vessels – is also necessary for the formation of a solid tumor; without vascular growth, the tumor mass is restricted to within a tissue-diffusion distance of approximately 0.2 mm. Results support that H2O2 has an important function in angiogenesis. For instance, angiopoietin-1 plays an important role in angiogenesis and it has been found that its angiogenic effect is mediated by H2O2. It is recognized that the metastatic spread of primary tumors accounts for approximately 90% of all cancer deaths. The process by which cells from a localized tumor invade adjacent tissues and metastasize to distant organs is therefore the most clinically relevant processes involved in carcinogenesis. H2O2 has been found to modulate the activity of several processes (e.g., cell ▶ motility, ▶ migration, ▶ adhesion) and molecules (e.g., ▶ MET, ▶ matrix metalloproteinases) involved in tumor invasion and metastasis. Indeed, studies carried out in animal models have revealed that the targeted delivery of catalase can inhibit tumor metastasis.

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Although the transcription factor HIF-1 plays an important role in the physiological control of cell function, HIF-1 overexpression is commonly observed in most human cancers and has been associated with increased patient mortality in several cancer types. HIF-1 increases the transcription of a variety of genes that code for proteins involved in processes intimately related to cancer, including apoptosis resistance, angiogenesis or invasion, and metastasis. H2O2 can both activate HIF-1 and mediate the activation of HIF-1 caused by different stimuli; in fact, the presence of enzymes that reduce the cellular levels of H2O2 prevents the activation of HIF-1 caused by different triggers (e.g., hypoxia, TNF alpha). The key role of H2O2 in carcinogenesis is also supported by experimental data that have demonstrated that H2O2 can cause and mediate cell malignant transformation. For instance, it has been reported that the expression of Nox1 (a homologue of gp91phox, the catalytic moiety of the O2 •-generating NADPH oxidase of phagocytes) in normal NIH3T3 fibroblasts resulted in cells with malignant characteristics that produced tumors in athymic mice. These transformed cells showed a 10-fold increase in H2O2 levels. When catalase was overproduced in these transformed cells, H2O2 concentration decreased and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. Clinical Relevance Despite some important advances in cancer therapy, the number of cancer deaths has not decreased in the last three decades. New strategies to control this disease are required. It is recognized that cancer chemoprevention (the use of chemicals to prevent, stop, or reverse the process of carcinogenesis) is an essential approach to controlling cancer. Since H2O2 seems to have an important role in carcinogenesis, a chemical capable of preventing or decreasing excessive cellular levels of H2O2 might be useful in cancer chemoprevention. Some complementary and alternative medicine practitioners have used H2O2 and other

Hydrogen Peroxide

“hyperoxygenation” therapies for the treatment of cancer. In 1993, the American Cancer Society studied the available literature and found no evidence that treatment with H2O2 and other “hyperoxygenation” therapies was safe or resulted in objective benefit in the treatment of cancer. Today it is accepted that the direct administration of H2O2 is not an appropriate strategy for the treatment of cancer, as H2O2 can produce toxicity by oxidizing macromolecules such as DNA, proteins, or lipids. Indeed, as discussed above and represented in Fig. 2, a large body of research strongly supports that H2O2 can produce carcinogenic effects. However, an increasing number of reports indicates that a sufficient increase in the cellular levels of H2O2 may be an effective therapeutic strategy. For instance, it is recognized that many anticancer drugs currently used in the clinic produce their antitumor activity by inducing apoptosis in cancer cells, and H2O2 is an effective inductor of apoptosis in cancer cells. In addition, some studies have shown that specific concentrations of H2O2 can kill cancer but not normal cells; it has been proposed that the increased levels of H2O2 found in tumor cells may account for their increased susceptibility to H2O2. This selectivity for cancer cells is in accordance with animal experiments that have shown that the use of H2O2-generating systems can deliver H2O2 to sites of malignancy and produce anticancer effects with little toxicity to the host. The therapeutic potential of H2O2 is also supported by the fact that the anticancer activity of several drugs commonly used in the clinic (e.g., paclitaxel [▶ taxol], arsenic trioxide) is mediated, at least in part, by an increase in the cellular levels of H2O2. Data have also shown that high concentrations of vitamin C (only achievable through the i.v. route) can produce selective killing of cancer cells through a H2O2-dependent mechanism. Therefore, although the direct administration of H2O2 does not seem an appropriate anticancer approach, any strategy capable of increasing the levels of H2O2 in cancer cells might be therapeutically useful. Accumulating evidence suggests that the modulation of the cellular levels of H2O2 may be an important approach for the development of cancer

2-Hydroxyoleic Acid

chemopreventive and therapeutic strategies. Chemicals with antioxidant properties may reduce the cellular levels of this oxidant and produce cancer chemopreventive effects; indeed, most cancer chemopreventive agents have antioxidant properties. Chemicals with prooxidant properties may induce a sufficient increase in the cellular levels of H2O2 and produce cell death in cancer cells; these drugs may produce a chemotherapeutic effect. Many chemicals (e.g., vitamin C, vitamin E, b-carotene, ▶ curcumin, ▶ sulforaphane, ▶ epigallocatechin, etc.) induce either an antioxidant or prooxidant effect mostly depending on the concentration at which they reach the cells. Low concentrations of these agents would therefore produce chemopreventive effects, while high concentrations would produce chemotherapeutic effects. However, these agents may produce carcinogenic effects when used at concentrations that increase the cellular levels of H2O2 but not sufficiently to induce cell death (Fig. 2). Antioxidant/prooxidant drugs may act as cancer-preventive, therapeutic, or carcinogenic agents mostly depending on their dose and route of administration.

2173

References American Cancer Society (1993) Questionable methods of cancer management: hydrogen peroxide and other ‘hyperoxygenation’ therapies. CA Cancer J Clin 43:47–56 Arnold RS, Shi J, Murad E et al (2001) Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci U S A 98:5550–5555 Burdon RH (1995) Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 18:775–794 Lopez-Lazaro M (2007) Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and therapy. Cancer Lett 252:1–8 Szatrowski TP, Nathan CF (1991) Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 51:794–798

See Also (2012) Oxidative phosphorylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2730. doi:10.1007/978-3-642-164835_4308 (2012) Reduction/Oxidation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3214. doi:10.1007/978-3-642-16483-5_5009

Cross-References ▶ Adhesion ▶ Angiogenesis ▶ Apoptosis ▶ Carcinogenesis ▶ Curcumin ▶ DNA Oxidation Damage ▶ Epigallocatechin ▶ Flavonoids ▶ Hypoxia ▶ Invasion ▶ Matrix Metalloproteinases ▶ MET ▶ Metastasis ▶ Migration ▶ Motility ▶ Oxidative Stress ▶ Reactive Oxygen Species ▶ Sulforaphane ▶ Taxol

14-Hydroxydaunorubicin ▶ Adriamycin

2-Hydroxyoleic Acid Maitane Ibarguren, Paula Fernández-García, Laura Arbona, Xavier Busquets and Pablo V. Escribá Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain

Synonyms Minerval; 2OHOA; 2-OHOA; NaCHOleate

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Definition 2-Hydroxyoleic acid (2OHOA) is a first-in-class ▶ Membrane-Lipid Therapy anticancer drug that specifically activates sphingomyelin (SM) synthase (SMS), a new therapeutic target in the fight against cancer. SMS activation by 2OHOA causes profound membrane lipid remodeling in cancer cells but not in normal cells, altering the organization of membrane microdomains. Normalization of the lipid composition/structure of cancer cell membranes provokes changes in the type and abundance of extrinsic membrane proteins that receive and propagate cell signals. These regulatory effects induce cell cycle arrest, cell differentiation, and programmed cell death of cancer cells.

Characteristics Structure 2OHOA has 18 C atoms with a cis double bond between carbons 9 and 10 (Fig. 1). It is classified as a monounsaturated omega-9 fatty acid, abbreviated as 18:1n-9 or 18:1 cis-D9. It has a hydroxyl moiety on the a-carbon that prevents its rapid degradation through b-oxidation, consequently increasing its half-life with respect to its nonhydroxylated analogue, oleic acid.

2-Hydroxyoleic Acid, Fig. 1 Structure of 2OHOA

2-Hydroxyoleic Acid

Mechanism of Action The presence of 2OHOA in membranes and the activation of SMS induce important changes in the composition and structure of membranes. The presence of 2OHOA in membranes provokes a reorganization of the lipid microdomains, increasing cell membrane fluidity and reducing the lipid bilayer liquid ordered–to–liquid disordered (Lo/Ld) ratio in vitro from ca. 50 to ca. 40%. The presence of 2OHOA in membranes augments the propensity of membrane lipids to organize into nonlamellar HII structures, promoting the subsequent recruitment of protein kinase C (PKC) to the plasma membrane. This event is followed by the activation of CDK inhibitors like p21CIP1 and p27Kip1, cyclin-CDK inhibition, retinoblastoma protein hypophosphorylation, and E2F-1 knockdown. E2F1 is a critical transcription factor that regulates several genes involved in cell proliferation, including dihydrofolate reductase (DHFR). The downregulation of DHFR impairs DNA synthesis and results in cell cycle arrest. 2OHOA also activates SMS, causing significant reductions in the membrane phosphatidylcholine and phosphatidylethanolamine content and increasing the SM in cancer cell membranes. This modification also changes the balance of membrane raft:nonraft microdomains. In human glioma cells, such an alteration induces a dramatic translocation of Ras from the plasma membrane to the cytoplasm, the subsequent inhibition of the mitogen-activated protein kinase (MAPK) pathway, and the PI3K/Akt and cyclin D-cdk4/6 pathways, causing cell cycle arrest and inducing differentiation. In glioma cells, 2OHOA also induces autophagic cell death (see below), and in human leukemia cells, and possibly in other cancer cell lines, it induces apoptosis. Indeed, this synthetic fatty acid causes the clustering of the Fas death ligand receptor (FasR) to defined membrane areas in Jurkat cells, inducing the ligandfree activation of extrinsic (caspase-8-mediated) apoptosis specifically in cancer but not in normal cells. Again, the high levels of SM in these tumor cells may account for these effects. The huge demand for ceramide to produce sphingomyelin on exposure to 2OHOA causes an important alteration in sphingolipid

Hypericin

metabolism, which has been associated with the induction of endoplasmic reticulum (ER) stress in brain cancer (glioma) but not in noncancer MRC-5 cells. In fact, 2OHOA induces several key effectors of the ER stress/unfolded protein response (P-eIF2a, ATF4, and CHOP) in cancer cells alone. This sustained ER stress is most likely involved in programmed autophagic (type 2) cell death. Hitherto, both apoptosis and autophagy have been proposed to be responsible for the specific death of cancer cells upon 2OHOA treatment. The induction of autophagy has been described in glioma cells and could be the result of ER stress/ UPR activation. By contrast, apoptosis has been reported in human leukemia (Jurkat) cells and could be the result of modifications to the composition of the plasma membrane that leads to Fas receptor clustering and ligand-free activation of apoptosis. Clinical Trials A phase I/IIa clinical trial to test the safety and efficacy of 2OHOA (Minerval) in adult patients with advanced solid tumors, including malignant glioma (MIN-001-1203), has been ongoing since May 2013. Other trials have been programmed in the USA and Europe in 2015.

Cross-References

2175 apoptosis in Jurkat and other cancer cells. J Cell Mol Med 14:659–670 Marcilla-Etxenike A, Martín ML, Noguera-Salvà MA, García-Verdugo JM, Soriano-Navarro M, Dey I, Escribá PV, Busquets X (2012) 2-Hydroxyoleic acid induces ER stress and autophagy in various human glioma cell lines. PLoS One 7(10):e48235 Terés S, Lladó V, Higuera M, Barceló-Coblijn G, Martin ML, Noguera-Salva MA, Marcilla-Etxenike A, GarcíaVerdugo JM, Soriano-Navarro M, Saus C, GómezPinedo U, Busquets X, Escribá PV (2012) 2Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy. Proc Natl Acad Sci U S A 109:8489–8494

7-Hydroxystaurosporine ▶ UCN-01 Anticancer Drug

Hydroxysteroid Dehydrogenases ▶ Reductases

Hypercalcemic Type ▶ Ovarian Tumors During Childhood and Adolescence

▶ Membrane-Lipid Therapy

References

Hypericin

Barceló-Coblijn G, Martin ML, de Almeida RF, NogueraSalva MA, Marcilla-Etxenike A, Guardiola-Serrano F, Luth A, Kleuser B, Halver JE, Escribá PV (2011) Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy. Proc Natl Acad Sci 108:19569–19574 Ibarguren M, López DJ, Escribá PV (2014) The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health. Biochim Biophys Acta 1838:1518–1528 Lladó V, Gutiérrez A, Martínez J, Casas J, Terés S, Higuera M, Galmés A, Saus C, Besalduch J, Busquets X, Escribá PV (2010) Minerval induces

Constance L. L. Saw1 and Paul W. S. Heng2 1 Department of Pharmaceutics, Rutgers, The State University of New Jersey, Ernest Mario School of Pharmacy, Piscataway, NJ, USA 2 Department of Pharmacy, National University of Singapore, Singapore, Singapore

Synonyms Hypericum extract; Hypericum red

H

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Hypericin

Hypericin, Table 1 Comparison of 5-ALA and hypericin for detecting human superficial bladder carcinoma Characteristics Form

Stability

5-ALA Prodrug, needs to be converted to the active form 75–100% Very low specificity, 43–68.5% (with many false positives) Easily photobleached during process

Permeability across biological membrane

Charged molecule – difficult to cross biomembranes

Sensitivity Specificity

Definition Hypericin is a natural photosensitizer present in the plant Hypericum perforatum, commonly known as St. John’s wort. It belongs to the class of phenanthroperylenequinones and napthodianthrone and has a molecular weight of 504.45. St. John’s wort has been used to treat inflammation, bacterial and viral infections, and depression. Hypericin is used in photodynamic diagnosis (PDD) and ▶ photodynamic therapy (PDT) for diagnosis and treatment of cancers.

Characteristics Physical Properties Hypericin yields red fluorescence when excited with a specific wavelength of light by lasers such as 442 nm (He–Cd), 488 nm (Ar), or 543 nm (He–Ne) and light by xenon-arc lamp with a band-pass filter of 380–450 nm or 375–400 nm. It has a high extinction coefficient near 600 nm. The two main maximum absorption and photoactivation peaks of hypericin occur near 550 and 600 nm. The two fluorescence peaks of hypericin are near 600 and 650 nm. Hypericin has very low water solubility. The aggregated hypericin is not fluorescent. It generates a high quantum yield of singlet oxygen and superoxides. Clinical Applications PDD of Cancers

The initial presentations of bladder cancer in 70–80% of the cases are superficial and limited

Hypericin Active form 82–94% High, 91–98.5% Greater stability, no significant photobleaching Hydrophobic – good permeability

to the urothelial lining of the bladder mucosa and submucosa. Visual differentiation between normal tissue and transitional cell carcinoma is relatively easy but not for carcinoma in situ or nonmalignant diseases such as cystitis due to radiotherapy or chemical or bacterial origins. They are often invisible to the naked eye. Fluorescence cystoscopy is a form of PDD of cancer and has significantly improved the diagnosis and early detection of cancer. In bladder cancer detection, hypericin has several advantages over conventional photosensitizer, 5-aminolevulinic acid (5-ALA, a prodrug) (Table 1). A human clinical trial had found that the use of hypericin showed higher sensitivity (82%) than just white-light cystoscopy (62%). This report justifies the use of hypericin-PDD for bladder cancer in clinical settings. Hypericin was also shown to accumulate in patients with stomach cancer and detected stomach cancer with 85% specificity. The synthetic hypericin has been tested clinically in patients with recurrent or progressive malignant gliomas who had received standard radiation therapy with or without chemotherapy. The rationale for postulating an antitumor effect for hypericin was originally related to its ability to inhibit protein kinase C, an enzyme that has been found to be correlated with glioma cell growth in vitro. In the phase 1 and 2 studies, hypericin was administered as an oral solution with doses ranging from 0.05 to 0.50 mg/kg, once each morning for up to 3 months. The treatment was reasonably well tolerable although some adverse events were observed which was expected from the pharmacology of hypericin such as photosensitivity reaction, erythema, and skin burning sensation.

Hypericin

The mean maximum tolerated dose was 0.40 0.098 mg/kg daily. The findings were promising as it provided stabilization or a slight (T transversions in ▶ lung cancer of smokers (tobacco-related lung cancer), and exposure to aristolochic acid and A>T mutations in upper-tract urinary cancers. Specific mutation patterns in TP53 may thus help identify environmental exposures that may be involved in ▶ carcinogenesis. Database Structure and Content The IARC TP53 database contains manually curated data that are extracted from peer-reviewed publications and public genomic databases. The database is a relational database that integrates data on somatic mutations in sporadic cancers, on germ line mutations in familial cancers, on TP53 gene status of cell lines, on polymorphisms in human population, on predicted and experimentally-assessed functional

IARC TP53 Database

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I

IARC TP53 Database, Fig. 1 Structure and contents of the IARC TP53 database

impacts of mutations, and on experimentallyinduced mutations. It is organized around a “gene variation module” to which six modules are connected: a “somatic module,” a “germ line module,” a “mouse-model module”, a “function module,” a “polymorphism module,” an “induced mutations module” (Fig. 1). The database design and content allow, for every single gene variation, to retrieve data on its functional and structural impact and data related to its expression in a somatic, cell line, or germ line context. Details on database contents and annotations are available at http://p53.iarc.fr/Manual.aspx. The “gene variation module” contains all possible single-nucleotide substitutions in the coding sequence and splice sites of TP53, plus all other sequence alterations that have been reported in human cancers, each alteration being a unique entry. Gene variations are described at the DNA and protein levels and are annotated with structural and functional information related to the position of the mutation in the protein sequence (functional domain, residue function, structural

motif, solvent accessibility of residue). For missense mutations, classifications based on the predicted or experimentally assessed functional impacts, and whether isoforms (10 isoforms are described for TP53) are affected are also available. The “polymorphism module” includes common gene variations found in healthy individuals and reported in publications or extracted from SNP databases, with links to dbSNP for information on population frequency and disease associations. The “somatic and germ line modules” contain data related to the somatic or germ line expression of mutations, respectively, with common set of dictionaries used to annotate pathological, clinical, and patient information. TP53 gene status of human cell lines is also provided with links to the ATCC catalog (cell line provider). The “function module” contains experimental data obtained on the activities and properties of p53 mutant proteins when transfected and overexpressed in human or yeast cells. Each entry in this module corresponds to the results of a set of experiments performed with one mutant in

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IARC TP53 Database

IARC TP53 Database, Fig. 2 Search interface for the IARC TP53 Database. Search options specific to each dataset are available (top panel). Search results are displayed with various graphical outputs (lower panel)

IARC TP53 Database

a specific cell type. Experimental assays that have been included were performed in yeast or human cells and were designed to (i) measure the transactivation activities of mutant proteins on reporter genes placed under the control of various p53 response elements, (ii) test the capacity of mutant proteins to induce cell cycle arrest or apoptosis, and (iii) exert dominant-negative effect over the wild-type protein, be temperature sensitive, or display various activities that are independent and unrelated to the wild-type protein (gain of function). A specific vocabulary has been implemented to describe experimental results in a standard format. The detailed annotation system used in all modules is described in the online user guide at http://p53.iarc.fr/Manual.aspx.

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on the data included in the database. Studies from which data are extracted have diverse designs and diverse ways of reporting mutations and related information. This diversity requires an effort of standardization of annotations and affects database analysis. It thus important that users are aware of the limitations and possible biases that may affect their analysis of the database. The IARC TP53 Database is a free service to the scientific community, and contributions from researchers and journal editors are most welcome to help us develop this important resource for cancer research.

Cross-References Web Site and New Search Tools The IARC TP53 web site (http://www-p53.iarc.fr/) provides a search interface for the core database and includes a comprehensive user guide, a slide show on TP53 mutations in human cancer, protocols and references for sequencing TP53 gene, and links to relevant publications and entries to bioinformatics and cancer databases. The database search interface allows the download of all datasets and proposes various tools for the selection, analysis, and downloads of specific sets of data according to user’s query (Fig. 2). Mutations reported in specific types of cancers and/or population groups can be analyzed with graphs that display their type by base substitutions (mutation patterns), codon distribution, position within the 3D structure of p53 DNA-binding domain and predicted or observed effect on protein (function patterns). Other graphs can be drawn to display the types of tumor associated with specific mutations expressed in a somatic or germ line context and to display the prevalence of mutations found in specific tumor types and/or population groups. A tool dedicated to the analysis of TP53 gene status in cancer cell lines is also available. Recommendation to Users The IARC TP53 Database is mainly based on peerreviewed publications. Trends in reporting and publishing mutations have thus a strong influence

▶ Carcinogenesis ▶ Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma ▶ Li-Fraumeni Syndrome ▶ Lung Cancer ▶ Molecular Pathology ▶ TP53 ▶ Tumor Suppressor Genes

References Hernandez-Boussard T, Montesano R, Hainaut P (1999) Sources of bias in the detection and reporting of p53 mutations in human cancer: analysis of the IARC p53 mutation database. Genet Anal 14:229–233 Olivier M, Hollstein M, Hainaut P (2010) TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol. 2(1):a001008 Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 Database. Hum Mutat 28:622–629 Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310 Vousden KH, Lane DP (2007) p53 in health and disease. Nat Rev Mol Cell Biol. 8(4):275–83

See Also (2012) CpG. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 990. doi:10.1007/978-3-642-16483-5_1360

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ICF Syndrome

(2012) Missense Mutation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2330. doi:10.1007/978-3-642-16483-5_3761 (2012) Mutation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Polymorphism. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2954– 2955. doi:10.1007/978-3-642-16483-5_4673 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) SNP. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3460. doi:10.1007/978-3-642-16483-5_5395 (2012) Tobacco-Related Lung Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3722. doi:10.1007/978-3-642-16483-5_5849 (2012) Ultraviolet Radiation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3841. doi:10.1007/978-3-642-16483-5_6102

IgM, are present in their blood. It has been shown that negative selection, which is a process of excluding autoreactive B-cells, is impaired, resulting in blockage of B-cell maturation and subsequent immunodeficiency. This causes severe recurrent infections, which are often seen in early childhood, and leads to early death of many patients. Almost all ICF patients have severe respiratory infections, and more than half go through recurrent gastrointestinal infections. Pericarditis, ear infections, septicemia, and oral Candida infections have also been observed. The immunodeficiency in ICF patients ranges from agammaglobulinemia to a mild reduction in immune response. Most patients have a poor immune response with low or undetectable levels of immunoglobulins. Low levels of T-cells are observed in about half of the patients.

ICF Syndrome

Centromeric Instability

Motoko Unoki and Hiroyuki Sasaki Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

Definition Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by mild facial dysmorphism, hypo- or agammaglobulinemia, and branching of chromosomes 1, 9, and 16 after mitogen stimulation of lymphocytes. Detection of DNA hypomethylation at pericentromeric satellite-2 and satellite-3 repeats is used for definitive diagnosis of this syndrome.

Characteristics Clinical and Cytological Features Immunodeficiency

ICF patients only possess naive (immature) B-cells in their peripheral blood; no or reduced number of memory and plasma cells, which produce immunoglobulins such as IgA, IgG, and/or

Centromeric instability is a distinctive feature of ICF syndrome. Chromosomes 1, 9, and 16 of ICF patients show whole-arm deletions, chromosome breaks, stretching (decondensation), and multiradial chromosome configurations (connections of chromosomes via centromeric/pericentromeric regions) in mitogen (phytohemagglutinin: PHA)stimulated lymphocytes (Fig. 1). Centromeric regions, which form kinetochore structure, are composed of a-satellite repeats, and pericentromeric (juxtacentromeric) regions, which form constitutive heterochromatin, are composed of satellite-2 and satellite-3 repeats. It is thought that trimethylation at lysine 9 of histone H3 (H3K9me3) and DNA methylation play an important role in establishing and maintaining the pericentromeric heterochromatin structure. Chromosomes 1 and 16 are characterized by long satellite-2 repeats, and chromosome 9 has long satellite-3 repeats, and all ICF patients have DNA hypomethylation at satellite-2 and satellite3 repeats, causing centromeric instability of these particular chromosomes. ICF syndrome is classified into at least five types. One type only shows DNA hypomethylation of satellite-2 and satellite-3 repeats in the pericentromeric regions (ICF1; see later), and the rest of the types have DNA

ICF Syndrome

ICF Syndrome, Fig. 1 Cytogenetic abnormalities in three patients with type-1 ICF syndrome (ICF1). (a) Multiradial configurations with multiple p and q arms derived from chromosomes 1 and 16 from patient 1. (b) Multiradial configurations with multiple p and q arms derived from chromosome 1 (upper) and a stretching of

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chromosome 1q (lower) from patient 2. (c) Multiradial configurations with multiple p and q arms derived from chromosome 1 (upper) and a stretching of chromosome 1q (lower) from patient 3 (Cited from Shirohzu et al. 2002 John Wiley & Sons, Inc.)

ICF Syndrome, Table 1 Types of ICF syndrome Causative gene Frequency (%) DNA methylation of satellite-2 and satellite-3 (pericentromeric regions) DNA methylation of a-satellite (centromeric regions)

hypomethylation of a-satellite repeats in the centromeric regions in addition to the pericentromeric repeats (ICF2 and ICFX; see later) (Table 1). All types show nearly identical symptoms and can be distinguished only by DNA methylation levels of these repeats.

Type 1 DNMT3B 50% Hypo

Type 2 ZBTB24 25% Hypo

Type3 CDCA7 10% Hypo

Type 4 HELLS 10% Hypo

Type X Unknown 5% Hypo

Normal

Hypo

Hypo

Hypo

Hypo

defects, respectively. These defects include slow cognitive and motor development and psychomotor impairment (ataxic gait and muscle hypotonia). Other congenital abnormalities in ICF patients are highly variable among the patients. Causative Genes and Molecular Background

Facial Anomalies

The dysmorphic facial features of ICF patients are usually mild. The typical features are hypertelorism (widely spaced eyes), a broad flat nasal bridge, and epicanthic folds. Some patients also show micrognathia (small jaw), low-set ears, and macroglossia (protrusion or enlargement of the tongue). Other Clinicopathological Features

Approximately, one-third and one-fifth of ICF patients have mental retardation and neurologic

Type-1 ICF Syndrome (ICF1)

ICF patients having DNA hypomethylation only at satellite-2 and satellite-3 in the pericentromeric regions are classified into type-1 ICF syndrome (ICF1) (Table 1). Mutations in the DNA (cytosine-5)methyltransferase 3B (DNMT3B) gene located on chromosome 20q11.21 are the cause of this type. DNMT3B is a de novo DNA methyltransferase, which establishes cell-lineage-specific DNA methylation patterns during embryogenesis. It is noteworthy that mutations observed in ICF1

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patients are hypomorphic at least in one allele; many ICF1 patients possess homozygous hypomorphic mutations, but some ICF1 patients are compound heterozygotes having a null mutation in one allele and a hypomorphic mutation in the other allele. Because Dnmt3b null knockout mice show embryonic lethality with multiple tissue defects, DNMT3B null mutations may also be lethal in human. ICF syndrome model mice, which possess ICF1 hypomorphic mutations, show phenotypes that are reminiscent of ICF1 patients, including DNA hypomethylation of the satellite repeats, distinct cranial facial anomalies, and T-cell death. Most of the mutations found in ICF1 patients attenuate catalytic activity of DNMT3B. A mutation found in its PWWP domain, which is reported to interact with H3K36me3, might cause mislocalization of DNMT3B. Type-2 ICF Syndrome (ICF2)

About half of ICF patients having DNA hypomethylation at a-satellite repeats at centromeric regions in addition to satellite-2 and satellite-3 repeats at pericentromeric regions possess mutations in the zinc-finger and BTB domain containing 24 (ZBTB24) gene located on chromosome 6q21 (Table 1). These patients are classified into type-2 ICF syndrome (ICF2). Unlike hypomorphic mutations in the DNMT3B gene found in ICF1 patients, some ICF2 patients possess null mutation in the ZBTB24 gene, indicating that this protein is dispensable for embryogenesis and development at least in human. The ZBTB24 protein has a BTB domain and an AT-hook domain in its N-terminal region and eight C2H2type zinc-finger domains in its C-terminal region. Most of the mutations found in ICF2 patients cause partial or complete loss of zinc-finger domains of this protein, and the rest of the mutations, which do not cause loss of the domains, alter highly conserved cysteines in the C2H2 motif of the domains. The zinc-finger domains have been shown to be important for the protein to target pericentromeric heterochromatin regions. It is also shown that DNA methylation and DNA sequences of centromeric/ pericentromeric regions are not important for the

ICF Syndrome

localization of ZBTB24. Knockdown of ZBTB24 in mouse primary embryonic fibroblast (MEFs) using siRNAs leads to hypomethylation at centromeric repeats, suggesting that this protein is involved in the maintenance of DNA methylation of the repeats. Type-3 ICF syndrome (ICF3)

About quarter of ICF patients having DNA hypomethylation at both a-satellite and satellite2 and -3 repeats possess mutations in the cell division cycle-associated protein 7 (CDCA7) gene located on chromosome 2q31 (Table 1). These patients are classified into type-3 ICF syndrome (ICF3). CDCA7 possesses a four CXXCtype zinc finger domain and a c-Myc/14-3-3 interaction domain. All patients identified so far possess homozygous missense mutations near the first two CXXC motifs in the conserved carboxyterminal zinc finger domain. It is reported that CDCA7 is involved in neoplastic transformation, MYC-dependent apoptosis, and hematopoietic stem cell emergence, however its molecular function is unknown. Knockdown of CDCA7 using siRNAs in mouse primary MEFs leads to hypomethylation at centromeric repeats, suggesting that this protein is involved in the maintenance of DNA methylation of the repeats. Type-4 ICF syndrome (ICF4)

About another quarter of ICF patients having DNA hypomethylation at a-satellite and satellite2 and -3 repeats possess mutations in the helicase lymphoid-specific (HELLS) gene, whose alternative name is lymphoid specific helicase (LSH), located on chromosome 10q23 (Table 1). These patients are classified into type-4 ICF syndrome (ICF4). HELLS is a chromatin remodeling factor, which possesses a SNF2 family N-terminal domain and a helicase C-terminal domain. Mutations in the gene are various. Some patients carry null mutations in both alleles, while mutations in some other patients are found in its C-terminal region and do not destroy either the SNF2 family N-terminal domain or the helicase C-terminal domain, suggesting that the C-terminal region of the protein has some important function. It is reported that HELLS mediates de novo DNA

ICF Syndrome

methylation in plant (Arabidopsis thaliana) and mice through an interaction with Dnmt3b; the plant homologue of the gene is decreased in DNA methylation (DDM1). Genome wide loss of CpG methylation is observed in HELLS knockout mice. This gene is preferentially expressed in lymphoid cells in adult mice and has been shown to be important for normal lymphoid development. Others (Type X)

There are about 5% of ICF syndrome patients who do not have mutations in these four genes (Table 1). These patients possess DNA hypomethylation of a-satellite repeats in the centromeric regions in addition to that of satellite-2 and -3 repeats in the pericentromeric regions as ICF2, 3 and 4 patients. Causative genes for this type(s) are under investigation. Clinical Management Early immunoglobulin supplementation can improve the course of this disease as supportive care. However, causal therapy is still under development. Similarity Between ICF Syndrome and Human Cancers It is reported that pericentromeric heterochromatin of chromosomes 1 and 16 is frequently hypomethylated in a variety of human cancers such as ovarian epithelial carcinomas, breast adenocarcinomas, and Wilms tumors, as observed in ICF syndrome. The DNA hypomethylation observed in cancer cells could cause imbalance of chromosome arms and gene dosage. Actually, chromosomal rearrangements at the vicinity of pericentromeric heterochromatin are observed in many cancers. Multiradial chromosomes with multiple arms joined in the pericentromeric regions observed in ICF patients might be unstable intermediates in the formation of more stable pericentromeric rearrangements observed in cancer cells.

Cross-References ▶ Hypomethylation of DNA ▶ Methylation

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References de Greef JC, Wang J, Balog J, den Dunnen JT, Frants RR, Straasheijm KR, Aytekin C, van der Burg M, Duprez L, Ferster A, Gennery AR, Gimelli G, Reisli I, Schuetz C, Schulz A, Smeets DF, Sznajer Y, Wijmenga C, van Eggermond MC, van Ostaijen-Ten Dam MM, Lankester AC, van Tol MJ, van den Elsen PJ, Weemaes CM, van der Maarel SM (2011) Mutations in ZBTB24 are associated with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2. Am J Hum Genet 88:796–804 Ehrlich M, Jackson K, Weemaes C (2006) Immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF). Orphanet J Rare Dis 1:2 Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CM, Gartler SM (1999) The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci U S A 96:14412–14417 Jiang YL, Rigolet M, Bourc’his D, Nigon F, Bokesoy I, Fryns JP, Hultén M, Jonveaux P, Maraschio P, Mégarbané A, Moncla A, Viegas-Péquignot E (2005) DNMT3B mutations and DNA methylation defect define two types of ICF syndrome. Hum Mutat 25:56–63 Nitta H, Unoki M, Ichiyanagi K, Kosho T, Shigemura T, Takahashi H, Velasco G, Francastel C, Picard C, Kubota T, Sasaki H (2013) Three novel ZBTB24 mutations identified in Japanese and Cape Verdean type 2 ICF syndrome patients. J Hum Genet 58:455–460 Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257 Shirohzu H, Kubota T, Kumazawa A, Sado T, Chijiwa T, Inagaki K, Suetake I, Tajima S, Wakui K, Miki Y, Hayashi M, Fukushima Y, Sasaki H (2002) Three novel DNMT3B mutations in Japanese patients with ICF syndrome. Am J Med Genet 112:31–37 Thijssen PE, Ito Y, Grillo G, Wang J, Velasco G, Nitta H, Unoki M, Yoshihara M, Suyama M, Sun Y, Lemmers RJLF, de Greef JC, Gennery A, Picco P, KloeckenerGruissem B, Gungor T, Reisli I, Picard C, Kebaili K, Roquelaure B, Iwai T, Kondo I, Kubota T, van Ostaijen-Ten Dam MM, van Tol MJD, Weemaes C, Francastel C, van der Maarel SM, Sasaki H (2015) Mutations in CDCA7 and HELLS cause Immunodeficiency, Centromeric Instability and Facial 2 Anomalies Syndrome. Nat Commun. In press Ueda Y, Okano M, Williams C, Chen T, Georgopoulos K, Li E (2006) Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome. Development 133:1183–1192 Xu GL, Bestor TH, Bourc’his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Péquignot E (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402:187–191

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ICL670A ▶ Deferasirox

Idiopathic Myelofibrosis ▶ Primary Myelofibrosis

Idiotype Vaccination Maurizio Bendandi Department of Clinical Medicine, School of Medicine, Ross University, Roseau, Commonwealth of Dominica

Synonyms Anti-idiotype vaccination; Idiotypic vaccination

Definition Idiotype vaccination is an ▶ immunotherapy procedure based on the fact that, save for its very early stage of differentiation, each clone of B lymphocytes features a specific antibody on the cell surface. The most variable portion of this antibody is a unique feature of the corresponding clone. Its natural function is that of specifically recognizing an antigen, but it can also be used as an antigen (idiotype) itself, that is, as a potential vaccine target and tool. The most relevant context in which this second function is exploited is idiotype vaccination as a treatment for human ▶ B cell lymphoma.

Characteristics Field of Application The vast majority of B cell lymphomas consist of the clonal expansion of neoplastic B cells, all

ICL670A

featuring the same surface antibody and consequently the same idiotype. However, each patient’s tumor presents with a different, patient-, and tumor-specific idiotype. Therefore, an individualized, custom-made idiotype vaccine must be produced for each patient. Most of what we know about idiotype vaccination in human B cell lymphoma derives from studies conducted in an indolent subset called ▶ follicular lymphoma. Far less has been instead concluded so far as to whether or not the same approach could be successful in other B cell malignancies, such as ▶ mantle cell lymphoma, ▶ multiple myeloma, and ▶ chronic lymphocytic leukemia, or even in certain solid tumors, whenever an anti-idiotype monoclonal antibody structurally mimics tumor-associated antigens other than antibodies. Nevertheless, idiotype vaccination stands out as the most successful human cancer vaccine, since over the last 20 years has provided the first formal proofs of principle concerning biological efficacy, clinical efficacy, and clinical benefit of such a procedure. Formulation Although idiotypic vaccination has been tested in humans with different formulations, such as soluble protein idiotype associated with an immunogenic carrier and an immunologic adjuvant, ▶ dendritic cells pulsed with the soluble protein idiotype, or idiotype ▶ DNA vaccine, most clinical results and all proofs of principle have been obtained using the first of these three options. In particular, the most successful idiotype vaccine formulation consists of soluble protein idiotype, that is, the patient- and tumor-specific antigen, which is conjugated with keyhole limpet hemocyanin (KLH), the immunogenic carrier, and administered together with granulocyte-macrophage colony-stimulating factor (GM-CSF), the immunologic adjuvant. Production While KLH and GM-CSF are commercially available, soluble protein idiotype needs to be produced one patient at a time through either hybridoma or recombinant technology. In the

Idiotype Vaccination

former case, each patient’s tumor cells, which are capable of producing the idiotype, but not to release it, are fused with a cell line that vice versa can release an idiotype, but is unable to produce it. The hybridoma resulting from the fusion process features both functions, can be cultivated in vitro, and releases therapeutic amounts of the patient- and tumor-specific, soluble protein idiotype. In the latter scenario, the genetic information encoding for the idiotype is introduced by means of a vector inside mammalian, insect, plant, or bacteria cells, which will ultimately replace the hybridoma as a factory for soluble protein idiotype production. Clinical Aspects Idiotype vaccination has provided the first formal proof of biological efficacy in 1992, when scientists at Stanford University showed that patients with follicular lymphoma were capable of developing a specific, anti-idiotype immune response after receiving idiotype vaccination. Seven years later, researchers at the National Cancer Institute proved clinical efficacy of idiotype vaccination by showing that in most follicular lymphoma patients who had received it, the immune system had become capable of killing in vivo tumor cells that had survived prevaccine ▶ chemotherapy. Finally, in 2006, clinical benefit of idiotype vaccination was proved at the University of Navarra by showing that all follicular lymphoma patients who respond to the vaccine from an immunologic standpoint experience a significant prolongation of their disease-free survival. Idiotype vaccination’s procedure typically consists of a monthly, subcutaneous injection of 0.5 mg of idiotype conjugated with 0.5 mg of KLH, administered together with 125 mcg of GM-CSF. The same dose of GM-CSF alone is then given daily over the following 3 days at the same site of the complete vaccine formulation delivery. After five or six monthly doses, further boosts every 2–3 months are becoming increasingly popular and recommended. Idiotype vaccination’s side effects are mild and mostly local, if at all present. Flu-like symptoms are rare and self-limiting.

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Open Questions To date, a number of questions remain open with respect to idiotype vaccination. For instance, from a clinical standpoint, we do not know whether this form of active ▶ immunotherapy has the potential to cure or just to control the disease in lymphoma patients who respond to it. Nor do we know whether patients with B cell malignancies other than follicular lymphoma may benefit from it. The former question implies that it remains currently impossible to determine whether the administration of boosts should or could be stopped in patients who have responded to the vaccine from an immunologic point of view, irrespective of whether that immune response persists to be detectable, and remain free of disease. The latter question implies instead that only through well-designed ▶ clinical trials it might be possible to test idiotype vaccination in settings other than follicular lymphoma. In particular, it has to be reminded that typical randomized studies may often fail to serve the purpose of proving the clinical benefit of a customized form of active immunotherapy. Indeed, two of the three randomized clinical trials on idiotype vaccination carried out in the setting of follicular lymphoma failed to achieve their main endpoints likely owing to study design pitfalls, while the third study did confirm clinical benefit and yet failed to achieve regulatory approval due to an incomplete patient accrual. Similarly, surrogate endpoints such as those defining minimal residual disease also need to be used with caution. For instance, in the very case of follicular lymphoma, both ▶ BCL-2 rearrangement assessed by molecular fingerprinting through qualitative and quantitative polymerase chain reaction and tumor cell phenotype assessed through ▶ flow cytometry did not always correlate with immune responses and clinical outcome. As for the adequate timing for treating patients with this immunotherapeutic approach, it seems to have become ever clearer that the best clinical setting to use idiotype vaccination is a good quality clinical complete response. This prevaccine result can be achieved nowadays through a variety of old and new therapeutic approaches such as standard or high-dose chemotherapy and cold or

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passive immunotherapy using radioimmunoconjugates. However, it is likely that in lymphoma patients selected to later undergo idiotype vaccination, prevaccine treatment should privilege agents and procedures with the lowest immune suppressive potential. At the opposite side of the widening spectrum of possible applications of idiotype vaccination stands the anecdotal attempts conducted with healthy donors undergoing this totally safe procedure with the myeloma-specific soluble protein idiotype obtained from their sibling recipient before hematopoietic stem cell allotransplant. Clinical and biological results remain incompletely understood, though extremely appealing from a purely scientific point of view. Another crucial context in which idiotype vaccination specialists still struggle is that of defining the relevance of idiotype vaccine-induced humoral and cellular responses. In fact, it is not yet clear whether, though both desirable, just either of them is required for patients to experience clinical benefit. The question is of no little importance, considering that in nearly half of the patients with any vaccine-induced, idiotypespecific immune response, either type of immune response is not detectable. Currently, humoral responses, that is, those featuring vaccine-induced anti-idiotype antibodies, are assessed and monitored in the lab by a single, relatively standardized method, whereas at least half a dozen of nonstandardized techniques are used in different labs worldwide to assess and monitor vaccineinduced cellular responses.

Cross-References ▶ B-cell Lymphoma ▶ Bcl2 ▶ Chemotherapy ▶ Chronic Lymphocytic Leukemia ▶ Clinical Trial ▶ Dendritic Cells ▶ DNA Vaccination ▶ Flow Cytometry ▶ Follicular Lymphoma ▶ Immunotherapy

Idiotypic Vaccination

▶ Inflammatory Response and Immunity ▶ Mantle Cell Lymphoma ▶ Multiple Myeloma

References Bendandi M (2006) Clinical benefit of idiotype vaccines: too many trials for a clever demonstration? Rev Recent Clin Trials 1:67–74 Bendandi M (2009) Idiotype vaccines for lymphoma: proof-of-principles and clinical trial failures. Nat Rev Cancer 9:675–681 Bendandi M, Gocke CD, Kobrin CB et al (1999) Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat Med 5:1171–1177 de Cerio AL, Inogés S, Ai WZ, Villanueva H, Pastor F, Soldevilla MM, Soria E, Bendandi M (2013) Successful idiotypic vaccination following stem cell allotransplant in lymphoma. Leuk Lymphoma 54:881–884 Inogés S, Rodríguez-Calvillo M, Zabalegui N et al (2006) Clinical benefit associated with idiotypic vaccination in follicular lymphoma. J Natl Cancer Inst 98:1292–1301 Inoges S, de Cerio AL, Villanueva H, Pastor F, Bendandi M (2014) Idiotype vaccine production using hybridoma technology. Methods Mol Biol 1139:367–387 Kwak LW, Campbell MJ, Czerwinski DK et al (1992) Induction of immune responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotype expressed by their tumors. N Engl J Med 327:1209–1215

See Also (2012) Hematopoietic stem cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1645. doi:10.1007/978-3-642-16483-5_2619 (2012) Hybridomas. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1775. doi:10.1007/978-3-642-16483-5_2884 (2012) Idiotype. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1804. doi:10.1007/978-3-642-16483-5_2944 (2012) Monoclonal antibody. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Radioimmunoconjugates. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3148. doi:10.1007/978-3-642-16483-5_4917 (2012) Recombinant. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3205. doi:10.1007/978-3-642-16483-5_4991

Idiotypic Vaccination ▶ Idiotype Vaccination

Imatinib

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Definition

IDO ▶ Indoleamine 2,3-Dioxygenase

I-FLICE ▶ FLICE-Inhibitory Protein

It is a small molecular weight compound that inhibits tyrosine kinases including ABL, KIT (▶ Kit/stem cell factor receptor in oncogenesis), and PDGFR (▶ platelet-derived growth factor receptor). It has significant antitumor activity in ▶ chronic myeloid leukemia (CML) and ▶ gastrointestinal stromal tumors.

iFOBT Characteristics ▶ Fecal Immunochemical Test

IgE-Mediated Hypersensitivity ▶ Allergy

IHC ▶ Immunohistochemistry

IL-6 ▶ Interleukin-6

ILP1 ▶ XIAP

Imatinib Brian J. Druker Oregon Health and Science University Cancer Institute, Portland, OR, USA

Synonyms CGP57148; Imatinib mesylate; STI571; STI-571

Imatinib (US brand name: Gleevec, foreign brand name: Glivec, formerly STI571 or CGP57148) is a ▶ tyrosine kinase inhibitor with activity against all of the ABL tyrosine kinases including ▶ BCRABL1, ABL, v-ABL, and ARG (Abelson-related gene). Besides the ABL tyrosine kinase, other kinases inhibited by imatinib are the ▶ plateletderived growth factor receptor alpha (PDGFRA) and beta (PDGFRB) and KIT. Given the critical role of ▶ receptor tyrosine kinases in the regulation of cellular growth and known activating mutations that cause several cancers, it was hypothesized that specific inhibitors of these protein kinases might be effective anticancer agents. Beginning in the late 1980s, scientists at Ciba-Geigy (now Novartis), under the direction of N. Lydon and A. Matter, performed highthroughput screens of chemical libraries searching for compounds with kinase inhibitory activity. From this time-consuming approach, a lead compound was identified. Its inhibitory activity against the PDGFR was optimized by synthesizing a series of chemically related compounds and analyzing their relationship between structure and activity (▶ drug design). The most potent molecules in the series were dual inhibitors of the PDGFR and ABL kinases. Of the several compounds generated from this program, imatinib emerged as the lead compound for clinical development based on its superior in vitro selectivity against CML cells and its drug-like properties, including pharmacokinetic and formulation properties (pharmacokinetics; pharmacodynamics).

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Imatinib

BCR-ABL1 and CML as a Target for Imatinib CML is characterized by the presence of the ▶ BCR-ABL1 ▶ fusion gene and protein. It arises from a reciprocal translocation between the long arms of chromosomes 9 and 22 (▶ chromosomal translocations) that generates a shortened chromosome 22 termed the Philadelphia chromosome. This was the first consistent chromosome abnormality associated with a human cancer. The (9;22) translocation results in fusion of the ABL ▶ oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (BCR), giving rise to a chimeric BCR-ABL1 gene. This fusion gene is transcribed and translated into a protein that functions as a constitutively activated tyrosine kinase. BCR-ABL1 induces a CML-like syndrome when introduced into bone marrow cells of mice (▶ mouse models), confirming its causative role in the human disease. All of the transforming activities of BCR-ABL1 are dependent on its tyrosine kinase activity; thus, a specific inhibitor of this kinase would be predicted to be an effective and selective therapeutic agent for CML (Fig. 1). In a pivotal set of ▶ preclinical testing, imatinib was shown to suppress the proliferation of BCR-ABL1-expressing cells in vitro and in vivo. In colony-forming assays of peripheral blood or bone marrow from patients with CML, imatinib caused a 92–98% decrease in the number of BCR-ABL1 colonies formed, with minimal inhibition of normal colony formation.

ATP

ATP

ST1571

ADP PO4

Bcr-abi tyrosine kinase

TYR

Substrate

CML

Bcr-abi tyrosine kinase

TYR

Substrate

CML

Imatinib, Fig. 1 Schematic representation of the mechanism of action of the BCR-ABL tyrosine kinase and its inhibition by STI571 (imatinib)

Activity of Imatinib in CML Given the central role of the BCR-ABL1 tyrosine kinase in causing CML and the favorable preclinical profile of imatinib, CML was selected as the initial disease in which to test imatinib. In a standard dose-escalation Phase I study (▶ Clinical Trial) conducted in patients with CML, imatinib was well tolerated. Significant clinical benefits were observed at doses of 300 mg per day and above. In patients with chronic-phase disease who were interferon resistant or intolerant, 53 of 54 (98%) had their previously abnormal blood counts return to normal, typically within 4–6 weeks after beginning imatinib. Ninety percent of these responses lasted beyond 1 year. In patients with myeloid ▶ blast crisis, the most advanced phase of the disease, 21 of 38 (55%) patients responded, with 18% having responses lasting beyond 1 year. Imatinib rapidly advanced through Phase II and III testing for patients with CML and was FDA approved in May 2001. It is now the standard initial therapy for patients with CML. A 5-year update of imatinib as initial therapy for patients with CML demonstrates an overall survival of 89%. At 5 years, an estimated 93% of patients remain free from disease ▶ progression to advanced phases of the disease: accelerated phase or blast crisis. Most of the side effects of imatinib are classified as mild to moderate and include low blood counts, ▶ myelosuppression, fluid retention, diarrhea, nausea, muscle and joint aches, and skin rashes. Mechanisms of Relapse/Resistance to Imatinib Response rates to imatinib in CML patients with chronic-phase disease are quite high and, thus far, have been durable. Response rates are also quite high in patients with advanced-phase disease, but relapses, despite continued therapy with imatinib, are common. In the largest studies of resistance or relapse, several consistent themes have emerged. In the majority of patients who respond to imatinib but subsequently relapse while remaining on therapy, the ▶ BCR-ABL1 kinase has been reactivated. ▶ BCR-ABL1 kinase activity was analyzed by assessing tyrosine

Imatinib

phosphorylation of CRKL, a direct substrate of the BCR-ABL1 kinase, and the major tyrosinephosphorylated protein in samples from patients with CML. In these studies, between 50% and 90% of relapsed patients have a BCRABL1 point mutation located in one of over 40 different amino acids scattered throughout the ABL kinase domain. Some other patients have ▶ amplification of BCR-ABL1 at the genomic or transcript level. In contrast, in patients with primary resistance – that is, patients who do not respond to imatinib therapy – BCR-ABL1independent mechanisms are most common. In patients who relapse as a consequence of reactivation of the BCR-ABL1 kinase, the BCR-ABL1 kinase remains a good target. Alternate ABL kinase inhibitors are already in clinical trials (▶ nilotinib, dasatinib), and dasatinib is FDA approved for patients with CML with imatinib resistance. There remains at least one imatinib-resistant mutation, T315I, that is not inhibited by either drug. Activity of Imatinib in Other Diseases Given the success of imatinib in CML, it was logical to try imatinib in other diseases where activated tyrosine kinases targeted by imatinib have causative roles. Thus, imatinib also has significant activity in patients with ▶ acute lymphoblastic leukemia who are BCR-ABL1 positive. Another tumor in which imatinib has shown activity is ▶ gastrointestinal stromal tumor (GIST). GISTs are mesenchymal neoplasms that can arise in any organ in the gastrointestinal tract or from the mesentery or omentum. The majority of GISTs express KIT, and in 90% of cases, KIT activation is linked to mutation, usually involving exon 9 or 11. Published data suggest that the response rate of GISTs to single- or multi-agent ▶ chemotherapy is less than 5%. In contrast, the response rate to imatinib as a single agent in patients with advanced GIST was 53–65% with another 19–36% of patients having disease stabilization. Mutational status helps predict response to imatinib in GIST patients. Patients whose tumors contain the most common activating KIT mutation exon 11 have a significantly higher partial response rate (83.5%) to imatinib therapy than

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did patients whose tumors had no detectable mutations in KIT. Patients whose tumor harbored a KIT exon 9 mutation did less well than those with an exon 11 mutation but responded better (response rate of 48.7%) and had a longer timeto-treatment failure than those with no detectable mutation. Similar to the situation in CML, many imatinib-resistant tumors have acquired kinase mutations in the kinase domain of KIT. However, resistance mechanisms in GISTs are more heterogeneous than those seen in chronic-phase CML as some tumors actually lose KIT expression and other tumors become imatinib resistant without acquisition of secondary kinase mutations. Again, similar to the situation with CML, novel KIT kinase inhibitors are being tested in patient with imatinib-resistant GIST, and one of them, sunitinib, has been FDA approved for this indication. Imatinib also has activity in neoplasms that are caused by oncogenic activation of PDGFRs (▶ platelet-derived growth factor receptors). This includes the subset of patients with chronic myelomonocytic leukemia that results from fusion of the EVT6 (TEL) and PDGFRB genes. Similarly, dermatofibrosarcoma protuberans, a low-grade sarcoma of the dermis, is characterized by a (17;22) translocation involving the COL1A1 and PDGF-B genes, which results in overproduction of fusion COL1A1-PDGF-BB ligand and consequent hyperactivation of PDGFRB. Patients with this tumor also respond to imatinib. Hypereosinophilic syndrome is another example of an imatinib-sensitive disease. In this case a PDGFRA fusion gene product (FIP1L1-PDGFRA) is the activated target. Conclusion The development of imatinib and its clinical application demonstrate an emerging paradigm in cancer therapy where the tumor is defined by molecular genetic abnormalities instead of the tissue of origin. It further demonstrates that effectiveness of cancer therapy when treatments target events critical to the growth and survival of specific tumors (▶ Personalized Cancer Medicine).

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Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Amplification ▶ BCR-ABL1 ▶ Blast Crisis ▶ Chemotherapy ▶ Chromosomal Translocations ▶ Chronic Myeloid Leukemia ▶ Clinical Trial ▶ Drug Design ▶ E T V6 ▶ Fusion Genes ▶ Gastrointestinal Stromal Tumor ▶ Kit/Stem Cell Factor Receptor in Oncogenesis ▶ Mouse Models ▶ Myelosuppression ▶ Nilotinib ▶ Oncogene ▶ Personalized Cancer Medicine ▶ Platelet-Derived Growth Factor ▶ Preclinical Testing ▶ Progression ▶ Receptor Tyrosine Kinases ▶ STI-571 ▶ Tyrosine Kinase Inhibitors

References Druker BJ (2006) Circumventing resistance to kinase-inhibitor therapy. N Engl J Med 354:2594–2596 Druker BJ, Tamura S, Buchdunger E et al (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566 Druker BJ, Talpaz M, Resta DJ et al (2001) Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344:1031–1037 Druker BJ, Guilhot F, O’Brien SG et al (2006) Five-year follow- up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 355:2408–2417 Heinrich MC, Corless CL, Demetri GD et al (2003) Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21:4342–4349

See Also (2012) ABL. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 14. doi:10.1007/ 978-3-642-16483-5_15

Imatinib (Gleevec) (2012) Dasatinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1060. doi:10.1007/978-3-642-16483-5_1518 (2012) Dermatofibrosarcoma Protuberans. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1087. doi:10.1007/978-3-642-164835_7044 (2012) Exon. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1360. doi:10.1007/978-3-642-16483-5_2059 (2012) FDA. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1386. doi:10.1007/978-3-642-16483-5_2136 (2012) KIT. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1945–1946. doi:10.1007/978-3-642-16483-5_3228 (2012) Mutation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3911 (2012) Pharmacodynamics. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2840. doi:10.1007/978-3-642-164835_4495 (2012) Pharmacokinetics. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2845. doi:10.1007/978-3-642-16483-5_4500 (2012) Philadelphia Chromosome. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2864. doi:10.1007/978-3-642-164835_4520 (2012) Platelet-Derived Growth Factor Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2910. doi:10.1007/9783-642-16483-5_4612 (2012) Reciprocal Translocation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3204. doi:10.1007/978-3-642-164835_4989 (2012) Sunitinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3562. doi:10.1007/978-3-642-16483-5_5575 (2012) Tyrosine Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3822. doi:10.1007/978-3-642-16483-5_6079

Imatinib (Gleevec) ▶ Receptor Tyrosine Kinase Inhibitors

Imatinib Mesylate ▶ Imatinib

Immune Escape

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adaptation of tumor cells to evade the immune systems or developing a microenvironment that Imidazo[5,1-d]-1,2,3,5-tetrazine-8carboxamide, 3, 4-dihydro-3-methyl- suppresses the immune system. 4-oxo▶ Temozolomide

Immediate Early Stress Response ▶ Stress Response

Immune Escape Jiahua Qian1, Tamara Floyd2, Yufei Jiang2, Anat Ohali2, Raed Samar2, Hong Yang2 and Samir N. Khleif3 1 Qiagen, Frederick, MD, USA 2 Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 GRU Cancer Center, Augusta, GA, USA

Definition Immune escape is one of the hallmarks of cancer development and ▶ metastasis. It is characterized by the lack of ability of the immune system to eliminate transformed cells prior to and after tumor development.

Characteristics Several mechanisms have been proposed and tested to explain cancer immune escape. It is now evident that both the host as well as the tumor play important roles in this phenomenon. The host’s contribution is manifested by the host’s ability to recognize to antigens expressed by tumor cells, a phenomenon known as “host ignorance.” It happens because of defects in both the innate and adaptive arms of the immune system. The tumor’s contribution is manifested by the

Host Ignorance The innate arm of the immune system forms a first line of defense against cancers. ▶ Macrophages, ▶ dendritic cells, and monocytes are essential in eliminating cancers. Another type of cell involved in the elimination of cancers is NK cells. These cells are capable of recognizing and attacking cells that have downregulated MHC class I expression. This phenomenon is known as “missing self-recognition.” NK cells can effectively eliminate cells that have been altered due to cellular transformation, infection, or other carcinogens and cannot present new antigens. If these players involved in the ▶ innate immune system are defective, cancer cells may escape tumor immunosurveillance from not only innate immunity but also ▶ adaptive immunity as NK cells, macrophages, and NK T cells play a role in the induction and enhancement of ▶ adaptive immune responses. The adaptive arm of the immune system has also been shown to play a crucial role in recognizing and eliminating cancer cells. However, several defects have been shown to hamper the ability of this arm to combat tumor cells. 1. Defects in Antigen-presenting cell (APC). To elicit an effective immune response, antigens must be processed first by the APCs and presented to the immune system. Dendritic cells (DCs) are professional APCs and play an important role in regulation of adaptive immune response. Many tumor patients have shown defects in the DC compartment in terms of cell number and/or altered function. Tumorinfiltrating DCs have defective surface expression of HLA and B7 costimulatory molecules and thus are likely to induce anergy rather than to stimulate tumor-specific T cells. Additionally, the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO), which has been implicated as one mechanism that helps to maintain maternal tolerance toward the fetus during pregnancy, was recognized as

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a potential mechanism of tolerance in malignancies. Expression of IDO on APCs was observed in tumor-draining lymph nodes, which may be indicative of the creation of a tolerogenic microenvironment. The molecular mechanism by which IDO suppresses T cells is still being elucidated. It was suggested to be mediated by depletion of the essential amino acid tryptophan and by the generation of pro-apoptotic metabolites. 2. Negative immune regulation by suppressive cells. There are several types of suppressive cells in the immune system, including ▶ regulatory T cells, myeloid suppressor cells (MSCs) and NKT cells. They exist to keep the immune response under control and prevent autoimmunity. In addition, these cells have been shown to inhibit immune responses against tumor cells. (i) Regulatory T (Treg) cells are a subpopulation of CD4+T cells. Tregs are crucial for controlling autoreactive ▶ T cell responses. At least two distinct subsets of Tregs have been identified: natural and inducible Tregs. Natural Tregs are T cells that arise in the thymus. They require direct cell contact with their target cells in order to exert their regulatory functions. Inducible Tregs arise in the periphery and exert their regulatory functions by the soluble cytokines IL-10 and TGF-b. The exact mechanisms by which Tregs exert their regulatory functions remain largely unknown. However, there is sufficient evidence, both in humans and in rodents, demonstrating that Tregs play crucial roles in suppressing antitumor responses. (ii) Myeloid suppressor cells were first identified by the expression of surface marker CD11b and Gr-1. Their suppressive activity was shown by inhibiting lymphocyte proliferation and inducing apoptosis in CD8 T cells. (iii) NKT cells are a unique sub-lineage of T cells. However, in contrast to conventional and regulatory T cells that recognize peptides in the context of MHC class I and II molecules, NKT cells recognize glycolipids presented by CD1d, a nonclassical antigen-presenting molecule. This difference in recognition means that NKT cells can recognize a class of antigens

Immune Escape

that conventional T cells cannot recognize. NKT cells have been shown to promote as well suppress immune responses to cancer. Currently, it is still unclear what factors determine their function. 3. Defective antitumor T cells. Another factor that leads to a failed immune response to tumor is cell dysfunction in tumor-infiltrating lymphocytes (TILs). In a number of studies, TILs have been shown to have defective antitumor killing effect. This dysfunction may result from the ineffective granule exocytosis. TILs also have been reported to have defective cytokine production and low expression of B7.1 and B7.2 costimulatory molecules, suggesting anergy could occur. Tumor Cell Adaptation To escape immune surveillance, tumor cells deploy several strategies. 1. Downregulation of MHC class I expression on cell surface. The presentation of antigen by MHC class I is crucial for immune responses. By downregulating MHC class I molecules on the surface, tumor cells may become elusive targets for T cells, thereby escaping the destruction by cytotoxic T lymphocytes (CTLs). 2. Suppression of the immune system. Tumor cells have several “counterattack” methods to fight the immune system. These include: (i) secretion of immunosuppressive cytokines such as IL-10, TGF-b, IL-13, etc. IL-10 interferes with the induction of antitumor responses. It has been shown to block dendritic cell-mediated priming of T cells into CTL effectors in vitro, and its expression in serum appears to be associated with negative prognosis in certain cancers. ▶ TGF-b is another cytokine that inhibits activation, proliferation, and activity of T lymphocytes. This ▶ cytokine is often found at high levels in malignancies and is associated with a poor prognosis as well as lack of response to tumor immunotherapy. Both cytokines can be produced by the tumor cells themselves or by infiltrating stroma cells. Interestingly, TGF-b has also been shown to

Immunochemical Fecal Occult Blood Test

drive expansion of regulatory T cells, thus bringing together two evasion mechanisms. (ii) Upregulation of tumor cells of Fas ligand on the surface to induce apoptosis of tumorkilling effector cells. The death-inducing FAS/APO-1/CD95 ligand (FasL/CD95L) has been reported to be expressed on many human tumors of various origins. Expression of FasL in tumors implies that cancer cells are resistant to Fas-induced cell death, which preserves them from extermination by cytotoxic T cells and actively induces death of effector T cells. In addition to local defense, accumulating evidence suggests that the FasL expression may also be relevant for tumor progression and formation of metastasis. It was further described that tumor cells are able to release membrane vesicles (MV) or exosomes containing FasL which induce ▶ apoptosis of activated T cells. This apoptosis-inducing pathway, via the release of FasL-positive MV, may indeed play a significant role in eliminating the most effective component of the antitumor T-cell response and provides an explanation for the observed spontaneous T cell apoptosis in the peripheral circulation of cancer patients. (iii) Inhibition of effector cells by inhibitory ligands including PD-L1, CTLA-4, and LAG-3. Evidence suggests that tumor cells may evade T and NK cell recognition by receptor-ligand interaction. CTLA-4 is a receptor predominantly found on T lymphocytes interacting with CD86 ligand. CTLA-4 is related to CD28 on T lymphocytes, but plays an opposite role. CTLA-4 may act at the level of TCR or CD28 signaling to inhibit T cell activation. Several studies provide evidence that CTLA-4 can upregulate TGF-b expression and can attenuate CD28 signals on Treg expansion and survival. The precise mechanism of T cell inactivation by CTLA-4/CD86 signaling, however, remains uncertain. The PD-1 – PD-L pathway has been postulated to regulate the immune response in both lymphoid and non-lymphoid organs. PD-Ls are expressed in many tumors such as ▶ ovarian cancer, ▶ esophageal cancer, kidney cancer, and ▶ brain cancer. It is suggested that tumor associated PD-Ls may promote T cell apoptosis and

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thus affect immune responses. Another inhibitory ligand associated with MHC class II interactions is LAG-3 (lymphocyte activation gene-3). LAG-3-MHC class II could enhance the cross-talk between T cell and DC. In human cells, LAG-3 serves as a negative regulator of activated T cells. A comprehensive understanding of these inhibitory pathways will facilitate the design of effective ▶ immunotherapy in the future.

References Diefenbach A, Raulet D (2002) The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol Rev 188:9–21 Gajewski TF, Meng Y, Harlin H (2006) Immune suppression in the tumor microenvironment. J Immunother 29:233–240 Lang K, Entschladen F, Weidt C et al (2006) Tumor immune escape mechanisms: impact of the neuroendocrine system. Cancer Immunol Immunother 55:749–760 Munn DH (2006) Indoleamine 2,3-dioxygenase, tumorinduced tolerance and counter regulation. Curr Opin Immunol 18:220–225 Rivoltini L, Canese P, Huber V et al (2005) Escape strategies and reasons for failure in the interaction between tumour cells and the immune system: how can we tilt the balance towards immune-mediated cancer control? Expert Opin Biol Ther 5:463–476

Immune Responses to Autoantigens ▶ Autoimmunity and Cancer

Immune-Mediated Suppression ▶ Immunosuppression and Cancer

Immunochemical Fecal Occult Blood Test ▶ Fecal Immunochemical Test

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Immunocytochemistry ▶ Immunohistochemistry

Immunoediting Yvonne Paterson Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Definition Changes in the immunogenicity of tumors due to the antitumor response of the immune system that result in the emergence of immune-resistant variants.

Characteristics History The concept of immunoediting is predicated on the insight that the immune system can recognize tumor cells. The notion that the immune system monitors the host, not only for pathogen invasion but also for neoplastic changes, arose early in the history of immunology and was first proposed by Paul Ehrlich in 1909 and then resurrected 50 years later by Burnet and Thomas. These early immunologists proposed that the immune system recognizes cells that have undergone neoplastic changes and eliminates them before they can form tumors, a concept known as immune surveillance. However, although this notion has been around for nearly a century, it was not until the twenty-first century that it was unequivocally demonstrated in murine models when Schreiber and colleagues, in 2001, examined the incidence of adenomas and carcinomas in aging wild-type mice compared to mice lacking the recombinase-activating gene-2 (RAG2). The RAG2 gene controls the ▶ V(D)J recombination of genes required to generate B cell and T cell antigen-specific receptors. In its

Immunocytochemistry

absence, or the absence of its partner in this process, RAG1, the development of lymphocytes that bear these receptors is aborted. Thus in mice that lack RAG1 or RAG2, T cells, B cells, and NKT cells fail to develop and the mouse has no adaptive immune response. The Schreiber lab showed that these immunodeficient mice had a higher incidence of spontaneous cancer and were more susceptible to carcinogen-induced sarcomas. These tendencies were increased in mice lacking not only the RAG2 gene but in addition the STAT-1 gene, which controls the interferon gamma ▶ signal transduction pathway. Thus both adaptive immune effector cells and the multifunctional lymphokine IFN-g were shown to play a clear role in immunosurveillance. However, in the process of studying immunosurveillance, the Schreiber group made the interesting observation that a high proportion of tumors that emerged in carcinogen-treated RAG2-deficient mice failed to grow in wild-type mice indicating that within the population of tumor cells that arose in the immunodeficient mice were cells that could be recognized and eliminated by an intact immune system. In contrast tumors that arose in wild-type mice were less immunogenic and would grow when transplanted into either wild-type or RAG2deficient mice. Schreiber termed this process by which the immunological environment alters the immunogenicity of tumors “immunoediting.” The Role of Interferons and Other Immune Components A large body of work provides evidence that both ▶ adaptive immunity and ▶ innate immunity play a role in controlling the immunogenicity of tumors that develop in the mouse. Most of these studies have focused on the formation of carcinogeninduced sarcomas in mice genetically manipulated to lack the expression of genes required for the generation of lymphocytes and cytokines. Thus in terms of innate immune cells, gd (gamma delta) T cells, ▶ natural killer cells, and NKT cells have been shown to be involved in controlling tumor growth as have conventional ab (alpha beta) T cells, the hallmark of the adaptive cellular immune response. The importance of cytotoxic lymphocytes, such as T cells and NK cells, is highlighted by the inability of mice that lack the

Immunoediting

specific toxic molecules perforin and ▶ tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), expressed by cytotoxic cells, to control both carcinogen-induced and spontaneous tumors. That at least a subset of the lymphocytes that recognize and control tumor growth are MHC class I-restricted, tumor-specific, ▶ cytotoxic T cells (CTLs) is evidenced by the fact that mice lacking the LMP2 (low-molecular mass protein 2) ▶ proteasome subunit, involved in processing antigen for recognition by CTL, develop spontaneous uterine tumors. The adaptive immune response has evolved to ignore its own tissues, by eliminating self-reactive lymphocytes during their development, in order to avoid ▶ autoimmunity or “horror autotoxicus” as Paul Ehrlich termed it. Thus the participation of CTLs in tumor immunoediting implies that tumors must express tumor-associated antigens. The original observation of immune editing identified a key role for the type II interferon, IFN-g, in the process. This pleiotropic cytokine is the product of lymphocytes of both the innate (gd T cells, NK and NKT cells) and adaptive (CD4+ MHC class II-restricted helper T (Th) cells and CD8+ MHC class I-restricted CTLs) immune response. It seems likely that both innate and adaptive lymphocytes are the source of the IFN-g that shapes the immune response to tumors. The effects of IFN-g on the immunogenicity of tumors could occur through multiple processes since it is known as a key regulator of adaptive and innate immune responses. There is a great deal of evidence that some of its effects are mediated through host cells in addition to tumor cells. However, given that many tumor cells express IFN-g receptors, it can directly interact with tumor cells, and there is evidence that it stimulates tumor cells to increase MHC class I expression, downregulate ▶ angiogenesis, and promote the infiltration of CTLs into the tumor mass by releasing chemokines. Another cytokine, IL-12, has also been shown to promote tumor immunoediting. IL-12 is intimately involved in the regulation of IFN-g production early in the immune response and probably mediates its effects through its influence on IFN-g expression. In addition to IFN-g, the ▶ type I interferons have a profound influence on tumor growth. Early

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studies in mice using type I interferons for tumor ▶ immunotherapy have been translated into several clinical applications for these cytokines in the therapy of ▶ melanoma, ▶ CML, ▶ follicular lymphoma, ▶ hair cell leukemia, and ▶ Kaposi sarcoma. There are several important differences in mice and humans between the type II interferon, IFN-g, which is the sole molecular species, and the type I interferons, IFN-a, for which there are at least 12 variants, and IFN-b, which is unique. Unlike IFN-g, which is the product only of cells of the lymphocytic lineage, type I interferons can be produced by all nucleated cells when stimulated by products of viral infection and also by infection with some bacteria. As such they are a first line of defense against pathogen invasion of the host. In addition, although tumor cells can express receptors for type I interferons, it appears that unlike IFN-g, the type I interferons do not act directly on tumor cells and mediate their antitumor effects through host cell responses. Determining exactly which cells are involved requires further study, but they appear to be cells of the hematopoietic lineage. Similar to IFN-g, there are several points at which type I interferons might influence the immune response to tumors since they can activate ▶ dendritic cells, ▶ macrophages, and ▶ NK cells and are involved in the priming and survival of T cells. In addition, similarly to IFN-g, they can act on stromal cells within and surrounding tumors to downregulate angiogenic factors. Finally, there is evidence that type I interferons may inhibit the transformation of normal cells by upregulating the expression of the tumor-suppressor molecule, ▶ p53. The importance of this class of cytokines in shaping the immune response to tumors was confirmed by studies in mice lacking the IFN-a receptor. Carcinogen-induced sarcomas were found to arise more frequently in these mice and were also shown to be more immunogenic when transplanted into wild-type mice. The Role of Induced Immune Pressure The editing of tumors in response to immune pressure is exacerbated when the immune system has been specifically primed to a tumor-associated antigen. In a ▶ mouse model for ▶ breast cancer in

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which ▶ HER-2/neu, a member of the ▶ epidermal growth factor receptor family, is overexpressed on breast tissue, resulting in the emergence of breast tumors, it was shown that mice immunized with ▶ cancer vaccines expressing fragments of HER-2/ neu could control tumor outgrowth for a significant length of time but eventually succumbed. When HER-2/neu was isolated from the emerging tumors, they were found to have accumulated a significant number of mutations in the precise region of the molecule the particular vaccine targeted. In addition, it was verified for one of the fragments that each mutation occurred in a region recognized by CTLs and that this mutation abrogated CTL recognition. This is a clear demonstration of how the host immune system can sculpt a tumor-associated antigen to evade the immune system. Transplanted, syngeneic mouse tumors have also been shown to mutate in a region of an antigen expressed by the tumor and recognized by CTLs after adoptive transfer of T cells that recognize that region. Evidence of Immunoediting in Human Cancer Although there is an abundance of evidence for immunoediting in murine models of cancer, the evidence that this occurs in human cancer is largely circumstantial. It has long been known that cancer patients develop immune responses to their own tumors. Indeed, both tumor-specific T cells and antibodies isolated from cancer patients have been harnessed to identify tumorassociated antigens by methods such as ▶ SEREX. In addition the ability of a patient to mount a response to their tumors, particularly if there is evidence of tumor infiltration of immune effector cells, is a strong predictor of a favorable prognosis. That the immune system sculpts tumor immunogenicity in tumors that arise in cancer patients is supported by the emergence of tumors in patients undergoing antigen-specific immunotherapy that have downregulated the tumor antigen to which the therapy is directed or components of the cellular machinery that generates the cell surface complex of ▶ HLA I and antigen recognized by CTLs. These include LMP proteasome subunits and transporter molecules that chaperone antigen peptides from the cytosol to the Golgi apparatus

Immunoediting

for loading onto HLA class I molecules. Sometimes the HLA I molecule itself is lost, usually by mutation of the beta2 microglobulin subunit, common to all HLA class I molecular complexes. In some cases, however, lack of HLA class I on the surface of a tumor is due not to mutational events in the HLA class I genes themselves but to downregulation of expression of the protein. This can often be reversed by cytokines, such as IFN-g, but it is interesting to note that human tumors have been shown to arise that lack the IFN-g receptor. The phenomenon of HLA class I loss from the surface of tumor cells has been documented in numerous clinical cancer vaccine trials for ▶ melanoma, ▶ prostate carcinoma, and HER-2/neu positive tumors. In addition, even in the absence of immunotherapy, HLA class I expression has been shown to be lost or downregulated in all types of tumors especially in patients with advanced disease. Indeed, the frequency of deletion or downregulation of these cell surface molecules has been found to be as high as 15% in primary melanoma lesions and 50% in primary ▶ prostate carcinoma lesions. A further piece of evidence for immunoediting in human cancer is that patients that are seriously immunosuppressed, for example, post organ transplant, have a higher incidence of cancer than healthy individuals. Finally, the emergence of so-called checkpoint inhibitors of immunoinhibitory molecules such as CTLA-4, PD-1, or PD-L1 to treat cancer, particularly melanoma, by augmenting the natural immune response of the patient against his or her own tumor also supports the existence of immunosurveillance in human cancer.

Cross-References ▶ Adaptive Immunity ▶ Angiogenesis ▶ Breast Cancer ▶ Cancer Vaccines ▶ Chronic Myeloid Leukemia ▶ Cytotoxic T Cells ▶ Dendritic Cells ▶ Epidermal Growth Factor Receptor

Immunohistochemistry

▶ HER-2/neu ▶ HLA Class I ▶ Immunotherapy ▶ Innate Immunity ▶ Interferon-Alpha ▶ Kaposi Sarcoma ▶ Macrophages ▶ Mouse Models ▶ Natural Killer Cell Activation ▶ Prostate Cancer Clinical Oncology ▶ Proteasome ▶ SEREX ▶ Signal Transduction ▶ Uterine Leiomyoma ▶ V(D)J Recombination

References Chang CC, Campoli M, Ferrone S (2005) Classical and nonclassical HLA class I antigen and NK Cell-activating ligand changes in malignant cells: current challenges and future directions. Adv Cancer Res 93:189–234 Mittal D, Gubin MM, Schreiber RD, Smyth MJ (2014) New insights into cancer immunoediting and its three component phase – elimination, equilibrium and escape. Curr Opin Immunol 27:16–25 Naidoo J, Page DB, Wolchok JD (2014) Immune modulation for cancer therapy. Br J Cancer 111(12):2214–2219. doi:10.1038/bjc.2014.348 Neeson P, Paterson Y (2006) Effects of the tumor microenvironment on the efficacy of tumor immunotherapy. Immunol Invest 35:359–394 Singh R, Paterson Y (2007) Immunoediting sculpts tumor epitopes during immunotherapy. Cancer Res 67:1887–1892

2215 (2012) Interferon gamma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1888. doi:10.1007/978-3-642-16483-5_3092 (2012) LMP2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2068. doi:10.1007/978-3-642-16483-5_3401 (2012) Major histocompatibility complex. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2137. doi:10.1007/978-3-642-16483-5_3500 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Perforin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2814. doi:10.1007/978-3-642-16483-5_4443 (2012) Recombinase activating gene-2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3208. doi:10.1007/978-3-642-16483-5_4995 (2012) STAT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3502. doi:10.1007/978-3-642-16483-5_5481 (2012) T cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3599. doi:10.1007/978-3-642-16483-5_5645 (2012) Tumor-associated antigen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3807–3808. doi:10.1007/978-3-642-16483-5_6017 (2012) Tumor necrosis factor-related apoptosis-inducing ligand. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6043

Immunoglobulin Rearrangements ▶ V(D)J Recombination

See Also (2012) Autoimmunity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 312. doi:10.1007/978-3-642-16483-5_478 (2012) B cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 331. doi:10.1007/978-3-642-16483-5_508 (2012) Follicular lymphoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1441. doi:10.1007/978-3-642-16483-5_2240 (2012) Gamma delta (γδ) T cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1493. doi:10.1007/978-3-642-16483-5_2313 (2012) Hairy cell leukemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1625. doi:10.1007/978-3-642-16483-5_2553 (2012) IL-12. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1807. doi:10.1007/978-3-642-16483-5_2959

Immunohistochemistry Clive R. Taylor1 and Richard J. Cote2 1 Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA 2 Department of Pathology, Miller School of Medicine, University of Miami, Miami, FL, USA

Synonyms IHC; Immunocytochemistry; Immunohistology; Molecular morphology

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Definition Immunohistochemistry (IHC) is a technique to detect and localize specific proteins in tissue sections by labeled antibodies that bind specifically to the investigated antigen.

Characteristics History Since the introduction of microscopy into the diagnosis of diseases by Hughes Bennett, Rudolf Virchow, James Paget, and others in the mid-1800s, pathologists have searched for better and more specific stains. In 1934, Marrack was the first to employ modified antibodies to visualize cholera and typhoid agents. Nearly a decade later, Coons (1941) used a primary antibody labeled with a fluorescent dye, fluorescein isothiocyanate, and utilized it on human tissue in an effort to localize antigens in the tissue. However, specialized dark-field microscopy and the use of frozen sections were required for the fluorescent dyes, and as a result the technique remained primarily a research tool until other labels were developed. In 1974, Taylor and Burns demonstrated that IHC could be performed on routinely processed tissue using immunoperoxidase techniques with chromogenic dyes. This step enabled the use of IHC in routine clinical laboratories. Techniques Immunohistochemistry combines the principles of immunology with histochemistry and involves two basic steps: first, an antibody binds to its specific target antigen at the cellular location in the tissue sample. The antigen-antibody binding is then detected by labeling techniques. IHC therefore not only enables pathologists to detect whether or not particular antigens are present within a given tissue but also allows marking its cellular location. There are two major types of antibodies in use: polyclonal antibodies or antisera and monoclonal antibodies. Polyclonal antibodies are produced by an injection of an antigen or antigen fragments into a host animal. The most commonly used host

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animals are rabbit, horse, mouse, and goat. If a small antigenic molecule or antigen fragment (hapten) is used as immunogen, immunogenicity is typically enhanced by coupling the hapten with a larger molecule such as keyhole limpet hemocyanin. In the resulting immunological reaction, host lymphocytes are activated, and ultimately plasma cells initiate production of antibodies. As antigens contain various immunogenic determinants, the resulting antiserum (or polyclonal antibody) will be a heterogeneous mix that recognizes several epitopes. Such potentially cross-reacting specificities are unwanted for use in IHC and must be removed by further purification. By way of contrast, monoclonal antibodies are developed by the hybridoma technique or through molecular engineering. In the hybridoma technique, activated (potentially antibody-secreting) B-lymphocytes are isolated from the immunized animal (in the past usually mouse, now increasingly rabbit) and fused with cultured myeloma cells which have limitless proliferative potential. Each of the resulting hybrid cells (hybridoma) produces a single type of antibody, thus being “monoclonal” in nature. The detection methods include the “direct” and the “indirect” alternatives. In the direct conjugatelabeled-antibody method, the label (such as a fluorescent agent or an enzyme) is chemically attached to the primary antibody. The localization of a fluorescent label is detected by immunofluorescence microscopy, or if the label is an enzyme (typically, horseradish peroxidase) coupled to antibody, then it is detected by its action on a chromogenic substrate. The indirect method uses an unlabeled primary antibody and a labeled secondary antibody (second layer) directed against the primary antibody. Several secondary antibody molecules bind to each primary antibody molecule resulting in signal enhancement, thus in increased sensitivity. Furthermore, methods involving a third layer for signal amplification can be employed. These include the PAP (peroxidase-antiperoxidase) method, the biotin-avidin mediated procedures (such as those employing the avidinbiotin conjugate or ABC procedure, biotinstreptavidin systems), immunogold, and increasingly

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polymer-based labels and catalyzed signal amplification, polymer-based labels being the preferred approach at the time of writing. Alkaline phosphatase may be used as an alternative to horseradish peroxidase. Several efforts have been made to improve the sensitivity, the reliability, and the standardization of IHC. A major step was the ongoing development of the antigen retrieval technique by ShanRong Shi and colleagues. The antigen retrieval process breaks protein cross-linkages formed during formaldehyde fixation of a tissue and enables these proteins to be accessible for antibodies. Antigen retrieval is performed by exposing tissue to heat for various lengths of time in retrieval buffer solutions with specific pH. Several protocols are available but often have to be adjusted for optimal results which can be standardized for various temperatures and durations in a “test battery” approach. Today preanalytic variatio, in sample collection and fixation increasingly is recognized as a cause of variability in IHC results; calling for better control systems and internal controls (internal reference standards). Several automated immunostaining systems (platforms) are commercially available and increasingly have replaced manual methods. While they significantly increase the reproducibility and standardization, alone they certainly do not guarantee optimal results. Quality control issues as well as the need for the validation of the results apply for staining carried out with both the automated systems and manual IHC. Interpretation of IHC staining results necessitates a highly trained pathologist and even among experts is subject to interobserver discrepancies. The development of automated image analysis systems addresses this problem. The currently available systems can assess the intensity as well as the percentage positivity of antigenic markers. The limitations of the human eye to differentiate colors restricts the use of multiple markers on the same slide. The spectral imaging technology allows to differentiate different chromogens reporting on multiple markers by serially, digitally “erasing” a given chromogen image from the microscope image by a specially designed software. Digital whole slide imaging and analysis for selected IHC

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application has been approved in many countries and is likely to become the standard, especially when quantification is required. Uses IHC plays an essential role in modern pathology. Its most common use is the identification of tumor origin and tumor type by tissue-specific markers. By IHC a pathologist can specify tumor entities and evaluate tumors of unknown origin as well as stage lymph nodes and bone marrow for the presence of metastatic disease. Biomarkers have been identified that can predict disease prognosis (predictive markers), and especially treatment response, thereby serving to select specific therapy (so-called companion diagnostics, or advanced personalized diagnostics). Use in Research In a research setting, the expression of relevant markers is investigated. This demands studies on well-defined samples across various tumor stages and clinical outcome. The tissue microarray technique allows an acceleration of such studies in a high-throughput manner. Tissue microarrays (TMA) consist of compendia of 0.6 mm cylindrical biopsy cores from paraffin-embedded tissue that are transferred to defined array coordinates in a recipient block. These multitissue-core blocks can then be processed similar to a routine singletissue block. A difference from the conventional IHC is the high level of standardization since all slides in one TMA experiment are incubated together, ensuring identical reagent concentrations and incubation temperatures. However, it must be remembered that in TMAs the multiple tissue cores may have been subjected to different sample preparation (fixation) steps, which may introduce unsuspected variability. Clinical Uses (Examples) Diagnosis and Tumor Classification

Intermediate Filaments The presence of intermediate filaments, which function as cytoskeletal components in both normal and malignant cells, is useful in the initial classification and diagnosis of

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neoplasms. Five major classes of intermediate filaments exist: Cytokeratin (CK), vimentin, desmin, neurofilament, and glial fibrillary acidic protein (GFAP). Most neoplasms show a predominant expression of one or more of these intermediate filaments. Based on their molecular weight and isoelectric point, the cytokeratins are subdivided into more than 20 types. Carcinomas are typically CK positive, whereas sarcomas, melanomas, and lymphomas are generally vimentin positive. Tumors of myogenic origin characteristically express desmin and/or muscle actins and vimentin. Glial tumors are predominantly positive for GFAP. Nuclear Transcription Factors Transcription factors are proteins involved in the regulation of gene expression. By binding to promoter elements upstream of genes they either facilitate or inhibit transcription. Though not exclusively found in a specific tumor type, transcription factors are highly tissue specific and can be useful in determining the primary site of a tumor. Cell Surface Membrane Markers Proteins expressed at the cell surface may be of great value in recognition of cell lineage, where it may not otherwise be apparent, as in poorly differentiated malignancies. Malignant lymphoma is the area par excellence where cell surface (CD) markers, along with other biomarkers, are now considered essential to accurate diagnosis and classification. Carcinoma Essentially all cells of epithelial origin express cytokeratins, which are therefore highly sensitive markers for carcinomas. However, other tumors such as mesotheliomas and nonseminomateous germ cell tumors also stain positively (usually less intensely) with some antibodies to cytokeratin subtypes. More specific markers and subtyping of keratins must be applied to further differentiate CK-positive cells and to assess the site of origin of the carcinoma. The profile of cytokeratin subtypes is useful to determine the tumor type. For example, hepatocellular carcinomas are positive for CK antigens that can be detected by antibodies AE3 and CAM5.2 but

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are negative for antibody AE1, which is directed against a different set of CK. The use of CK7 and CK20 profile has proven a useful tool in distinguishing cellular origin. Nuclear transcription factors are useful in the detection of carcinomas. Thyroid transcription factor-1 (TTF-1) is found in the thyroid and lung. CDX-2 is specific for colorectal epithelium. For some tissue types, tissue-specific tumor markers (e.g., ▶ prostatespecific antigen (PSA) for prostate, or thyroglobulin for thyroid) or tissue-associated markers (e.g., gross cystic disease fluid protein 15 (GCDFP-15) and mammaglobin in breast, uroplakins for transitional urothelial cells, OC 125 for ovarian cells, synaptophysin for neuroendocrine lesions, etc.) are available. It is important to remember that these markers are often not uniquely found in the specified tissue. For instance, the detection of PSA has also been described (rarely) for samples of salivary glands and breast. Melanoma For melanomas, which usually are cytokeratin negative and vimentin positive, a sensitive (but nonspecific) tissue marker, S-100, is available. HMB-45, Melan A, and tyrosinase are used to confirm the diagnosis. Sarcoma The common IHC pattern for all forms of sarcoma includes vimentin positivity. Generally speaking, sarcomas are CK negative, but some may yield positive reactions especially for low molecular CKs following antigen retrieval and highly sensitive labeling techniques. Additional stains are then employed to identify the tumor type. For example, ▶ Rhabdomyosarcoma and ▶ Leiomyosarcoma express markers that are typical for muscular tissue such as desmin and muscle-specific actin. Lymphoma Lymphomas show a wide variety of morphological appearances, making IHC especially important in their diagnosis. Membranous staining for CD45 can typically be seen in leucocytes. CD3 (T cells) and CD20 (B cells) are more specific markers and are used to further confirm lymphomas. ▶ CD antigens are essential in subclassifying lymphoid tissue, and more than 40 are in common use.

Immunohistochemistry

Infectious Agents Since the early days of its development, IHC has been used for the detection of infectious agents. Nowadays, microbiological cultures are the gold standard, but for pathogens that are difficult to culture such as cytomegalovirus, mycobacteria, toxoplasmosis, pneumocystis carinii, histoplasma capsulatum, ▶ Helicobacter pylori and human papillomavirus IHC methods are at least as effective. Tumor Stage and Occult Metastases

The most important factor determining outcome of patients with cancer is the presence of regional and/or systemic dissemination (metastases). IHC is a highly sensitive way to detect such occult metastases in blood, lymph nodes, and bone marrow. For example, normal lymph nodes, bone marrow, or blood do not contain cells with epithelial antigens, thus the finding of CK-positive cells suggests metastases of a carcinoma. The presence of occult metastases has been shown to influence clinical outcome for several cancer types such as breast, lung, and prostate cancer. Lymph node metastases occult to routine pathological workup have been described for several tumors including breast cancer in up to 20% of patients and in more than 10% of patients with prostate cancer. The reported sensitivity of this technique ranges from the detection of 1 epithelial cell in 10,000 to 2–5 epithelial cells in a million hematopoietic cells. Prognosis

Mutations and overexpression of oncogenes and ▶ tumor suppressor genes play a vital role in tumorigenesis. The presence or absence of their protein products may predict the biological behavior of tumors more accurately than clinical and pathological criteria. Since antibodies are available for many of these gene products, tumors with different prognosis can be differentiated by means of IHC. The most widely investigated proteins include p53, the protein product of the Retinoblastoma gene (pRb), p27, ▶ p21, and ▶ p16. RB gene alterations resulting in reduced expression are known to characterize tumors with a higher risk for metastatic disease. p21, p16, and p27 are the three major ▶ cyclin-dependent kinase inhibitors

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which negatively regulate pRb activity and are normally required for cell cycle suppression. The loss of p27 expression is associated with colon, breast, prostate, and gastric cancer progression. p53 plays an important role in cell cycle progression, and apoptosis in response to ▶ DNA damage, and is expressed by all human cells. Normal (wild-type) p53 protein has a short half-life of only 6–30 min because of its ubiquitin actionmediated degredation dictated by its binding with another regulatory protein, ▶ MDM2. Therefore p53 does not accumulate in normal cells. Overexpression of p53 (by altered p53 gene or because of ineffective ▶ MDM2 protein) in the nuclear compartment therefore indicates a dysfunctional p53 pathway, a characteristic of tumors. Alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote cancer progression, and simultaneous overexpression has been linked to worse prognosis. Treatment Response: Companion Diagnostics

An increasingly important application of IHC is to identify specific targets for therapy. Consequently, this will allow better selection of patients who will benefit from certain treatment modalities. In many tumor types, treatment decisions already are influenced or determined by molecular findings. An early example is ▶ hormonal therapy for breast cancer patients depending on estrogen and progesterone expression of the tumor. Because of its vital role in the apoptotic pathway p53 alterations are likely to influence response to chemotherapy. p53 alterations confer increased chemosensitivity on tumors such that combining agents with different actions may have synergistic effects on tumor cell killing. Targeted Therapy

In breast cancer Her-2/neu overexpression was initially seen as an indicator of poor prognosis. However, it has been shown that the ▶ HER-2/ neu ▶ receptor tyrosine kinase can serve as a therapeutic target. Treatment with the monoclonal antibody ▶ trastuzumab (brandname: Herceptin) which is directed against this protein is effective only for patients whose tumors show overexpression of Her-2/neu. This was effectively

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the first targeted therapy with its “companion diagnostic” (in 1998), and detection of Her-2 has since been extended and approved for other tumor types, such as gastric carcinoma. Large numbers of other biomarkers that are predictive of response to specified targeted therapies have since been identified in many types of cancer, including lung, breast, colon, and melanoma. These “tests” represent “companion diagnostics” and are the fastest-growing area of IHC (Gu and Taylor 2014; Taylor 2014). However, because some degree of quantification is required, there is greatly increased pressure for improved standardization of IHC, including preanalytic steps, and rigorous control systems, and ideally quantifiable controls for calibration of results (Torkalovic et al. 2015; Taylor 2015). Summary Immunohistochemistry has allowed for identification of tumor origin, tumor prognosis, and likelihood of response to therapy for an increasing array of tumors. The ability to determine which tumors are most likely to progress (and thus need further therapy) coupled with the ability to predict specifically the response of individual tumors to chemotherapeutic agents and the ability to identify specific targets of therapy will have a profound effect on the way treatment decisions for patients with cancer are made. It is not difficult to envision the day when drug selection is based on the presence of specific targets and on the resistance patterns of individual tumors to specific agents. Treatment decisions will become less organ based and will reflect the biology of the tumors. This has already helped us to approach patient-specific (as opposed to diseasespecific) management of care.

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▶ Hormonal Therapy ▶ Leiomyosarcoma ▶ p21 ▶ Prostate-Specific Antigen ▶ Receptor Tyrosine Kinases ▶ Rhabdomyosarcoma ▶ Trastuzumab ▶ Tumor Suppressor Genes

References Gu J, Taylor CR (2014) Practicing pathology in the era of big data and personalized medicine. Appl Immunohistochem Mol Morphol 22:1–9 Hawes D, Taylor CR, Cote R (2003) Immunohistochemistry. In: Weidner N, Cote R, Suster S, Weiss L (eds) Modern surgical pathology, 1st edn. Saunders, Philadelphia, pp 57–80 Mitra AP, Lin H, Cote RJ et al (2005) Biomarker profiling for cancer diagnosis, prognosis and therapeutic management. Natl Med J India 18(6):304–312 Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem Mol Morphol 9(2):107–116 Taylor CR (2014) Predictive biomarkers and companion diagnostics. The future of immunohistochemistry – ‘in situ proteomics’, or just a ‘stain’? Appl Immunohistochem Mol Morphol 22:555–561 Taylor CR (2015) Quantitative in situ proteomics; a proposed pathway for quantification of immunohistochemistry at the light microscope level. Cell Tissue Res 360:109–120 Taylor CR, Cote RJ (2006) Immunomicroscopy: a diagnostic tool for the surgical pathologist, 3rd edn. Saunders, Philadelphia Torkalovic EE et al (2015) Standardization of positive controls and introduction of immunohistochemistry critical assay performance controls (iCPAPS) in diagnostic immunohistochemistry: recommendations from the International Ad Hoc Committee. Appl Immunohistochem Mol Morphol 23:1–18

See Also

Cross-References ▶ CD Antigens ▶ CDKN2A ▶ Cyclin-Dependent Kinases ▶ DNA Damage ▶ Helicobacter Pylori in the Pathogenesis of Gastric Cancer ▶ HER-2/neu

(2012) Antibody. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 208. doi:10.1007/978-3-642-16483-5_312 (2012) CD20. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 693. doi:10.1007/978-3-642-16483-5_922 (2012) CD45. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 702. doi:10.1007/978-3-642-16483-5_931 (2012) Cytokeratins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1051. doi:10.1007/978-3-642-16483-5_1472

Immunoliposomes (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1291–1292. doi:10.1007/978-3-642-16483-5_1958 (2012) Germ cell tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905 (2012) Lymph node metastases. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2116. doi:10.1007/978-3-642-16483-5_3444 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Plasma cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2900. doi:10.1007/978-3-642-16483-5_4598 (2012) PRb. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2967. doi:10.1007/978-3-642-16483-5_4708 (2012) PSA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3111–3112. doi:10.1007/978-3-642-16483-5_6738 (2012) Synaptophysin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3592. doi:10.1007/978-3-642-16483-5_6921 (2012) Thyroglobulin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3687. doi:10.1007/978-3-642-16483-5_5805 (2012) Tumor markers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3796. doi:10.1007/978-3-642-16483-5_6036 (2012) TTF-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3787. doi:10.1007/978-3-642-16483-5_6005 (2012) Uroplakins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3851. doi:10.1007/978-3-642-16483-5_6121

Immunohistology ▶ Immunohistochemistry

Immunoliposomes Roland E. Kontermann Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany

Definition Are liposomal drug formulations possessing antibody molecules conjugated to the liposomal

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surface. This allows for a ▶ targeted drug delivery to tumor cells or other tumor-associated structures (active targeting).

Characteristics Liposomes are vesicular particles composed of a lipid bilayer enclosing a hydrophilic inner phase. Liposomes can be used as carrier systems for cancer drugs (▶ drug delivery systems) by encapsulating hydrophilic drugs into the interior or by incorporating lipophilic drugs into the lipid bilayer. Liposomes have normally a size (diameter) of 100–200 nm. Several liposomal formulations of therapeutic drugs are approved for cancer therapy, e.g., liposomal doxorubicin (Doxil/Caelyx, Myocet) (▶ liposomal chemotherapy). Delivery of these drugs to tumors is a passive process, and efficacy depends on long circulation and enhanced permeability and retention in the tumor tissue (EPR effect). New formulations of liposomal drugs (e.g., Doxil/Caelyx) have polyethylene glycol (PEG) chains incorporated into the lipid bilayer to further increase stability and pharmacokinetic properties and to decrease elimination of the liposomes by phagocytic cells. The coupling of antibody molecules to the lipid surface allows for an active targeting by recognition of antigenic structures expressed by the tumor. Thus, immunoliposomes are designed to increase selectivity and efficacy of liposomal drugs. Immunoliposome Types and Antibody Formats Immunoliposomes are generated by chemically coupling antibodies or antibody fragments to the liposomal surface. Besides the whole antibody molecules (e.g., IgG molecules), antibody fragments such as Fab’ fragments or single-chain Fv fragments can be utilized for the generation of immunoliposomes. The use of whole antibodies is less favorable since these immunoliposomes are recognized by phagocytic cells via Fc receptors. Consequently, Fab’ or scFv’ fragments are the formats of choice to generate immunoliposomes. Immunoliposomes can be classified depending on

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the position of coupled antibodies and the liposomal composition. In type I immunoliposomes, the antibodies are coupled directly to the lipid bilayer either in the absence (type Ia) or presence of PEG chains (type Ib). Coupling of the antibodies to the distal end of incorporated PEG chains results in type II immunoliposomes (Fig. 1a). Coupling of antibodies is facilitated by the use of functionalized lipids, e.g., lipids possessing an amino-reactive succinimidyl moiety or a sulfhydryl-reactive maleimide group. Antibody or antibody fragments can be generated by established hybridoma technology and biochemical means or by genetic engineering as

Type la

a Fab

PEG

Type lb

b

IgG

Type lI

Fab′

scFv′

Immunoliposomes, Fig. 1 Classification of immunoliposomes and antibody formats. (a) Three types of immunoliposomes can be distinguished. Type I immunoliposomes have the antibody molecules coupled directly to the lipid bilayer, either in the absence (type Ia) or presence (type Ib) of polyethylene glycol (PEG) chains. In type II immunoliposomes, the antibodies are coupled to the distal end of PEG chains. (b) Antibody formats used for the generation of immunoliposomes. The two variable domains (VH, VL) forming the antigen-binding site are shown in dark and light gray

recombinant molecules. For example, Fab’ fragments can be produced by enzymatic cleavage of IgG molecules with pepsin resulting in F(ab’)2 fragments, which after reduction are separated in Fab’ fragments exposing a free sulfhydryl group at the end of the molecule (Fig. 1b). Furthermore, the implementation of antibody engineering allows for the generation of small antibody molecules with desired properties, e.g., scFv molecules exposing a genetically introduced cysteine residue at the C-terminus used for site-directed coupling (Fig. 1b). Drugs and Targets Immunoliposomes can be combined with a wide variety of different drugs. Besides small compounds and chemotherapeutic drugs, immunoliposomes can also be used for the delivery of nucleic acids (e.g., ▶ siRNA) or therapeutically useful peptides and proteins (Fig. 2). Encapsulation of drugs into liposomes alters their pharmacokinetic properties, e.g., reduces rapid renal elimination of small molecular weight drugs. In addition, drug encapsulation has been shown to reduce side effects and to increase stability of the drug within the body. Several modes of action have been described for immunoliposomes. Delivery to extracellular structures may lead to increased accumulation of liposomes in the tumor tissue and slow release of drug, which then can enter the target cell. Alternatively, binding of immunoliposomes to cell surface receptors results in internalization of immunoliposomes (▶ endocytosis) and intracellular release of the drug. Several studies have shown that internalization of immunoliposomes leads to increased cytotoxicity and may also bypass drug-resistance mechanisms. The main targets of immunoliposomes are the molecules expressed by tumor cells. Thus, various immunoliposomal formulations of chemotherapeutic drugs (e.g., doxorubicin, vincristine) have been generated using antibodies against tumorassociated antigens including CD19, CD20, Her2/neu, ▶ epidermal growth factor receptor (EGFR), and disialoganglioside GD2 for therapy of lymphoma and solid tumors. In addition, research has been focused on targeting of tumor

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Small compounds (e.g. contrast agents, radionuclides) Chemotheraperapeutic drugs (e.g. doxorubicin, vincristine, paclitacel) Nucleic acids (antisense oilgonucleotides, siRNA, ribozymes, DNA)

Tumor cells

Encapsulated drug

Targeted drug deliver

Tumor vasculature Tumor stroma

Peptides & proteins (e.g. cytokines, toxins, enzymes)

Immunoliposomes, Fig. 2 Active compounds combined with immunoliposomes. Immunoliposomes allow for a targeted delivery of various kinds of active molecules,

blood vessels (vascular-targeting agents) as well as targeting of extracellular tumor stroma components or tumor stroma fibroblasts, which are more easily accessible for circulating liposomes. Efficacy of immunoliposomes is influenced by several factors. Besides lipid composition, which has an influence on stability and release of drug, and the antibody format used, efficacy depends also on the kind of target molecule as well as its density on the cell surface and sensitivity of target cells for the encapsulated drug.

References

including small compounds, chemotherapeutic drugs, nucleic acids, and peptides and proteins

Immunophenotypic Determinants ▶ CD Antigens

Immunoprevention Pier-Luigi Lollini and Patrizia Nanni Laboratory of Immunology and Biology of Metastasis, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

Allen TM, Cullis PR (2004) Drug delivery systems: entering the mainstream. Science 303:1818–1822 Drummond DC, Meyer O, Hong K et al (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 51:691–743 Kontermann RE (2006) Immunoliposomes for cancer therapy. Curr Opin Mol Ther 8:39–45 Park JW, Benz CC, Martin FJ (2004) Future directions of liposome- and immunoliposome-based cancer therapeutics. Semin Oncol 31:196–205 Sapra P, Tyagi P, Allen TM (2005) Ligand-targeted liposomes for cancer treatment. Curr Drug Deliv 2:369–381

Synonyms

Immunological Fecal Occult Blood Test

Characteristics

▶ Fecal Immunochemical Test

Immunoprophylaxis of cancer

Definition Prevention of cancer onset or of early cancer development and ▶ progression by means of immunological treatments, such as vaccines, antibodies, or cytokines.

Immunoprevention of cancer can be applied to tumors caused by viruses and other infectious

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agents or to tumors unrelated to infectious agents. In both cases, the aim is the same; however, the underlying concepts and the advancement of clinical development are different. Prevention of viral tumors is based on vaccines against viral antigens, whereas immunoprevention of tumors unrelated to infectious agents targets antigens expressed by early neoplastic cells. Immunoprevention of Viral Tumors About 16% of all human tumors are directly caused by infectious agents or indirectly by persisting inflammation accompanying chronic infection. In such cases, the application of immunological strategies to prevent infection is a type of primary cancer prevention because it aims at removing a risk factor that can cause cancer (▶ cancer causes and control). The first success in this direction was the demonstration that vaccination programs implemented in the 1980s against ▶ hepatitis B virus (HBV) significantly reduce the incidence of hepatitis virus-associated ▶ hepatocellular carcinoma. Current vaccines are highly effective (90–95%) in preventing chronic HBV infection. Studies in countries where HBV infection is frequent demonstrated that vaccination reduced by half infantile hepatocellular carcinoma incidence and mortality. HBV is responsible for one third (developed countries) to two thirds (less developed countries) of all cases of hepatocellular carcinoma, and ▶ hepatitis C virus (HCV) causes about one fourth. HCV vaccines (under development) and new antiviral drugs (already available) are expected to contribute a further substantial decrease in the worldwide incidence of hepatocellular carcinoma. Human papillomaviruses (HPV) are the most prevalent carcinogenic viruses in humans; various types of HPV cause more than half a million new cases of ▶ cervical cancer and other tumors worldwide. The first human vaccine against HPV was approved in 2006 in the USA, EU, and various other countries. Two vaccines made of viruslike particles (VLP) are now available, a quadrivalent vaccine against HPV types 6, 11, 16, and 18 (upgraded to 9-valent), and a divalent vaccine against HPV 6 and 18. Clinical trials demonstrated 89–100% protection of vaccinated women from

Immunoprevention

persistent HPV infection, 100% protection from histologic evidence of cervical cancer, and no serious adverse events. Vaccines are thus expected to have a major impact on the incidence and mortality of HPV-related cancers. Critical issues are: (i) Current vaccines do not include all HPV types causing cervical cancers worldwide; hence, screening programs should not be abandoned until crossprotection by current vaccines has been demonstrated or novel multivalent vaccines have been developed. (ii) Vaccines are presently very expensive, and only a few national or regional health systems are prepared to subsidize the costs. (iii) HPV vaccination programs are routinely proposed only to the female population, but males are also at risk and represent a viral reservoir; hence, it would be reasonable (immunologically, if not economically) to vaccinate the entire population. (iv) Political issues are further complicated by the need to vaccinate prepuberal girls against a sexually transmitted virus, an issue that elicits considerable ethical debate in some countries. (v) The follow-up of vaccinated persons is still too short to estimate the long-term duration of immunity and the need for periodic boosts. (vi) Current vaccines prevent infection but are devoid of therapeutic activity in infected individuals. Attempts toward the development of vaccines against ▶ Helicobacter pylori, the third major infectious risk factor of human cancer, until now did not produce a credible candidate for mass vaccination; however, bacterial eradication with antibiotics and other drugs is now a standard and very effective practice. On the whole, it can be estimated that a fullscale deployment of the approaches described above for HBV, HCV, and HPV could lead to the immunoprevention of two thirds of infectious human cancers, or more than 10% of all human cancers. This is a very important result that, however, leaves open the question of what immunoprevention can do for the majority of human cancers not caused by infectious agents. Immunoprevention of Tumors Unrelated to Infectious Agents Immunoprevention of noninfectious tumors is currently at the level of ▶ preclinical testing and

Immunoprevention

early clinical trials. This type of immunoprevention targets early neoplastic cells; hence, it can be classified as a kind of secondary cancer prevention because it aims at preventing the evolution of incipient tumors into clinically evident, symptomatic masses. The ▶ Immune Surveillance of Tumors theory posits prevention of tumor onset as a fundamental function of the immune system, on a par with prevention of infection. In fact, cancer incidence in knockout mice lacking ▶ adaptive immunity and ▶ innate immunity is much higher than in immunocompetent mice. However, the very existence of progressive, malignant tumors demonstrates that the efficiency of spontaneous immune surveillance is lower than 100%. Thus, the aim of cancer immunoprevention is to enhance immune surveillance of tumors by means of treatments that elicit protective antitumor immune responses and/or decrease immunosuppressive components. Immune targeting of ▶ preneoplastic lesions or of early neoplastic cells has several advantages with respect to conventional cancer ▶ immunotherapy, which instead must necessarily target advanced tumors. The efficacy of immune defenses is higher against smaller tumor deposits, a property shared by most antitumor approaches, including chemotherapy. Nascent neoplastic lesions are less protected from immune effectors by ▶ stromagenesis and ▶ angiogenesis, tumor progression caused by the accumulation of multiple genetic alterations is still at an early stage, and genomic instability has not yet generated a wide array of heterogeneous tumor variants, eventually leading to the selection of immunoresistant phenotypes. Demonstrations of cancer immunoprevention were mainly obtained in cancer-prone genetically modified mouse models ▶ transgenic mouse and in some ▶ chemical carcinogenesis systems. Three different strategies were effective in inhibiting and/or delaying tumor onset in mice: (i) monoclonal antibodies directed against membrane tumor antigens; (ii) immunostimulants, such as recombinant ▶ cytokines (interleukin12), plasmids containing CpG sequences (▶ CpG islands), or bacterial derivatives

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(a-galactosylceramide); and (iii) vaccines containing whole cells, recombinant proteins, synthetic peptides, or plasmids encoding the target antigen. Using standard categories borrowed from immunotherapeutic jargon, the first approach can be classified as passive immunoprevention, because it is based on the administration of a preformed immunological “drug” directly acting on tumor cells; the second as active, antigen-nonspecific immunoprevention; and the third as active, antigen-specific immunoprevention. Early attempts provided proof-of-principle demonstrations that stimulation of the immune system in healthy, cancer-prone hosts can effectively delay or reduce tumor incidence later in life. Second-generation studies aimed at increasing preventive efficacy through the combination of different strategies, much as it happened in ▶ chemotherapy. Various combinations of antigen-specific vaccines and powerful immunostimulants completely prevented tumor onset in very aggressive mouse models of cancer development; thus, demonstrating that cancer immunoprevention can effectively halt a genetic predisposition to cancer. Microscopic analysis of tumor-free aged mice showed that tumor progression is blocked at the stage reached when vaccination begins. For effective immunoprevention of tumor onset, vaccination must start before tumor progression reaches specific critical stages, such as in situ carcinoma. Immune Mechanisms of Cancer Prevention

Highly active vaccines, like those yielding complete cancer immunoprevention, elicit simultaneously many overlapping immune responses in immunocompetent mice; thus, vaccination of mice with selective immunodeficiencies is the only way to dissect protective mechanisms from less important immune components. The most important immune mechanisms at work in cancer immunoprevention comprised both T cell responses, including the release of cytokines (gamma interferon, IFN-gamma) and, less frequently, cytotoxic T lymphocytes (CTL), along with antibodies whenever the target antigen was expressed on the cell surface.

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The importance of antibody responses clearly distinguishes cancer immunoprevention from cancer immunotherapy, the latter being mainly based on CTL rather than antibodies. The largely different timescales involved justify this discrepancy, because cancer immunoprevention requires a long-term protection from tumor onset, ideally extending for the entire life of the host, whereas a rapid destruction of tumors or metastases is the goal of cancer immunotherapy. CTL activity must be of short duration to avoid severe toxicity for the host, whereas protective antibodies persisting for a long time are harmless. The same dualism applies to viral immunity, in which acute infection is mostly resolved by CTL, whereas long-term immunity from reinfection and protection elicited by vaccination are provided by neutralizing antibodies. The direct interaction of cytokines like IFN-gamma and of antibodies with early neoplastic cells results in multiple molecular blocks of cell growth and tumor progression combining cytostatic and cytotoxic mechanisms. Inhibition of cell proliferation is a logical part of the set of antiviral activities of IFN-gamma that can directly block tumor cell growth; furthermore, it inhibits the release of matrix metalloproteinases involved in tumor cell invasiveness (▶ invasion) and induces the production of chemokines with antiangiogenic activity. Antibodies binding to surface tumor antigens mediate tumor cell killing via complement-mediated cytotoxicity and antibodydependent cell-mediated cytotoxicity (ADCC). Whenever the target antigen is involved in mitogenic signaling, antibodies can directly inhibit tumor cell proliferation without the need of further molecules or cells of the immune system, practically acting as “receptor antagonists.” In the case of the HER-2 oncogene, specific antibodies induced by preventive cancer vaccines inhibit dimerization of surface HER-2 proteins (a key step required for the initiation of signal transduction) or their proteolytic cleavage to constitutively active isoforms. Furthermore, antibodies induce HER-2 internalization and recycling, eventually leading to a complete depletion of HER-2 surface expression. In HER-2-addicted cells (▶ oncogene addiction), a prolonged loss of HER-2 expression

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and signaling blocks cell proliferation and can trigger apoptosis. Target Antigens of Cancer Immunoprevention

The inhibitory mechanisms targeting HER-2 can be ineffective against most other tumor antigens, either because the target is not a surface molecule and antibodies cannot bind tumor cells or because tumor progression leads to a loss of antigenprocessing machinery (including major histocompatibility complex molecules), required for antigen recognition by T cells. The latter is a very frequent event affecting 80–90% of all human tumors. For cancer immunoprevention, it was proposed that the immune recognition of ideal target antigens should persist even if defects in antigen processing impede T cell recognition; hence, the antigen should be expressed on the cell surface and recognized by antibodies. A second desirable property is a direct involvement of the target antigen in oncogene addiction or in the maintenance of tumorigenicity, as is the case of HER-2, because this prevents tumor escape from immune defenses through loss of antigen expression, again a frequent phenomenon affecting most tumor antigens. Tumor antigens fulfilling both requirements were named oncoantigens. Members of this new class of tumor antigens ideally suit the concepts of cancer immunoprevention, in particular for what concerns the need of persistent, lifelong antitumor responses; however, oncoantigens make also attractive new targets for cancer immunotherapy because they are not prone to relevant mechanisms of immunoresistance and therapeutic failure. Clinical Developments

The mass of preclinical data demonstrating the efficacy of cancer immunoprevention in mouse models warrants the translation of this approach to humans; however, this requires a precise definition of the subjects who can benefit from this type of intervention and consequently of the design of clinical trials. Immunoprevention of hereditary cancer syndromes would be a straightforward translation from preclinical models; however, the definition of suitable target antigens in most hereditary tumors is currently lacking and

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will require adequate immunological studies. Analogous problems are faced by the application of cancer immunoprevention to other human groups at increased risk of cancer due to carcinogenic exposures and/or to the presence of preneoplastic lesions. In addition, the lower level of risk (as compared to hereditary cancer) implies the recruitment of a large number of subjects. A MUC1 vaccination study in humans with colon adenomas showed that immunoprevention is indeed feasible in humans, and further clinical trials are expected to verify whether a significant reduction in cancer onset can be achieved.

▶ autoimmunity. However, the use of transgenic mouse models implies that, in most instances, protective immune responses were actually directed against transgenic products rather than against endogenous molecules, thus minimizing “real” autoimmunity. Therefore, early clinical trials of cancer immunoprevention in humans will require intensive analyses to discover early signs of autoreactive immune responses and a special attention to the long-term risks of triggering autoimmunity.

The Risks of Cancer Immunoprevention The lack of severe adverse effect registered in the clinical trials leading to the approval of HPV vaccines, along with similar results from worldwide HBV vaccination programs, indicates that the immunoprevention of virus-related cancers will share with other vaccines for infectious diseases an intrinsic high level of biosafety. The main reason is that the target antigens are not expressed by normal tissues of the host; hence, the risk of triggering autoimmune reactions is very low. On the contrary, many tumor antigens unrelated to infectious agents are also expressed by some normal cells during life. In some cases, normal cells express a cross-reacting molecule with aminoacidic or glycosylation differences from the tumor version, but more frequently the antigens are structurally identical, and the only differences are either quantitative or topographical. The implication is that some autoimmune responses will frequently accompany successful prophylactic or therapeutic vaccination, even though development of autoimmune diseases is not an automatic consequence. A major difference between cancer therapy and prevention is that both physicians and patients accept inherently high risks of serious adverse effects when dealing with an existing lifethreatening disease, whereas preventive treatments to be administered for long periods to healthy individuals need to minimize not only severe but also mild adverse reactions. Preclinical studies of cancer immunoprevention did not reveal major risks of

Cross-References ▶ Adaptive Immunity ▶ Angiogenesis ▶ Cancer Causes and Control ▶ Cervical Cancers ▶ Chemical Carcinogenesis ▶ Chemotherapy ▶ CpG Islands ▶ Cytokine ▶ Helicobacter Pylori in the Pathogenesis of Gastric Cancer ▶ Hepatitis B Virus ▶ Hepatitis C Virus ▶ Hepatocellular Carcinoma ▶ Immunosurveillance of Tumors ▶ Immunotherapy ▶ Innate Immunity ▶ Invasion ▶ Oncogene Addiction ▶ Preclinical Testing ▶ Preneoplastic Lesions ▶ Progression ▶ Stromagenesis ▶ Transgenic Mouse

References Cavallo F, De Giovanni C, Nanni P et al (2011) 2011: the immune hallmarks of cancer. Cancer Immunol Immunother 60:319–326 de Martel C, Ferlay J, Franceschi S et al (2012) Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol 13:607–615

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2228 Finn OJ (2008) Cancer immunology. N Engl J Med 358:2704–2715 Kimura T, McKolanis JR, Dzubinski LA et al (2013) MUC1 vaccine for individuals with advanced adenoma of the colon: a cancer immunoprevention feasibility study. Cancer Prev Res (Phila) 6:18–26 Lollini PL, Cavallo F, Nanni P et al (2006) Vaccines for tumour prevention. Nat Rev Cancer 6:204–216 Lollini PL, Nicoletti G, Landuzzi L et al (2011) Vaccines and other immunological approaches for cancer immunoprevention. Curr Drug Targets 12:1957–1973 Schiller JT, Castellsagué X, Garland SM (2012) A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine 30S:F123–F138

Immunoprophylaxis of Cancer

Immunosuppression and Cancer Nicole M. Haynes Cancer Therapeutics Program, Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia

Synonyms Immune-mediated suppression

See Also (2012) Autoimmunity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 312. doi:10.1007/978-3-642-16483-5_478 (2012) Cancer-prone genetically modified mouse models. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 635. doi:10.1007/978-3642-16483-5_813 (2012) Oncoantigen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2609. doi:10.1007/978-3-642-16483-5_4216 (2012) Primary cancer prevention. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2985. doi:10.1007/978-3-642-164835_4731 (2012) Secondary cancer prevention. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3347. doi:10.1007/978-3-642-16483-5_5198 (2012) Virus-like particles. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3924–3925. doi:10.1007/978-3-642-16483-5_6199

Immunoprophylaxis of Cancer ▶ Immunoprevention

Immunoproteasome ▶ Proteasome

Immunoreceptor ▶ Chimeric Antigen Receptor (CAR)

Definition Cancer-induced immunosuppression is a survival/ defense mechanism used by cancer cells to escape elimination by the immune system. Immunosuppression is the act of inhibiting or dampening the functional activity of immune cells.

Characteristics A highly dynamic relationship exists between cancer and the immune system, which can shape the evolution of the disease (Schreiber et al. 2011). For a cancer to survive and grow, it must overcome immune-mediated barriers to oncogenesis. It can do this by hijacking host regulatory mechanisms that function to maintain normal immune cell homeostasis and self-tolerance. Cancer cells can suppress the generation of anticancer immune responses by: (i) losing or downregulating key communicative links with the immune system, (ii) secreting factors that dampen or alter the functional activity of immune cells, and (iii) influencing the recruitment of regulatory immune cells that function to shut down ongoing or the priming of new immune responses. The extent to which a cancer will rely on these immunosuppressive mechanisms to promote its survival is dependent on the tissue in which it arises [immune celldeprived (e.g., brain, bone) versus immune cellrich (e.g., skin, colon) tissues], as well as the genomic stability of the disease, which can influence the immunogenicity of the cancer

Immunosuppression and Cancer

microenvironment and breadth of the immune cell repertoire capable of recognizing and mounting a response against the cancer. Communicative Barriers Inhibiting Anticancer Immunity The immune system uses highly sophisticated cellular surveillance mechanisms to recognize and eliminate diseased or dying cells within the body (Schreiber et al. 2011). Natural killer (NK) cells, which play an integral role in initiating innate immune responses to cancer, rely on the delivery of both stimulatory and inhibitory signals to regulate their activity. Under normal physiological conditions, NK cell activity is negatively regulated by inhibitory receptors that recognize and bind to self major histocompatibility complex (MHC) class I molecules on normal tissues. Ligands to NK stimulatory receptors are usually poorly expressed on healthy tissue. Conversely, unhealthy or stressed cells, including cells undergoing malignant transformation, often upregulate NK cellactivating ligands [MICA/B, ULBP1-6 (human ligands for the NKG2D receptor); RAE-1, H60, MULT-1 (mouse ligands for the NKG2D receptor), and CD112, CD155 (ligands for the DNAM1 receptor)] and lose expression of MHC class I, thus providing signals for NK cells to become activated and display effector functions. Cancer cells can evade elimination by NK cells through: (i) downregulating or shedding the expression of activating ligands, (ii) upregulating inhibitory ligands, (iii) losing expression of adhesion ligands (e.g., ICAM1, CD112, CD155), and (iv) developing resistance to apoptosis. Adaptive immune surveillance constitutes another powerful branch of the immune system that can mount exquisitely specific responses against cancer-associated self-antigens and antigens derived from mutated tumor-specific proteins. Central to the development of adaptive immunity is the activation of cytotoxic CD8+ T lymphocytes, a process that is dependent on the engagement of the T-cell receptor (TCR) by peptide/MHC class I complexes and provision of co-stimulatory (e.g., CD28:B7 signaling) and accessory (e.g., interleukin (IL)-2) signals by

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professional antigen-presenting cells (e.g., dendritic cells; DCs). Although there is a significant body of evidence to suggest that most, if not all-human tumors are antigenic, T-cell-mediated anticancer immunity is often compromised by the loss of peptide/MHC complexes and/or co-stimulatory molecules from the surface of cancer cells. Cancer cells can also directly break down the communicative network between T cell and DCs by actively suppressing the antigen-presenting capabilities of tumorinfiltrating DCs. By secreting immunosuppressive factors such IL-10, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-b, and prostaglandin E2 (PGE2), cancer cells can keep DCs in a functional immature or tolerogenic state. Tumor-infiltrating DCs are also vulnerable to elimination through exposure to gangliosides (e.g., GM3, CD2), glycoproteins (e.g., MUC2 mucins), and neuropeptides within the tumor microenvironment (Dudek et al. 2013). The ability of cancer cells to modulate their immunogenic status (i.e., their capacity to provoke immune responses) can lead to the outgrowth of tumor cell variants that are no longer susceptible to control by the immune system. Tumor escape may also result from the induction of immune cell exhaustion or anergy. The generation of dysfunctional or unresponsive immune cells, in the context of cancer, can be caused by the long-term exposure of immune cells to suboptimally presented tumor antigens and immunosuppressive factors. Such conditions can (i) induce the upregulation of checkpoint inhibitory molecules on immune cells that function to increase the signaling threshold of activating receptors and induce the contraction of immune responses and (ii) increase the vulnerability of immune cells to death. Checkpoint Inhibition of Anticancer Immune Responses A major obstacle to the induction of protective anticancer immune responses are negative regulators of immune cell function, which exist within secondary lymphoid organs and peripheral tissues to maintain self-tolerance and prevent autoimmunity. Inhibitory immune checkpoints that have

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been demonstrated to regulate the strength and duration of anticancer immune responses include cytotoxic T-lymphocyte-associated protein 4 (CLTA-4), programmed death 1 (PD-1; CD279), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), lymphocyteactivating gene-3 (LAG-3), B7 family inhibitory ligands – B7-H3, B7-H4, and B7-H5 (VISTA) – B and T-lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin (Ig) and ITIM domain (TIGIT), and killer inhibitory receptors, which can be divided into two classes based on structure, killer cell immunoglobulin-like receptors (KIRs) and C-type lectin receptors (Shin and Ribas 2015). Some of these checkpoint inhibitory pathways that are proving to be promising therapeutic targets for the clinical treatment of cancer are discussed in more detail below. Cytotoxic T-Lymphocyte-Associated Antigen-4 (CTLA-4)

T-cell expression of CTLA-4 is induced by TCR engagement. This inhibitory receptor regulates T-cell activation by outcompeting the T-cellassociated co-stimulatory receptor CD28 for its ligands, B7-1 (CD80) and B7-2 (CD86) on DCs. By limiting the delivery of positive signals to T cells through CD28, CTLA-4 can reduce a T cell’s ability to produce cytokines and proliferate. The inhibitory actions of CTLA-4 dominate within secondary lymphoid organs (e.g., spleen, lymph nodes) during the priming phase of a T-cell response. T regulatory cells also express CTLA4 at high levels and use the receptor to elicit suppression within secondary lymphoid organs by: (i) blocking conventional T-cell access to the B7 ligands on APCs, through direct binding of the ligands and inducing their downregulation, and (ii) removing the B7 ligands from APCs by transendocytosis, a process driven by TCR engagement. Reverse signaling by CTLA-4, via B7 ligands, can also induce the release of indoleamine 2,3-dioxygenase (IDO) from APCs, an enzyme that catalyzes the conversion of tryptophan into immunosuppressive metabolites that promote T regulatory cell activity and suppress T effector cells. Antibody-mediated blockade of

Immunosuppression and Cancer

CTLA-4 may promote anticancer immune responses by freeing B7 ligands for stimulation of CD28, depleting T regulatory cells via antibody-dependent cell-mediated cytotoxicity (ADCC), or altering the suppressive activity of T regulatory cells. Clinically, anti-CTLA-4 therapy [ipilimumab (also known as MDX-010, MDX-101, BMS-734016)] has demonstrated significant survival benefits compared with chemotherapy; however, only about 20% of patients benefit long term. Given the capacity of antiCTLA-4 treatment to promote diffuse and nonspecific T-cell activation, immune-related adverse events are commonly associated with the blockade of CLTA-4. Programmed Death-1 (PD-1)

PD-1 is an inducible co-inhibitory receptor, which is expressed on activated T cells, T regulatory cells, NK T cells, B cells, and APCs, and plays an important role in maintaining peripheral immune tolerance. It has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-L1 is constitutively expressed on APCs and is induced in peripheral tissues following cellular activation and exposure to pro-inflammatory cytokines such as interferon (IFN)-g. Tumor cells and tumor-associated stroma can also express PD-L1, which has been demonstrated to confer immune resistance and protect tumor cells from T-cell-mediated apoptosis. Indeed in human tumors, PD-L1 expression has been associated with poor prognosis. PD-L2 expression is restricted to myeloid cells. PD-1 engagement of its ligands can signal inhibition via inhibitory motifs within its intracellular domain that recruit phosphatases and in turn dephosphorylate key TCR signaling intermediates. Targeted blockade of PD-1 or PD-L1 has been demonstrated to reinvigorate the anticancer activity of tissueresident, antigen-educated T cells and reverse the suppressive phenotypes of myeloid and plasmacytoid DCs in preclinical models of cancer. Clinically, anti-PD-1 [nivolumab (BMS-936558; MDX-1106) and pembrolizumab (MK-3475)] and anti-PD-L1 (BMS-936559) antibodies have been tested in a diverse array of advanced PD-L1+ solid cancers with promising success.

Immunosuppression and Cancer

T-Cell Immunoglobulin and Mucin DomainContaining Protein-3 (TIM-3)

TIM-3 is another inducible checkpoint inhibitory receptor that plays an important role in maintaining peripheral immune tolerance by limiting T-helper 1 (Th1) immune responses. Within mouse and human cancers, TIM-3 is often co-expressed with PD-1 and has been demonstrated to mark the most suppressed or dysfunctional populations of T cells. Through binding its ligand, galectin-9 (C-type lectin), which is expressed on tumor cells, T regulatory cells, and tumor-infiltrating myeloid cells, TIM-3 can negatively regulate the proliferative activity of T cells and their production of cytokines [e.g., IL-2, tumor necrosis factor (TNF)-a, and IFN-g]. Galectin-9 can signal inhibition to T cells by inducing the phosphorylation of the intracellular domain of TIM-3, which triggers the release of human leukocyte antigen B (HLB-B)-associated transcript 3 (Bat3) and the subsequent accumulation of the catalytically inactive form of lymphocyte-specific tyrosine kinase (Lck). In addition to CD8 and CD4 T cells, TIM-3 is also expressed on tumor-associated T regulatory cells. TIM-3-positive T regulatory cells have been reported to be more suppressive than TIM-3negative T regulatory cells, a phenomenon potentially linked to their increased production of the immune-regulatory cytokine IL-10. Antibodymediated blockade of both TIM-3 and PD-1 can reinvigorate exhausted T cells and reprogram the suppressive activity of TIM-3+ T regulatory cells. Upregulation of TIM-3 expression on tumorassociated DCs may also interfere with the alarmin function of high-mobility group box 1 (HMGB1). By binding HMGB1, TIM-3 can inhibit HMGB1 capture of nucleic acids released from dying tumor cells, limiting the activation of innate immune responses in tumor tissue. Further to this, it has been reported that the coordinated actions of TIM-3 expression in cancer cells and myeloid cells may regulate tumorigenicity. Lymphocyte-Activating Gene 3 (LAG3)

LAG3 is expressed on activated T cells, NK cells, B cells, and plasmacytoid DCs. It has been documented to directly inhibit the effector

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functions of CD8 T cells and enhance the suppressive activity of T regulatory cells; however, the exact mechanisms by which it mediates its inhibitory actions remains unclear. The only known ligand of LAG3 is MHC class II molecules, which are up regulated on some epithelial cancers but are also expressed on tumor-infiltrating macrophages and DCs. While LAG3-targeted antibodies do not block LAG3-MHC class II interactions, they can reengage the anticancer activity of T cells in vitro and in vivo. The contribution of LAG3 to the development of immunosuppression within tumor microenvironments was also linked with its ability to bind the immuneregulatory lectin, Galectin-3, a phenomenon potentially linked with the highly glycosylated state of LAG3 within the tumor microenvironment, which can promote the cross-linking and activation of the LAG3-signaling complex and suppression of effector CD8 T-cell function. T cells and stromal cells are the major source of Galectin-3 in tumors. Attempts to block the inhibitor actions of LAG3 have included the use of a LAG-3-Ig fusion protein (Immutep, IMP321, Paris). Given the high level of redundancy that exists in eliciting immunosuppression, the concomitant targeting of multiple checkpoint inhibitory pathways will likely be necessary to achieve durable clinical responses. The use of agonistic agents to stimulatory/activating receptors including ICOS (CD278) and 4-1BB (CD137) are also being explored as a mechanism of expanding existing anticancer immune responses and shifting the balance of power in favor of cancer immunity over immunosuppression. Regulatory Immune Cells that Contribute to Tumor Escape Immunosuppression within the tumor microenvironment is also created by the recruitment of inhibitory immune cells such as T regulatory cells (Li et al. 2015), myeloid-derived suppressor cells (MDSCs; Solito et al. 2014), and M2 macrophages (Ostuni et al. 2015). These regulatory cells often accumulate within tumors in response to inflammatory triggers associated with hypoxia (owing to the unique metabolic properties of

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tumor cells and aberrant vascular network within tumors) and release of danger signals from dying tumor cells. The wound healing and/or tissue repair response that ensues can ultimately fuel, rather than limit, tumor progression and negatively regulate anticancer immune responses. T Regulatory Cells

T regulatory cells are pivotal to the maintenance of peripheral immune tolerance and control of immune responses to pathogens and cancer. They are a subset of CD4+ T cells that express the IL-2 receptor a-chain, CD25, and the forkhead box P3 transcription factor FOXP3, which is necessary and sufficient for their suppressive activity. T regulatory cells can be categorized into two groups: thymus-derived T regulatory cells (also known as naturally occurring T regulatory cells) and peripherally derived T regulatory cells (also known as induced T regulatory cells). In tumors, the accumulation of T regulatory cells can be promoted by tumor cell production of chemokines and pro-inflammatory mediators, such as CCL28 and VEGF, or activation of latent TGF-b that can aid in the conversion of CD4+ T cells into suppressive T regulatory cells. T regulatory cells can mediate their suppressive functions via direct interaction with DCs through the binding of CTLA-4 to its B7 ligands. Such interactions can block the delivery of stimulatory signals to T effector cells, induce the secretion of IDO, and inhibit DC maturation. T regulatory cells also secrete immunosuppressive cytokines including IL-10, TGF-b, and IL-35 and consume IL-2, which is critical to the activation and proliferation of T effector cells. Another mechanism by which T regulatory cells can limit the generation of anticancer immune response is by driving the production of adenosine within the tumor microenvironment. Under conditions of cellular stress, initiated by hypoxia or ischemia, this can induce the expression of ectonucleotidases CD39 and CD73 on T regulatory cells that can convert extracellular adenosine triphosphate (ATP) to adenosine. By signaling through the A2A receptor, adenosine can directly disable the cytotoxic effector function of CD8+ T cells and NK cells and

Immunosuppression and Cancer

inhibit Th1 CD4+ T-cell responses. It can also enhance the suppressive activity of myeloid cells and promote the expansion of T regulatory cells and MDSCs, which express the A2A and A2B adenosine-reactive receptors. Myeloid-Derived Suppressor Cells (MDSCs)

MDSCs are a tolerogenic and immunesuppressive population of immature myeloid cells. High numbers of tumor-associated MDSCs have been shown to correlate with poor prognosis. MDSCs can be broadly categorized into two subsets: granulocytic MDSCs (G-MDSCs or PMN-MDSCs; defined in mice as CD11b+ Gr1+ Ly6Clow Ly-6G+ CD49d and in humans as LIN CD11b+ HLA-DR CD33+ CD15+) and monocytic MDSCs (Mo-MDSC; define in mice as CD11b+ Gr-1Int Ly6Chi Ly6G CD49d+ and in human as CD33+ CD14+ HLA-DRlow/). However, the phenotype and mechanism of action of MDSCs is largely dependent on the cancer type and environmental conditions. Of the two MDSC populations, Mo-MDSCs are often found in greater numbers than G-MDSC in tumors. Factors that can influence MDSC accumulation and suppressive function within tumors include proinflammatory chemokines (CXCL1, CXCL2, CCL2), cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-a, TGF-b], adenosine, growth factors [hepatocyte growth factor (HGF), VEGF], and STAT3, which has been reported to induce the expression of the pro-inflammatory proteins S100A8 and A9 that can arrest MDSC maturation. Immune suppression by G-MDSCs is mediated via reactive oxygen species (ROS), while Mo-MDSCs function primarily by expressing nitric oxide synthase (NOS2) and arginase (ARG1) and by generating reactive nitrogen species (RNS). MDSCs can suppress T-cell responses through the secretion of catabolic enzymes that can deplete specific amino acids, leading to the production of ROS, such as hydrogen peroxide (H2O2). This can cause the subsequent inhibition of CD3z chain expression in T cells, ultimately reducing the capacity of the TCR to recognize peptide/MHC complexes, leading to the induction of cell-cycle arrest.

Immunosuppression and Cancer

Tumor-Associated Macrophages (TAMs)

TAMs are an abundant population of terminally differentiated leukocytes in solid cancers, derived from blood monocytes that are recruited to tumors by malignant and stromal-derived chemokines including: CCL2, colony-stimulating factor-1 (CSF-1), CXCL12 (SDF-1), VEGFA, and semaphoring 3A (SEMA3A). TAM can also arise through the differentiation of Mo-MDSC in response to hypoxic conditions within the tumor. The abundance of TAM in tumors often correlates with poor prognosis and response to therapy in most human cancers. TAMs can promote tumor survival by secreting VEGF, which supports tumor vascularization and enables blood-borne cellular metastasis. They are also poor inducers of anticancer immune responses due to their ability to secrete immunosuppressive factors such as IL-10, TGF-b, and PGE2, which can promote the accumulation and suppressive activity of T regulatory cells. By producing IL-10, TAMs negatively regulate the production of the immune stimulatory cytokine IL-12 by myeloid cells. This can indirectly stimulate the differentiation of T-helper 2 immune cells that release IL-4 and IL-13 and in turn further support the generation of protumorigenic/anti-inflammatory, “M2” TAMs. Similar to MDSC, TAMs can also inhibit T-cell function by the production of ARG1. Tumor and stromal cells are major sources of signals that control TAM functions. They produce TGF-b and PGE2, which can promote the differentiation of macrophages and secrete factors such as macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and GM-CSF that can stimulate myelopoiesis. Conclusion For a cancer to survive and progress to clinic detection, it must overcome tumor intrinsic (cell autonomous) and extrinsic (immune-mediated) barriers to oncogenesis. Cancers deploy multiple immunosuppressive mechanisms that can affect the phenotype and function of immune cells, alter the antigenic landscape of the cancer, and protect against cell death. As we learn more about the complex interactions that exist between cancer

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and the immune system, we are starting to devise promising strategies to break down the immunosuppressive barrier within tumors and support the capacity of the immune system to eliminate cancer. Indeed, the blockade of multiple immune checkpoint inhibitory receptors has now successfully translated to oncology practice. As yet it is not clear as to whether therapeutic programs to deplete tumor-associated regulatory cells is a feasible goal; however, this may be a secondary effect of some commonly used anticancer agents including 5-FU, cyclophosphamide, gemcitabine, and trabectedin. Ultimately, by selectively blocking immunosuppressive barriers within tumors, the full therapeutic power of important first-line and experimental anticancer therapies will be unleashed.

Glossary Adaptive Immunity Acquired or learned immunity. The adaptive immune system comprises of circulating white blood cells including T lymphocytes [CD4+ (helper T cells) and CD8+ (cytotoxic/killer cells)] and B lymphocytes, which give rise to antibodyproducing plasma cells. Such cells express antigen-specific receptors formed by gene rearrangements. Adaptive immune responses are directed against specific antigens and afford protection against reexposure to the same antigens through the development of immunological memory. Cancer A disease characterized by the abnormal growth and division of a population of cells that have the potential to invade or spread to multiple organs/tissues of the body. Chemokines A group of chemotactic cytokines that provide directional cues for the movement of immune cells. Cytokines A class of small immune-regulatory proteins that are secreted by cells as a means of communication and coordinating immune responses. Genomic Stability Refers to the integrity of the genetic material of a cell. Genomic stability is often lost in cancer cells due to the accumulation of abnormal changes or mutations in the

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DNA or chromosomes of a cell. Such changes can cause loss of DNA or the mis-expression of genes that can drive cancer development. Immune System Is a complex network of biological structures and cells designed to protect against disease. Immunogenicity The degree to which a substance possesses the ability to provoke an immune response. Innate Immunity The innate immune system is the body’s first line of defense against foreign pathogens. It comprises of circulating white blood cells including: natural killer (NK) cells, macrophages, dendritic cells (DCs), basophils, neutrophils, and eosinophils. Such cells express germ line-encoded receptors that are not directed against specific antigens. Innate immune responses do not give rise to immunological memory.

Immunosurveillance of Tumors

See Also (2012) Adaptive immunity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 42–43. doi:10.1007/978-3-64216483-5_74 (2012) Cytokine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1051. doi:10.1007/978-3-642-16483-5_1473 (2012) Immune system. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1815. doi:10.1007/978-3-642-16483-5_2980 (2012) Immunogenecity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1819. doi:10.1007/978-3-642-16483-5_2989

Immunosurveillance of Tumors Chengcheng Jin The David H. Koch Institute of Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

Cross-References ▶ Cancer ▶ Chemokines ▶ Innate Immunity

References Dudek AM, Martin S, Garg AD, Agostinis P (2013) Immature, semi-mature, and fully mature dendritic cells: toward a DC-cancer cells interface that augments anticancer immunity. Front Immunol 4:438 Li Z, Li D, Tsun A, Li B (2015) FOXP3+ regulatory T cells and their functional regulation. Cell Mol Immunol 12(5):558–565. doi:10.1038/cmi.2015.10 Ostuni R, Kratochvill F, Murray PJ, Natoli G (2015) Macrophages and cancer: from mechanisms to therapeutic implications. Trends Immunol 36:229–239 Schreiber RD, Old LJ, Smyth MJ (2011) Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331:1565–1570 Shin DS, Ribas A (2015) The evolution of checkpoint blockade as a cancer therapy: what’s here, what’s next? Curr Opin Immunol 33:23–35 Solito S, Marigo I, Pinton L, Damuzzo V, Mandruzzato S, Bronte V (2014) Myeloid-derived suppressor cell heterogeneity in human cancers. Ann N Y Acad Sci 1319:47–65

Definition Tumor immunosurveillance is defined as an extrinsic tumor-suppressive mechanism whereby the immune system identifies and eliminates cancerous and/or precancerous cells.

Characteristics Development of the Cancer Immunosurveillance Theory The concept of cancer immunosurveillance was first proposed by Frank Macfarlane Burnet and Lewis Thomas in 1957 who speculated that lymphocytes acted as sentinels in recognizing and eliminating continuously arising, nascenttransformed cells before they manifested disease. However, this theory had been under a longstanding debate due to the absence of strong experimental data illustrating immunological eradication of cancerous lesions in vivo until late 1990s. Then, key findings from the first sets of

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experiments using mice with genetically defined mutations demonstrated that deficiencies in critical components of the immune system (effector molecules such as IFN-gamma and perforin; cellular compartments such as lymphocytes) increased the development of transplanted and carcinogen-induced tumors. Since then, the cancer immunosurveillance concept regained tremendous public and research attentions. Emerging evidence from both basic research with animal models and clinical studies with human subjects supports the important role of immune system in controlling cancer development. Now, it is generally accepted that the immune system not only protects the host from virusinduced tumors by eliminating or suppressing viral infections but also is able to specifically recognize and destroy tumor cells on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress. Furthermore, taking into account the fact that despite this immunosurveillance mechanism, tumors can still develop in immunocompetent hosts, an updated concept of tumor immunoediting has been formulated. Basically, the immune system can shape the immunogenicity of evolving tumors by selecting for neoplastic cells that are less immunogenic or more resistant to destruction by antitumor immunity. The process of tumor immunosurveillance and immunoediting can be divided into three phases: elimination, equilibrium, and escape. Basic Mechanism The basic of cancer immunosurveillance is that tumor cells express certain molecules that differentiate them from their nontransformed counterparts and thereby can be recognized by the immune system. For adaptive immune system, these molecules are antigens resulting from the expression of mutated genes or ectopic expression of normal gene (in aberrant location or timing or overexpressed). A number of tumor antigens have been discovered in humans, including melanocyte differentiation antigens, mutated p53 antigen, overexpressed HER-2, and temporally

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mis-expressed cancer/testis (CT) antigens (such as MAGE and NY-ESO-1). For innate immune system, the best characterized signals are stressinduced expression of NKG2D ligands on the surface of premalignant cells which can be recognized by germ line-encoded receptors on natural killer (NK) cells. Various cell types of both innate and adaptive immune system, including ab and gd T cells, NKT cells, and NK cells, have been shown to play important roles in tumor immunosurveillance and immunoediting. They can directly kill tumor cells via production of perforin and TRAIL, and a number of cytokines including IFN-g, type I IFNs, and IL-12 have been shown to promote the activity of these cytotoxic immune cells. Mice lacking such effector cells or molecules have been shown to exhibit increased incidence of spontaneous as well as carcinogen-induced tumor development. Elimination In this step, the immune system is alerted to the presence of developing oncogenic lesions and eliminates the transformed cells before they emerge to tumor. If the elimination is complete, all tumor cells can be successfully eradicated without progression to the next stage; if only a portion of tumor cells are cleared in this phase, the remaining tumor cells can further undergo immunoediting process and proceed to the “equilibrium” and possibly final “escape” stages. The mechanisms by which the immune system recognizes preemergent tumor cells have not been well understood thus far. It is suggested that damage-associated molecular pattern molecules (DAMPs) may be released from dying tumor cells or from damaged tissues of early oncogenic lesion, leading to the activation of germ lineencoded pattern recognition receptors on macrophages and dendritic cells. Type I IFNs and stress signals induced during early tumor development can also activate dendritic cells and NK cells respectively. Stimulation of antigen-presenting cells can then promote the induction of antitumor adaptive immunity.

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Equilibrium A temporary state of equilibrium between the immune system and the developing tumor can be generated if the elimination step is incomplete and the residue tumor cells are kept from outgrowth by the adaptive immune system. However, dormant tumor cells can continue to accumulate further mutations under the selection pressure of immune system. In some cases they will acquire changes that resist or suppress antitumor immunity and eventually allow them to escape the control of immune system. Escape In the escape phase, the immune system is no longer able to contain tumor growth, resulting in progressively outgrowing of tumor cells that have acquired the ability to avoid immune recognition and/or destruction. Many mechanisms have been uncovered to contribute to tumor escape. From the tumor side, genetic instability of tumor cells may lead to mutant cells with reduced immune recognition (commonly through a loss of antigen expression or downregulation of antigen processing/presenting function) or increased resistance to the cytotoxic effects of immunity (e.g., through induction of antiapoptotic pathways). On the other hand, establishment of an immunosuppressive state within the tumor microenvironment can also promote tumor escape. Recruitment of immunosuppressive cell types such as immature myeloid cells (myeloid-derived suppressive cells, MDSCs) and regulatory T cells can inhibit the activation of antitumor effector T cells.

Clinical Impact A number of clinical observations have been reported to support the notion of tumor immunosurveillance in humans. First, there is increased risk of tumor development in immunosuppressed patients such as organ transplant recipients who receive immunosuppressive drugs to

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prevent transplant rejection. Similarly, patients with acquired immunodeficiencies due to infection by human immunodeficiency virus 1 (HIV-1) or primary immunodeficiency are also more prone to cancer. For instance, patients with defective humoral immunity due to common variable immunodeficiency (CVID) had increased incidence of lymphoma and epithelial tumors of the stomach, breast, bladder, and cervix. Selective immunoglobulin A (IgA) deficiency was associated with a higher risk for gastric carcinomas, whereas patients with X-linked immunodeficiency with hyper-IgM had a high incidence of pancreatic and liver cancer. Second, infiltration of tumor-reactive lymphocytes in human primary tumors is associated with improved prognosis. For example, it has been shown that melanoma patients with high levels of CD8+ T cell infiltration survive longer than those whose tumors contain low numbers of lymphocytes. Last but not least, progress in cancer immunotherapy has revealed a very promising strategy to treat cancer which acts by potentiating the naturally occurring immune response of the patient, through blockade of the immune checkpoint molecules CTLA-4 and PD-1/PD-L1. Altogether, accumulating evidence supports that the immune system plays a critical role in protection against cancer.

References Burnet FM (1957) Cancer – a biological approach: I. The processes of control. II. The significance of somatic mutation. Brit Med J 1(5022):841–847 Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD (2002) Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3(11): 991–998 Swann JB, Smyth MJ (2007) Immune surveillance of tumors [Review]. J Clin Invest 117(5):1137–1146 Schreiber RD, Old LJ, Smyth MJ (2011) Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science 331(6024): 1565–1570

Immunotherapy

Immunotherapy Mehmet Kemal Tur1 and Stefan Barth2 1 Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Giessen, Germany 2 Institute of Infectious Disease and Molecular Medicine and Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa

Synonyms Biological therapy

Definition Immunotherapy is the treatment of cancer or inflammatory/autoimmune disease by inducing, enhancing, or suppressing an immune response. Immunotherapy can be nonspecific or antigenspecific. Nonspecific immunotherapy aims to enhance the overall host immune response, whereas specific immunotherapy targets the immune system against a particular tumor or increases tolerance toward a specific allergen. There are four main categories of specific immunotherapy: ▶ adoptive immunotherapy, antibody-based immunotherapy, cancer vaccine therapy, and allergen-specific immunotherapy. From these, adoptive and antibodybased immunotherapies are passive approaches, whereas cancer vaccine therapy and allergenspecific immunotherapy are active approaches.

Characteristics Despite advances in oncological research, cancer remains a leading cause of death throughout the developed world. Nonspecific approaches to cancer treatment, such as surgery, radiotherapy, and generalized chemotherapy, have been successful

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in the management of a distinct group of leukemias and slow-growing solid cancers. However, many solid tumors show considerable resistance to such approaches, and the prognosis in these cases is correspondingly poor. Immunotherapy is an emerging alternative area of cancer treatment. Cancer immunotherapy includes both passive and active strategies. Passive immunotherapy involves the ex vivo creation of established tumor-immune elements (antibodies, immune cells) that are administered to patients to mediate antitumor activity directly or indirectly and which do not stimulate the host immune system. In contrast, active immunotherapy induces a tumor-specific immune response in the patient, leading to the production of specific immune effectors (antibodies and T-cells). Historically, cancer immunotherapy has focused on nonspecific immune stimulants. Pioneering work began more than 140 years ago, when Wilhelm Busch observed that tumors show temporary regression during an infection. Two decades later, Sir William Coley developed and improved this therapeutic concept by vaccinating a large number of sarcoma patients with attenuated mixed bacterial extracts (Coley toxin). Nonspecific Immunotherapy 1. ▶ Bacillus Calmette-Guerin (BCG) is the most effective intravesical nonspecific immunotherapeutic agent and is used for the prevention and treatment of superficial ▶ bladder cancer. The proposed antitumor mechanism of BCG involves activation of the immune system and the promotion of a local acute nonspecific ▶ inflammation in the bladder lumen. Immune cell activation in response to BCG is mediated by a family of transmembrane recognition receptors called ▶ Toll-like receptors (TLRs). Intravesical, BCG-induced inflammation facilitates the infiltration of a broad range of immune cells (▶ macrophages, lymphocytes and natural killer cells) and the activation of pro-inflammatory cytokines such as interleukin-1 (IL-1), ▶ interleukin-6, and tumor necrosis factor-alpha (TNF-a).

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2. ▶ Cytokines are low-molecular-weight, soluble proteins that regulate the innate and adaptive immune systems. The antitumor activity of cytokines is mediated by one of two general mechanisms: first, a direct antitumor effect and, second, indirect enhancement of the antitumor immune response. It has been hypothesized that both the cytokine-activated lymphocytes and their secretory products such as interferon-gamma and tumor necrosis factor-beta (TNF-b) may contribute to the lysis of tumor cells in vivo. The exogenous administration of interleukin-2 (IL-2) is efficient in a broad spectrum of experimental tumors, including sarcomas, carcinomas, hemoblastoses, melanomas, and hepatomas. In humans, IL-2 and interferon-a2b are approved for the treatment of advanced melanoma and for use with ▶ adjuvant therapy. Specific Immunotherapy 1. Adoptive immunotherapy involves the infusion of immunologically competent, ex vivoexpanded, donor-derived lymphocytes (DLI), which specifically destroy tumor cells by graftversus-leukemia (GvL) or graft-versus-tumor (GvT) effects. In addition, peripheral bloodderived lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TILs) derived from tumor sections have proven to be effective antitumor agents. To address MHC and exogenous cytokineindependent activation of antitumor effector functions, T-cells can be engineered to express ▶ chimeric T-cell receptors, chimeric antigen receptors (CARs). CARs are composed of a recognition unit (antibody fragment) and an intracytoplasmic signaling molecule. Such receptors can be used to target various types of effector cells including cytotoxic T cells toward any tumor-associated antigen for which there is a suitable antibody. 2. Antibody-based immunotherapy exploits the highly specific binding between antibodies and their corresponding tumor-associated antigens (TAAs), resulting in some significant clinical responses. Tumor-associated antigens are structures presented predominantly by

Immunotherapy

tumor cells, thereby allowing antibodies to distinguish tumors from nonmalignant tissue. Therapeutic monoclonal antibodies can destroy tumor cells directly by inducing ▶ apoptosis or indirectly through immunologic mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or ▶ complement-dependent cytotoxicity (CDC). In addition, the natural function of antibodies can be enhanced by conjugating them to toxins (▶ immunotoxins), radionucleotides (radioimmunoconjugates), liposomes (▶ immunoliposomes), and cytotoxic drugs. Host immune responses can be enhanced through the induction of anti-idiotypic antibodies or through the use of ▶ bispecific antibodies containing arms with different specificities. Monoclonal antibodies are the largest class of biotechnology-derived proteins, with 29 monoclonal antibodies already approved for clinical use. 3. ▶ Cancer vaccine therapy represents an active, systemic, tumor-specific immune response of host origin. It is used either to treat existing cancers (therapeutic vaccines) or to prevent cancer development (prophylactic vaccines). There are several types of cancer vaccine: isolated whole cell cancer vaccines or tumor cell lysates, protein- or peptide-containing vaccines, viral vector vaccines, and anti-idiotype vaccines. Following the administration of a vaccine antigen that resembles a specific target, the patient’s humoral and T-cell-specific immune response induces defense mechanisms to combat the target in vivo.

Cross-References ▶ Adjuvant Therapy ▶ Adoptive Immunotherapy ▶ Apoptosis ▶ Bacillus Calmette-Guérin ▶ Bispecific Antibodies ▶ Bladder Cancer ▶ Cancer Vaccines ▶ Chimeric Antigen Receptor (CAR) ▶ Chimeric Antigen Receptor on T Cells

Immunotoxins

▶ Complement-Dependent Cytotoxicity ▶ Cytokine ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶ Immunoliposomes ▶ Immunotoxins ▶ Inflammation ▶ Interleukin-6 ▶ Macrophages ▶ Natural Killer Cell Activation ▶ Toll-Like Receptors ▶ Tumor Necrosis Factor

References Schuster M, Nechansky A, Kircheis R (2006) Cancer immunotherapy. Biotechnol J 1(2):138–147 Waldmann TA (2003) Immunotherapy: past, present and future. Nat Med 9(3):269–277

See Also (2012) Allergen-Specific Immunotherapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 137. doi:10.1007/978-3-642-16483-5_188 (2012) Anti-Idiotypic Antibodies. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 213. doi:10.1007/978-3-642-164835_306 (2012) Coley Toxin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 895. doi:10.1007/978-3-642-16483-5_1259 (2012) Graft-Versus-Leukemia/Tumor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1597. doi:10.1007/978-3-642-16483-5_2503 (2012) Interleukin-1. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1892. doi:10.1007/978-3-642-16483-5_3095 (2012) Interleukin-2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1892. doi:10.1007/978-3-642-16483-5_3097 (2012) Lymphocytes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2123. doi:10.1007/978-3-642-16483-5_3455 (2012) Lymphokine-Activated Killer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2123. doi:10.1007/978-3-642-16483-5_3460 (2012) Monoclonal Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Prophylactic Vaccine Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3005. doi:10.1007/978-3-642-16483-5_4773 (2012) Therapeutic Vaccine Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3668. doi:10.1007/978-3-642-16483-5_5766

2239 (2012) Tumor-Associated Antigen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3807-3808. doi:10.1007/978-3-642-16483-5_6017 (2012) Tumor-Infiltrating Lymphocytes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3815. doi:10.1007/978-3-642-16483-5_6032 (2012) Vaccine Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3879. doi:10.1007/978-3-642-16483-5_6147

Immunotoxins Hendrik Fuchs1 and Stefan Barth2 1 Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany 2 Institute of Infectious Disease and Molecular Medicine and Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa

Synonyms Antibody toxin fusions or conjugates; Cytokine toxin fusions or conjugates; Growth factor toxin fusions or conjugates; Targeted toxins

Definition Immunotoxins (ITs) are chimeric molecules consisting of one component that binds to a disease-specific cell-surface target molecule and another that confers cytotoxicity. The two components are either joined covalently in vitro by chemical conjugation or expressed as a fusion protein generated by recombinant DNA technology. The binding moiety is usually a monoclonal antibody, a derivative thereof or a cytokine, ultimately of mammalian origin. In contrast, the cytotoxic moiety is a catalytically active protein-based toxin or enzyme and may derive from any source, including plants and bacteria as well as mammals. After selective binding to diseased cells, immunotoxins are internalized and released into the cytosol. The cytotoxic component then induces cell death through its catalytic activity.

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Characteristics Immunotoxins are used to achieve specific, passive immunotherapy, a strategy that has emerged as a potentially effective approach to combat cancer. The major advantages of immunotherapy include its noninvasive nature and the selective targeting of cancer cells, leaving normal cells unharmed. In order to succeed, this approach requires the isolation and preparation of an antibody that targets a tumor-associated antigen (TAA) and exhibits tumoricidal or tumoristatic activity. Murine, chimeric, humanized and human antibodies, and their respective antibody fragments, directed against tumor-associated antigens have been used for the diagnosis and treatment of certain human cancers. Unconjugated, toxin-conjugated, and radiolabeled forms of these antibodies have been used in such therapies. The early vision of Paul Ehrlich, who postulated the development of “magic bullets” for tumor-specific therapy in 1896, became true in 1970 when Moolten and Cooperband described the selective destruction of cells by antibodies conjugated to the bacterial diphtheria toxin. Subsequently, other potent cytotoxins were coupled to disease-specific immunologic ligands. Catalytic Activity Two groups of toxins have been used in clinical trials thus far: (a) ribosome-inactivating proteins (RIPs) derived from plants and (b) ADPribosylating proteins derived from bacteria. 1. Ribosome-inactivating proteins are subdivided into two classes. Class I RIPs, which include pokeweed antiviral protein, saporin, bouganin, momordin, and gelonin, comprise a single catalytic polypeptide. In contrast, class II RIPs, which include ricin and mistletoe lectin, consist of a catalytic A chain and a lectin-binding B chain that associates with specific cellsurface carbohydrate groups. The adenosine nucleosidase activity of the catalytic polypeptide cleaves a specific N-glycosidic bond in mammalian 28S RNA (adenine4324 in the rat sequence). Because this residue is required to

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bind the eukaryotic elongation factors eEF-1 and eEF-2, securing them in the 60S ribosomal subunit, its modification inhibits the binding of these factors resulting in the abrupt arrest of protein biosynthesis and ultimately in cell death. 2. Two bacterial ADP-ribosylating proteins have been mainly used to date. These are Pseudomonas aeruginosa exotoxin A (ETA) and diphtheria toxin (DT), the latter produced by Corynebacterium diphtheriae, a bacterium causing diphtheria that is a contagious disease of the throat. Both toxins catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) onto a modified histidine residue (diphthamide) in eEF-2. This prevents eEF-2 from interacting with its binding cavern in the 60S ribosomal subunit, also arresting protein biosynthesis and leading to cell death. Developments The first generation of immunotoxins was prepared by conjugating native, glycosylated plant toxins to tumor-specific antibodies. This resulted in some nonspecific toxicity due to the affinity of the B chain for common oligosaccharide groups, as well as binding between the glycosyl groups of the toxin and non-parenchymal mannose receptors on liver cells and cells of the reticuloendothelial system (RES). In the second generation of immunotoxins, tumor-specific antibodies were therefore conjugated to the deglycosylated, catalytically active subunit, resulting in increased activity and better tolerance both in animal models and phase I/II trials in cancer patients. In patients, the major nonspecific and dose-limiting toxicity associated with immunotoxins is vascular leak syndrome (VLS), characterized by fluid leakage from capillaries, a fall in serum albumin, fluid retention, edema, and weight gain. Baluna et al. identified a (x)D(y) motif present in both the ricin A chain and human interleukin-2 which caused nonspecific binding to integrin receptors on vascular endothelial cells. However, endothelial cell damage caused by high concentrations of immunotherapeutics can usually be managed by adequate hydration of patients. Major challenges for the continued development of chemically

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linked immunotoxins include better purification strategies for these heterogeneous compounds, reducing the production costs, and removing the nonspecific integrin-binding motif to reduce toxicity toward endothelial cells. The current third-generation immunotoxins consist of mutant versions of bacterial toxins such as ETA and DT (with the internal integrinbinding motifs deleted) genetically fused to disease-specific ligands. The ETA deletion mutant retains the original domain II (responsible for translocation) and catalytic domain III. After binding to its cell-surface receptor, the immunotoxin is guided to the trans-Golgi network and endoplasmic reticulum by its C-terminal KDEL-like sequence known as an ER retention signal. In these compartments, the immunotoxin is bound to the protease furin, located on the inner membrane, through its furin consensus site in domain II. After cleavage by furin, only the C-terminal portion of the toxin (including the catalytic domain) is actively translocated into the cytosol, where its catalytic activity causes the arrest of protein biosynthesis and cell death as described above. Initial data from phase I trials have become available, including the treatment of immunocompromised patients suffering from chemotherapy-resistant hairy cell leukemia with a Pseudomonas aeruginosa exotoxin A-based anti-CD22 immunotoxin (BL22) described by Kreitman et al. In this trial, 19 of 31 patients (61%) treated with up to 33 cycles per patient showed complete remission (CR), whereas six (19%) showed partial responses. Within the median follow-up time of 36 months, seven patients were still in CR. Such responses have been observed predominantly in patients with hematologic malignancies, whose immune systems were impaired by previous chemotherapy. The patients were therefore unable to identify the immunotoxin as a foreign protein and did not produce anti-immunotoxin antibodies, which would neutralize the drug even after one treatment cycle. The first immunotoxin approved by the Federal Drug Administration (FDA) was a fusion of the catalytic domain and translocation domain of diphtheria toxin with interleukin-2 and was called

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DAB389IL2, denileukin diftitox or Ontak. In a phase III trial of 71 cutaneous T-cell lymphoma patients, a 30% response rate was achieved, including 10% CR. In patients with functional immune systems, the immunogenicity of recombinant immunotoxins continues to be a major challenge. Possible solutions include the deletion of T- and B-cell epitopes, modification with polyethylene glycol, or the treatment of patients with immunosuppressive agents. The most obvious modification would be the complete humanization of the recombinant immunotoxins following the identification and integration of human enzymes that induce apoptosis when internalized. The first examples of these fourth-generation immunotoxins are under development and feature human RNases such as angiogenin, human proteases such as granzyme B, and human kinases. A major challenge when using these immunotoxins will be to guarantee that internalization and translocation occur with the same efficiency as bacterial toxins such as ETA. Further improvements have been achieved by the addition of small functional peptides that link the cytotoxic and disease-specific binding moieties. Cleavable peptides targeted by endogenous endosomal and cytosolic proteases facilitate the cellular release of the binding moiety, resulting in improved cytosolic uptake of the cytotoxic moiety and unidirectionality of the process. So-called Trojan peptides are able to cross membranes and to transport cargo with them at the same time. These membrane-penetrating peptides also serve to augment the efficiency of cytosolic drug transfer. Newest developments to enhance the endosomal escape of toxic proteins include photochemical internalization and the use of particular glycosylated triterpenoids from plants. By the end of 2014, more than 50 immunotoxins had been tested in clinical trials. The most promising results were observed for hematologic malignancies, while solid tumors often remained unaffected. Nevertheless, vigorous research into the development of novel ITs is a promising field of research for the future treatment of cancer.

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Cross-References ▶ Antibody-Directed Enzyme Prodrug Therapy ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶ Immunotherapy ▶ Lung Cancer Targeted Therapy ▶ Pancreatic Cancer Molecular Targets for Therapy ▶ Photodynamic Therapy ▶ Prostate Cancer Molecularly Targeted Therapies ▶ Renal Cancer Trends in Molecularly Targeted Therapies ▶ Ribosome-Inactivating Proteins ▶ Saporin ▶ Targeted Drug Delivery

Imprinting

See Also (2012) Diphtheria toxin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1124. doi:10.1007/978-3-642-16483-5_1636 (2012) Tumor-associated antigen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3807–3808. doi:10.1007/978-3642-16483-5_6017

Imprinting Christoph Plass German Cancer Research Center (DKFZ), Heidelberg, Germany

Definition References Baluna R, Rizo J, Gordon BE, Gethie V, Vitetta ES (1999) Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome. Proc Natl Acad Sci U S A 96:3957–3962 Barth S, Huhn M, Matthey B, Tawadros S, Schnell R, Diehl V, Engert A (2000) Ki-4(scFv)-ETA’, a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice. Blood 95:3909–3914 Gilabert-Oriol R, Weng A, Mallinckrodt Bv, Melzig MF, Fuchs H, Thakur M (2014) Immunotoxins constructed with ribosome-inactivating proteins and their enhancers: a lethal cocktail with tumor specific efficacy. Curr Pharm Des 20:6584–6643 Hristodorov D, Mladenov R, Schiffer S, Aslanian E, Huhn M, von Felbert V, Fischer R, Barth S, Thepen T (2015) Targeting CD64 mediates elimination of M1 but not M2 macrophages in chronic cutaneous inflammation. mAbs 7:853–62 Kreitman RJ, Wilson WH, Bergeron K, Raggio M, StetlerStevenson M, FitzGerald DJ, Pastan I (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Engl J Med 345:241–247 Tur MK, Huhn M, Thepen T, Stöcker M, Krohn R, Vogel S, Jost E, Osieka R, van de Winkel JG, Fischer R, Finnern R, Barth S (2003) Recombinant CD64-specific single chain Immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. Cancer Res 63:8414–8419

▶ Genomic imprinting describes a phenomenon in which a gene is expressed either from the paternal or from the maternal allele and thus discriminates these genes from the majority of genes that are expressed from both alleles.

Characteristics Normally, genes are expressed from both the maternal and the paternal allele. Genomic imprinting results in allele-specific expression of certain genes from either the paternal or the maternal allele. These genes are marked before fertilization in a way that either the maternal or the paternal allele is transcriptionally silenced in the offspring. One of the first indications that certain autosomal regions are subject to genomic imprinting came from mouse genetic studies using Robertsonian and reciprocal translocations. In these studies, uniparental duplications or deficiencies for certain chromosomal regions were analyzed. The failure of a disomy or duplication from one parent to complement a corresponding nullisomy or deficiency from the other parent constituted the genetic evidence for the occurrence of imprinting effects. In addition, embryos that contain either two copies of the maternal or the paternal genomes fail to survive in early development

Imprinting

indicating the complementary need for both the maternal and paternal genome. More than 25 imprinted genes have been identified in mice and humans, and there are estimates for about 100 imprinted genes in the mammalian genome. Certain characteristic features have been identified for imprinted genes. Most of the imprinted genes have important roles in early development. Interestingly, imprinted genes tend to occur in clusters suggesting a common regulatory mechanism. One of the best studied clusters of imprinted genes is located on mouse distal chromosome 7 (human 11p15.5), encompassing 1.5 Mbp and including the maternally expressed genes p57KIP2, Kvlqt1, Mash2, and H19 as well as the paternally expressed genes Ins2 and Igf2. It is now well accepted that imprinting could be regulated in a tissue-specific manner in a way that only some tissues express the gene from one allele, while others show biallelic expression. Here, unknown mechanisms exist that allow to bypass the regulation of imprinting. It is interesting to note that a number of imprinted genes encode for RNAs but do not have an open reading frame and are not translated. It is believed that these RNAs play a role in the regulation of the imprinting process. Interestingly, short GC-rich repeat sequences were identified in the vicinity of many imprinted genes usually located in or near so-called differentially methylated sites, ▶ CpG island-like sequences that are methylated on only one allele. Cellular and Molecular Aspects Regulatory mechanisms underlying genomic imprinting are under intense investigations in many laboratories but only incompletely understood. The features of the imprinting signal and the mechanism are unknown, but strong evidence suggests the involvement of DNA ▶ methylation. Several requirements for the underlying mechanisms can be postulated. First, the imprinting signal, or imprint mark, in the imprinted region must be established before fertilization. Second, the imprint mark must be an ▶ epigenetic modification and must directly or indirectly affect the transcription of a gene by silencing one allele and leaving the other active. Third, the imprint

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mark must be stable in mitosis and must be transmitted during cell division. Finally, the imprint mark must be reversible in a passage through the opposite germline. At present, DNA methylation is the only mechanism that conforms with the above requirements. Several lines of experimental data support the assumption that DNA methylation plays an important role in imprinted regulation. In mammals, DNA methylation occurs only at the cytosine residue of ▶ CpG dinucleotides. It was shown that DNA methylation in promoter regions can turn off the transcription of a gene. Most genes are subject to a process of demethylation directly after fertilization with most of the CpG sites unmethylated at the 16-cell stage. However, imprinted genes are exceptions in this demethylation process by maintaining small regions that show allele-specific methylation. DNA methyltransferase generates methylation patterns that are transmitted correctly following DNA replication and cell divisions. Studies of the expression of imprinted genes in DNA methyltransferase-deficient mutant mice indicated that normal level of DNA methylation is required for the control of allele-specific expression. Studies with transgenic mice suggested that methylation is the epigenetic modification underlying genomic imprinting. A direct correlation between paternal inheritance, transgene, ▶ hypomethylation, and tissue-specific expression of the transgene was shown, while the maternally derived copy is methylated and not expressed. Clinical Relevance Imprinted genes are involved in critical steps during normal embryonic development. A growing body of evidence implicates genomic imprinting in the pathogenesis of certain human disorders, inherited tumor syndromes, and sporadic tumors. At least ten genetic disorders have been found to be associated with genomic imprinting effects. In some cases, the trait is transmitted exclusively (or mainly) from one parent (either father or mother), or the disease is particularly severe when transmitted from one parent. In other cases, the disease is associated with uniparental disomies or parent-of-origin-specific aberrations.

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The best studied examples of imprinted genetic diseases are the Prader–Willi syndrome (PWS) and Angelman syndrome (AS). PWS is characterized by mild to moderate mental retardation; individuals are slow moving and overweight due to severe hyperphagia. Patients with AS showing severe mental retardation are thin and hyperactive and show disorders of movement and uncontrolled laughter. Both syndromes are linked to abnormalities on human chromosome 15q11–13. The first hint for a possible imprinting effect in these syndromes came from the finding that the deleted fragments in both syndromes are from opposite parental origins. In PWS the deletion occurred in the paternal copy and in cases of AS the maternal copy was deleted. Additional evidence came from the finding of maternal disomy of chromosome 15 in PWS patients and paternal disomies of chromosome 15 in AS. These data suggest that the PWS gene(s) is transcribed from the paternal allele only and the AS gene(s) is expressed from the maternal allele. Several imprinted genes were identified in the critical region for PWS/AS including paternally expressed SNRPN and maternally expressed UBE3A. There is also evidence that some of the imprinted genes have oncogenic or tumor suppressor function. Loss of tumor suppressor function of an imprinted gene could be achieved by loss of heterozygosity (LOH) involving the usually active copy, as shown for the cyclindependent kinase inhibitor, p57KIP2, in lung cancers; H19 in ▶ Wilms tumor; and NOEY2, a member of the ▶ RAS superfamily, in breast and ovarian cancers. Alternatively, uniparental disomy including the normally silent allele could lead to inactivation of an imprinted tumor suppressor gene. Activation of a growth-supporting gene such as IGF2 (▶ insulin-like growth factors) could occur by uniparental disomy involving the normally active copy. In addition, relaxation of imprinting control, also called loss of imprinting (LOI), could lead to biallelic expression and thus overexpression of an imprinted oncogene, as shown for IGF2 in Wilms tumor. The first evidence for the involvement of DNA methylation in LOI came from the finding of complete methylation of the CpG island located immediately

In Situ Breast Cancer

upstream of H19 transcription start site. Usually, this ▶ CpG island shows allele-specific methylation on the maternal allele. This epigenetic change correlated with LOI in IGF2 and silencing of H19. Another human disease is the ▶ Beckwith–Wiedemann syndrome (BWS) that is characterized by a number of growth abnormalities including gigantism. Between 5% and 10% of BWS patients are prone to Wilms tumor, adrenocortical carcinoma, hepatoblastoma, or embryonal rhabdomyosarcoma. Wilms tumors have been shown to exhibit preferential loss of maternal alleles at chromosome 11p. A cluster of at least seven imprinted genes was identified in 11p15.5 including the paternally expressed IGF2 and the maternally expressed H19. The most common abnormality in BWS patients is LOI of IGF2 without any detectable chromosomal abnormalities.

References Bartolomei MS, Tilghman SM (1997) Genomic imprinting in mammals. Annu Rev Genet 31:493–525 Cattanach BM, Kirk M (1985) Differential activity of maternally and paternally derived chromosome regions in mice. Nature 315:496–498 Falls JG, Pulford DJ, Wylie AA et al (1999) Genomic imprinting: implications for human disease. Am J Pathol 154:635–647 Nicholls RD, Saitoh S, Horsthemke B (1998) Imprinting in Prader–Willi and Angelman syndromes. Trends Genet 14:194–200 Reik W, Maher ER (1997) Imprinting in clusters: lessons from Beckwith–Wiedemann syndrome. Trends Genet 13:330–334

In Situ Breast Cancer ▶ Ductal Carcinoma In Situ

In Situ Cancer ▶ Dormancy

Indirubin and Indirubin Derivatives

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Definition

In Situ Carcinoma ▶ Carcinoma In Situ ▶ Dormancy

In Utero Exposure to Carcinogenic Agents

Indirubin is the parent compound of a spectrum of 20 ,3-bisindoles synthesized to improve the biological activity of this natural 20 ,3-bisindole lead structure. Indirubin and its isomers indigo and isoindigo are composed of two indolinone ring systems, linked through a double bond to make up 2,20 -(indigo), 3,20 -(indirubin) and 3,30 (isoindigo) bisindoles, respectively (Fig. 1).

▶ Transplacental Carcinogenesis

Characteristics

In Vitro Bystander Effect ▶ Bystander Effect

In Vitro Genetics ▶ Combinatorial Selection Methods

In Vivo Bystander Effect ▶ Bystander Effect

Indirect Effect ▶ Bystander Effect

Indirubin and Indirubin Derivatives Gerhard Eisenbrand and Karl-Heinz Merz Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany

Synonyms 20 ,3-Biindolinylidene-2,30 -dione: 3-(3-Indolinone2-ylidene)-indolin-2-one; 3-(1,3-Dihydro-3-oxo2H-indol-2-ylidene)-1,3 dihydro-2H-indol-2-one

History While indigo is one of the oldest dyes known, with a history of use dating back to bronze age, the two other isomers, especially indirubin, have gained reputation as components of traditional medications used against a variety of human diseases. The discovery of the anticancer activity of indirubin can be traced back to a traditional Chinese medication, consisting of 11 ingredients, mostly of herbal origin, with the name of Danggui Longhui Wan. The preparation is used traditionally for a variety of chronic and acute diseases including chronic myelocytic leukemia (CML). Chinese scientists achieved the identification of the active ingredient of this medication, Quing Dai, which corresponds to natural indigo, prepared from the leaves of indigoproducing plants. Furthermore, the Chinese scientists discovered that not only the blue dye indigo but a minor byproduct, the red-colored trace constituent indirubin, was the active antileukemic principle. In a clinical trial, synthetic indirubin was given orally to CML patients at dosages of 150–450 mg/day. A total of 314 patients participated in the study. Complete remissions were observed in 26%, partial remission in 33%, and some beneficial response in 28% of patients. Treatment was welltolerated, without major side effects. Studies exploring the mechanism of action reported a spectrum of relatively unspecific biological effects, not really convincing to fully explain the respectable anti-CML activity of the compound.

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Indirubin and Indirubin Derivatives

a

O

4⬘

5⬘ 6⬘ 7⬘

H N 2 1 3

3⬘ 2⬘ 1⬘ N H

7 6 5 4

O

b

5 6 O

4⬘ 5⬘ 6⬘ 7⬘

4 7

3⬘ 2⬘ 1⬘ N H

3

1 2 NH

O

c

5 6 O

4 7

HN 2⬘ 1⬘ 3⬘

3 2

1 NH

7⬘ 4⬘ 6⬘

O

5⬘

Indirubin and Indirubin Derivatives, Fig. 1 Structures and numeration of indigoid bisindoles. (a) Indigo, (b) indirubin (E211), and (c) isoindigo

Mechanism Research on indirubins gained momentum, when in 1999 it was discovered that such 3,20 bisindoles act as potent inhibitors of serine/threonine kinases, especially of ▶ cyclin-dependent kinases (CDKs) and of glycogen synthase kinase 3b (GSK3b). Later it was found that indirubins also inhibit ▶ receptor tyrosine kinases, such as ▶ vascular endothelial growth factor receptor (VEGFR) or ▶ Src kinase, and block Stat-3 signaling. Moreover, indirubins have also been discovered to activate the ▶ aryl hydrocarbon receptor transduction pathway. Thus, indirubins exert, in addition to their multimodal kinase inhibitory activity, further bimolecular effects contributing to anticancer activity. The relative kinase inhibitory profile of individual representatives of these 3,20 -bisindoles, as well as their pharmacokinetic properties, is markedly influenced by the type and pattern of substituents attached to the 3,20 -bisindole basic

scaffold. Within cancer targets, VEGFR2 and c-Src, as well as specific CDKs are of prime interest, since their respective partner proteins, activators or inhibitors, are aberrantly expressed in many human malignancies. Structure/Activity Chemical improvement of the parent molecule initially was driven by the aim to improve solubility and bioavailability without compromising anticancer activity. Indirubins act as ATP-competitive inhibitors of ATP-dependent kinases. Crystal structures of CDK2–indirubin complexes have been described in detail. It was shown that the flat disk shape of the 3,20 -bisindole molecule slides easily into the binding pocket and is tightly bound, mainly by virtue of hydrogen bonding and lipophilic interactions with the ATP-binding cleft, situated in the hinge region connecting the amino terminal with the carboxy terminal part of the kinase. The strong binding affinity of indirubin derivatives to the ATP site is practically not affected by additional binding of the activating partner cyclin A to CDK2. A superposition of the structures of the binding complexes of the indirubin derivative, E 226, and of a nonhydrolyzable ATP mimic, AMPPNP, with CDK2, unraveled molecular positions of the indirubin scaffold preferably to be exploited for chemical improvement of the molecule. Thus, positions 5 and 30 (arrows in Fig. 2) were identified as those of first choice for molecular modifications by attaching appropriate substituents. The chemistry to achieve the synthesis of such derivatives has been described in detail. A selection of results from comprehensive structure/activity studies is summarized in Tables 1 and 2. The results show that the inhibitory activity of the parent molecule, indirubin (E211), on isolated CDKs could be improved dramatically. While indirubin (E211) itself was somewhat less active than roscovitine, a standard purine-type of CDK inhibitor, several 30 or 5-substituted derivatives achieved IC50 values, especially toward CDK2/ cyclin A, CDK2/cyclin E, and CDK6/▶ cyclin D down to the low nanomolar range (Table 1). At the

Indirubin and Indirubin Derivatives

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Indirubin and Indirubin Derivatives, Fig. 2 Superposition of crystal structures of CDK2–E226 (yellow) and CDK2–AMPPNP (white). Green arrows point to 5- and 30 -position of indirubin scaffold (Reprinted from Davies et al. (2001), Structure 9:389; with permission from Elsevier)

same time, solubility in water and thus bioavailability could be improved by factors of up to about 400, as compared with the parent compound. In addition, the potency to inhibit human tumor cell proliferation was also strongly improved (Table 2). These novel compounds not only induce arrest in G1/S and G2/M phases of the cell cycle of various human tumor cells but also strongly trigger ▶ apoptosis and cellular necrosis by several mechanisms, including inhibition of antiapoptotic proteins, such as ▶ Mcl-1 and ▶ survivin, and induction of endoreplication. Thus, within this family of bisindoles, molecular permutations entail major effects on biological activities and allow generation of novel derivatives that lend themselves to clinical application. Since the major cellular targets of these

Indirubin and Indirubin Derivatives, Table 1 Inhibition of CDK/cyclin complexes (IC50-Werte, mM) by indirubins in comparison with roscovitine E-Nr E211 E226 E231

R1 O O NOH

R2 H So3  H

CDK1/ cyclin B 10a 0.055a 5.1  2.9b 0.18a

CDK2/ cyclin A 2.2a 0.035a 2.2  0.2b 0.44a

E729 Glc*

NO O

|| OCH3 || 5.6  1.3b || 0.8  0.1b || 0.09  0.01b || 0.4  0.1b E804 NO HO

7.9  0.4b 0.65a a

OH

|| H || 1.7  0.4b || 0.5  0.1b || 0.2  0.04b || 0.06  0.01b 3.5  1.3b 0.7a

1.0  0.4b 0.7a

>10b

Method: Meijer et al. (1997) Method: Jakobs et al. (2005) Glc, b-D-glucopyranosyl; –, not determined

b

CDK2/cyclin E 7.5a 0.15a 0.33  0.05b 0.25a

CDK6/cyclin D – – 0.08  0.03b

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Individualized Treatment

Indirubin and Indirubin Derivatives, Table 2 Solubility in water and human tumor cell growth inhibition of indirubin derivatives Substancea E211 E226 E231 E729 E804

Solubility in water (mg/L) > 100 0.12 42 1.6

LXFL-529 L 9.9 100 3.0 0.5 0.9

MCF-7 4.0 100 3.3 1.2 0.1

HCT116 – – – 0.5 8 years) and anatomical extent of colitis [standardized incidence ratio (SIR): 1.7, 95% confidence interval (CI): 0.8–8.2 for proctitis; SIR: 2.8, 95% CI: 1.6–4.4 for left-sided colitis; SIR: 14.8, 95% CI: 11.4–19.9 for pancolitis] appear as the most important and reproducible from one study to another. Other factors like family history of sporadic CRC (twofold higher risk), young age of IBD onset, backwash ileitis, and degree of inflammation in the involved colon are also considered as risk factors. Finally, association to primary sclerosing cholangitis (PSC), although rare (approximately 2–7.5% of IBD patients, more frequently in UC patients), appears as a very important risk factor and has conducted to a specific surveillance strategy in these particular patients since the cumulative incidence of patients presenting both UC and PSC was 33% at 20 years in a population-based Swedish study. By contrast, a few other factors have been suggested to be protective, such as folate and ursodeoxycholic acid, and, with the highest degree of evidence, treatment by 5-aminosalicylates (5-ASA). The goal of surveillance strategies (i.e., surveillance colonoscopy) is to detect by using a safe and effective intervention neoplasia at a curable stage (ideally as ▶ dysplasia). Dysplasia is defined as an unequivocally but noninvasive (intraepithelial) neoplasia. Despite the theory proposing that IBD-associated colon carcinogenesis progresses from no dysplasia to indefinite dysplasia and then to low-grade dysplasia (LGD), highgrade dysplasia (HGD), and finally invasive cancer, in reality, this conceptually useful model is by no means absolute. This uncertainty thrives on much of the controversy regarding treatment of LGD. Except in the case of dysplasia in DALM which usually is not too difficult to detect for experienced practitioners, macroscopic identification of flat dysplastic lesions (either of low or high grade) appears particularly difficult during conventional colonoscopic surveillance. Nevertheless, this early identification represents a major challenge, as the presence of HGD in the colonic epithelium is associated with concurrent, macroscopically undetectable CRC in 42–67% of

Inflammatory Bowel Disease-Associated Cancer

patients (in case of colectomy performed shortly after HGD diagnosis at colonoscopy) and to the finding of a synchronous CRC in up to 19% of patients in case of LGD diagnosis at colonoscopy. Considering these facts, recommendations for colonoscopic surveillance have been established. Screening colonoscopy should be initiated after 8–10 years of disease, including biopsies of each macroscopic lesion (with additional biopsies in the flat mucosa surrounding the DALM) and random biopsies of the macroscopically normal appearing mucosa (four-quadrant biopsies every 10 cm, some authors considering sampling every 5 cm in the rectosigmoid). In patients with PSC, colonoscopic surveillance should begin at the time of diagnosis of PSC and IBD. At each surveillance examination, the whole colon should be examined and biopsies processed in separate clearly identified specimen containers. New endoscopic technologies have been suggested to improve the diagnosis yield of early malignancy, especially dysplasia detection in flat mucosa. This is the case for high-resolution and magnifying endoscopy, chromoendoscopy (or dye endoscopy) based on vital staining with methylene blue or contrast staining with indigo carmine, magnifying chromoendoscopy, narrow-band imaging, or confocal laser endomicroscopy. These techniques open a new world of endoscopic imaging, but their usefulness for improving dysplasia detection in IBD needs to be better assessed before they will be recommended in routine screening. Despite the fact that chromoendoscopy increases the rate of detected flat lesions (which are not necessarily HGD or CRC), data indicate that random biopsies may still be appropriate in selected patients considered at high risk of HGD or CRC development (i.e., patients with associated PSC, with personal history of neoplasia, with a tubular colon, or with non-polypoid or endoscopically invisible or large lesions (≥ 1 cm) or indefinite dysplasia on a former colonoscopy). If no dysplasia is identified, surveillance examination is recommended every 1–3 years (some authors proposing a subsequent screening at 3 years between 8 and 20 years of disease, every 2 years between 20 and 30 years, and each year after 30 years of IBD diagnosis), except for IBD

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patients with concurrent PSC (surveillance colonoscopy recommended each year regardless of IBD duration). In case of LGD detection in flat mucosa, sample analysis by a second pathologist is recommended, and if LGD is confirmed, a surveillance colonoscopy should be performed 6 months later, although some authors propose to perform colectomy. Detection of HGD in flat mucosa indicates colectomy. Decision after LGD or HGD diagnosis in a polypoid lesion should integrate additional information: (i) if the polypoid lesion has been completely removed and no dysplasia has been detected elsewhere in the colon, a control colonoscopy with multiple random biopsies is recommended 6 months later; (ii) if this is not the case (incomplete removal and/or dysplasia detected in other biopsies), colectomy is indicated. A number of studies have examined the chemopreventive potential of several medications. Until now, 5-ASA has been the most thoroughly studied as a potential IBD-associated CRCpreventing agent. In fact, at least in vitro but also in some in vivo studies, 5-ASA has been shown among other effects to inhibit cell proliferation, to induce apoptosis, to act as potent RONS scavengers, and to enhance DNA repair. Although the optimal dose and duration of 5-ASA treatment to prevent IBD-associated CRC are unclear, data suggest that chronic systemic administration of 5-ASA at a dose of at least 1.2 g/day is the most likely to prevent IBD-associated CRC development. Small Bowel Adenocarcinoma in Crohn Disease The incidence of small bowel adenocarcinoma (SBA) is very low. In CD, its relative risk has been reported to be 20 to 30 times higher than in the general population. In a study, its cumulative risk in patients with ileal CD has been estimated to be 0.2% after 10 years of disease and 2.2% after 20 years, with a median age at diagnosis of 47 years compared with 68 years for patients with SBA de novo. It occurs in the ileal lesions of patients with CD more the 8 years after diagnosis arising from long-standing inflammation. Data indicate in these patients an incidence rate

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of 0.5 per 1000 patient-year. CD-associated SBA is difficult to diagnose and causes premature mortality in early-onset CD patients. Cancer Risk Associated to Immunosuppressive Therapy in IBD The efficacy of “classical” immunosuppressive drugs [azathioprine (a potent immunosuppressive drug that is converted to its active form in vivo and then kills rapidly proliferating cells, including lymphocytes responding to grafted tissues), 6mercaptopurine (6-MP), or methotrexate] as well as that of biological agents (i.e., in current practice, anti-TNFa antibodies such as infliximab) led to their increased use in IBD. This emphasizes the question of their potential carcinogenic effects, in particular considering the risk of lymphoma which has been previously reported in patients treated by azathioprine or 6-MP after renal or hepatic transplantation, although used at doses even higher than in IBD. Nevertheless, some general statements based on the most relevant studies in that field can be suggested: (i) the risk of primary intestinal lymphoma is significantly increased in CD patients, despite the absolute risk is low (0.1 per 1000 patients-year) - these lymphomas are mainly B-cell non-Hodgkin lymphomas usually developed in chronically inflamed intestinal lesions of middle-aged men after 8 years of disease; (ii) the absolute risk of lymphoma in the general IBD population appears extremely low; (iii) extra-intestinal non Hodgkin lymphoma risk in IBD patients treated by azathioprine or 6-MP is probably not more than two- to fourfold increased, although the respective responsibility of treatment and underlying disease has to be more accurately investigated; (iv) particular attention needs to be brought to the ▶ Ebstein-Barr virus (EBV)-positive lymphoma risk in azathioprine or 6-MP-treated IBD patients; (v) the highest absolute risk is reported in patients older than 65 years of age (5 per 1000 patientyears); and finally, (vi) the issue of lymphoma risk is likely to become more relevant in the future with the growing number of immunosuppressive and/or biological agents being used (or tested) in IBD. This has been emphasized by the report of non-EBV-related hepatosplenic T-cells

Inflammatory Bowel Disease-Associated Cancer

lymphomas - a usually very rare type of lymphoma - in young men after 2 years of combined purine analogs and therapeutic anti-TNFa monoclonal antibodies.

Cross-References ▶ Adducts to DNA ▶ APC/b-Catenin Pathway ▶ APC Gene in Familial Adenomatous Polyposis ▶ CpG Islands ▶ Crohn Disease ▶ Dysplasia ▶ Epstein-Barr Virus ▶ Helicobacter Pylori in the Pathogenesis of Gastric Cancer ▶ Interleukin-4 ▶ Interleukin-6 ▶ Lynch Syndrome ▶ Methylation ▶ Microsatellite Instability ▶ Prostaglandins

References Barral M, Dohan A, Allez M, Boudiaf M, Camus M, Laurent V, Hoeffel C, Soyer P (2016) Gastrointestinal cancers in inflammatory bowel disease: an update with emphasis on imaging findings. Crit Rev Oncol Hematol 97:30–46 Beaugerie L and Itzkowitz SH (2015) Cancers complicating inflammatory bowel disease. N Engl J Med 372:1441–1452 Elris K, Carrat F, Carbonnel F, Marthey L, Bouvier AM, Beaugerie L (2013) Incidence, presentation, and prognosis of small bowel adenocarcinoma in patients with small bowel Crohn’s disease: a prospective observational study. Inflamm Bowel Dis 19:1823–1826 Farraye FA (ed) (2006) Dysplasia and cancer in inflammatory bowel disease. Gastroenterol Clin North Am 35:517–734 Itzkowitz SH, Yio X (2004) Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol 287:G7–G17 Kwon JH, Farrell RJ (2005) The risk of lymphoma in the treatment of inflammatory bowel disease with immunosuppressive agents. Crit Rev Oncol Hematol 56:169–178

Inflammatory Breast Cancer Li HC, Stoicov C, Rogers AB et al (2006) Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers. World J Gastroenterol 12:363–371 Palascak-Juif V, Bouvier AM, Cosnes J et al (2005) Small bowel adenocarcinoma in patients with Crohn’s disease compared with small bowel adenocarcinoma de novo. Inflamm Bowel Dis 11:828–832 Viennot S, Deleporte A, Moussata D, Nancey S, Flourié B, Reimund JM (2009) Colon cancer in inflammatory bowel disease: recent trends, questions and answers. Gastroenterol Clin Biol 33 (Suppl. 3):S190–S201 Zaho LN, Li JY, Yu T, Chen GC, Yuan YH, Chen QK (2014) 5-aminosalicylates reduce the risk of colorectal neoplasia in patients with ulcerative colitis: an updated meta-analysis. PLoS ONE 9:e94208

Inflammatory Breast Cancer Fredika M. Robertson1 and Massimo Cristofanilli2 1 The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Division of Hematology and Oncology, Robert H Lurie Comprehensive Cancer Center, Chicago, IL, USA

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Characteristics Incidence of Inflammatory Breast Cancer (IBC) The incidence of IBC in the United States is 1–5% of all breast cancers; however, in the Maghreb countries, including Morocco, Algeria, Tunisia, Libya, and Mauritania, estimates are that IBC occurs in approximately 20% of patients with a breast cancer diagnosis. IBC occurs at a younger age than other types of breast cancer and is diagnosed more frequently in African Americans and Latinas than Caucasian women. Reports from the Surveillance, Epidemiology, and End Results (SEER) database reveal that the incidence of IBC has increased at a much greater rate than that observed for other types of non-IBC breast cancers during the past decade. Although primary IBC is less commonly diagnosed than other types of breast cancer, it is responsible for a disproportionate number of breast cancer-related deaths each year. Women diagnosed with IBC have a significantly shorter median survival time (2.9 years) than women with either non-IBC locally advanced disease (6.4 years) or women with non-T4 breast cancer (>10 years).

Definition Inflammatory ▶ breast cancer (IBC) is the most aggressive form of primary breast carcinoma and presents with very specific signs and symptoms, quite distinct from other types of non-IBC. IBC does not appear as a lump but rather appears as diffuse skin erythema, with swelling and edema involving more than two-thirds of the breast. The skin overlying the breast has a wrinkled/dimpled appearance defined as “peau d’orange,” with tenderness, induration, warmth, breast enlargement, and diffuseness of the tumor upon palpation (Table 1; Fig. 1a, b). The symptoms of IBC usually progress rapidly, and IBC has a distinct pathological hallmark characterized by extensive lymphovascular invasion by tumor cells, defined as tumor emboli, into the papillary and higher reticular dermis. Although the presence of dermal tumor emboli is not a requirement for a diagnosis of IBC, they are detected in approximately 75% of IBC patients and serve as a characteristic signature of IBC.

Screening Due to the diffuse nature of the tumor and the invasion into the skin overlying the breast, IBC is not easily detected by mammography and ultrasound but requires more sophisticated imaging approaches such as magnetic resonance imaging (MRI) and ▶ positron emission tomography (PET) to accurately diagnose and stage IBC. Since IBC occurs in young women and is often mistaken for an infection such as mastitis, which occurs in women who are breastfeeding, IBC patients are often misdiagnosed. This delay in accurate diagnosis combined with the rapid progression of this distinct type of breast cancer results in involvement of the axillary lymph nodes at the time of first accurate diagnosis. Since IBC is less commonly diagnosed, clinicians, including specialists in obstetrics/gynecology and dermatology and breast medical oncologists, are often unfamiliar with the signs and symptoms of IBC. Over the past 5 years, the awareness about IBC has been raised through the efforts of patient-initiated

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foundations, including the IBC Research Foundation and the IBC Foundation that focus on IBC education and awareness, to ensure that IBC is recognized and accurately diagnosed at its earliest stages. Multimodality Treatment The goal of management of any type of breast cancer is to maintain local control of the disease, to prevent the development of distant metastases, and ultimately to improve overall survival. These goals become even more important in the management of IBC, where long-term outcome remains inferior compared to other types of breast cancer. Historically, IBC was considered a uniformly fatal disease. Modalities of treatment comprising surgery with or without radiation therapy have typically not been associated with good long-term outcomes for IBC patients. The introduction of a multidisciplinary management Inflammatory Breast Cancer, Table 1 Clinical presentation of IBC Signs and symptoms of inflammatory breast cancer One breast becomes larger than the other Redness, rash, or blotchiness Pain or itchiness Thickening or dimpling of the skin “peau d’orange” Warmth or tenderness Lymph node swelling Flattening or discharge from the nipple

Inflammatory Breast Cancer, Fig. 1 (a) Photograph of patient with classic signs of IBC, including enlargement of one breast, with dimpled skin defined as peau d’orange

Inflammatory Breast Cancer

approach to IBC composed of a sequential process of preoperative chemotherapy followed by surgery and radiation therapy has modified the management of women with IBC resulting in survival outcomes far superior than those reported historically with local treatments alone. In this setting, preoperative chemotherapy is thus used to downstage the tumor so that a successful mastectomy can be performed. Response to preoperative chemotherapy has also been shown to be an important prognostic marker among women with breast cancer with attainment of pathological complete response considered to be an intermediate surrogate marker (▶ surrogate endpoint) of improved survival outcome. There was a higher 5-year disease-free and overall survival rate in the group of IBC patients who attained pathological complete response (82.5% and 78.6%, respectively) in the axillary lymph nodes compared to those who had evidence of residual disease (37.1% and 25.4%, respectively). A review of the 20-year experience of a cohort of 178 women with IBC treated on four prospective clinical trials at the University of Texas MD Anderson Cancer Center of all women who received a doxorubicin-based preoperative chemotherapy regimen followed by local therapy with radiation therapy with or without mastectomy reported 5- and 10-year overall survival rates of 40% and 33%, respectively, with an impressive 28% of patients reported to be alive and without disease at 15 years of follow-up.

(orange peel skin), with flattened nipple. (b) Photograph of patient with IBC who has redness and blotchiness of skin overlying the breast with swelling of involved breast

Inflammatory Breast Cancer

Furthermore, 15-year survival rates of 44%, 31%, and 7% were reported among patients who achieved a complete response, a partial response, and a less than partial response, respectively, thereby providing further evidence of the prognostic role of response to preoperative chemotherapy in this cohort. The effects of incorporation of taxanes, both ▶ paclitaxel and ▶ docetaxel, into the preoperative ▶ anthracycline-based regimens have also been evaluated. In studies comparing a cohort of 178 patients with IBC who were treated with FAC (5-▶ fluorouracil, ▶ adriamycin, ▶ cyclophosphamide) alone to 62 patients with IBC who received sequential FAC followed by paclitaxel, there were higher pathological complete response rates (25% vs. 10%, p = 0.012) as well as higher median overall and progression-free survival rates in the group of patients who received paclitaxel compared to those who did not. The peculiar clinical behavior of IBC has been evaluated in large retrospective series and compared to other locally advanced non-IBC cancer treated at single institution using similar multidisciplinary treatments. Those analyses confirmed the worse outcome of patients with IBC. The 5-year overall survival (OS) rate was 40.5% for the IBC group (95% CI, 34.5–47.4%) and 63.2% for the non-IBC LABC group (95% CI, 60.0–66.6%; p < .0001). Moreover, patients with IBC had much higher cumulative incidence of soft tissue recurrence compared to patients with locally advanced disease. The latter was further analyzed demonstrating a significant higher incidence of both locoregional (skin, lymph nodes) and distant soft tissue disease supporting the importance of ▶ lymphangiogenesis in the metastatic process of IBC. The introduction of biological therapies in combination with chemotherapeutic agents into the treatment paradigm of women with breast cancer has been the focus of research efforts and could represent a critical step in the development of more effective therapies for IBC. Several molecular determinants of IBC have already been identified that may serve as prime targets for future development of therapeutic agents. Of particular interest in both IBC and non-IBC tumors is the overexpression and/or

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gene ▶ amplification of HER2. In a cohort of 327 patients enrolled on a phase III prospective randomized clinical trial, which had as the objective to evaluate the addition of ▶ trastuzumab to an anthracycline- and taxane-based preoperative chemotherapy regimen for patients with HER2positive locally advanced breast cancer, 27% of the patients with HER2-positive disease had IBC, and with the addition of trastuzumab to preoperative chemotherapy, these patients had significantly increased the 3-year event-free survival rate compared to preoperative chemotherapy alone (70.1% vs. 53.3%, p = 0.0007). The conclusions reached from this study were that trastuzumab-based preoperative chemotherapy should be considered a standard option for patients with HER2-positive locally advanced breast cancer, including those patients with IBC. Lapatinib (Tykerb, GlaxoSmithKline), a reversible inhibitor of the HER1 (ErbB1) and HER2 (ErbB2) tyrosine kinases, is another targeted agent whose efficacy is being evaluated in women with locally advanced breast cancer, including those with IBC. In a cohort of 21 chemo naive patients who had HER2 positive IBC and who received a combination pre-operative lapatinib and paclitaxel, with a study protocol of 14 days of daily lapatinib (1500 mg) followed by lapatinib given in combination with weekly paclitaxel (80mg/m2) for 12 weeks. There was good tolerance to the regimen with an associated 95% clinical response rate. Combinations of lapatinib are currently being studied prospectively in larger cohorts of patients with IBC. Molecular Biology of IBC While IBC patients may have tumors that possess any combination of hormone receptors and activated ▶ oncogenes, IBC tumors are most often classified within the HER2-amplified and basallike breast cancer cluster, which are recognized to be resistant to conventional therapies. Other characteristics of IBC tumors include high expression of the tumor suppressor ▶ TP53 and overexpression of the ▶ epidermal growth factor receptor (EGFR), which are both associated with a poor prognosis. Targeted therapies such as

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lapatinib, which inhibit the tyrosine kinases of EGFR and HER2, are being evaluated for their clinical utility for the treatment of IBC. There are two primary molecular signatures of IBC that have been identified: 1. Tumor emboli as well as primary IBC tumors and circulating tumor cells isolated from IBC patients exhibit aberrant overexpression of the calcium-dependent transmembrane glycoprotein, ▶ E-cadherin, which is believed to represent one of the mechanisms by which IBC exhibits such an aggressive invasive phenotype and is responsible for the skin ▶ metastasis that is the hallmark of IBC. There are active investigations ongoing to identify targeted therapeutics that block E-cadherin. 2. Another signature of IBC is the overexpression of the transforming oncogene Rho C GTPase (▶ Rho family proteins), which is a member of the RAS homology (Rho) GTPase family of proteins involved in cytoskeletal reorganization during invasion as well as regulation of angiogenic growth factors and inflammatory cytokine production. The gain of Rho C GTPase in IBC patients is associated with loss of the tumor suppressor Wnt-induciblesignaling protein 3/CCN6 (WISP3/CCN6). CCN6 is a secreted protein that contains a binding motif similar to insulin-like growth factor binding protein-related peptides (IGFBP-rp9), suggesting a link between altered insulin-like growth factor (IGF) signaling and IBC. Studies in animal models suggest that Rho C GTPase is inhibited using RAS farnesylation inhibitors, and there are ongoing clinical trials to evaluate the utility of the RAS ▶ farnesylation inhibitor, tipifarnib (Zanestra), in IBC.

Inflammatory Breast Cancer

▶ Docetaxel ▶ E-Cadherin ▶ Epidermal Growth Factor Receptor ▶ Farnesylation ▶ Fluorouracil ▶ HER-2/neu ▶ Insulin-Like Growth Factors ▶ Lymphangiogenesis ▶ Metastasis ▶ Oncogene ▶ Paclitaxel ▶ Positron Emission Tomography ▶ Rho Family Proteins ▶ Surrogate Endpoint ▶ TP53 ▶ Trastuzumab

References Anderson WF, Schairer C, Chen BE, Hance KW, Levine PH (2005–2006) Epidemiology of inflammatory breast cancer (IBC). Breast Dis 22:9–23 Cristofanilli M, Valero V, Buzdar AU et al (2007) Inflammatory breast cancer (IBC) and patterns of recurrence: understanding the biology of a unique disease. Cancer 110:1436–1444 Hennessy BT, Gonzalez-Angulo AM, Hortobagyi GN et al (2006) Disease-free and overall survival after pathological complete disease remission of cytologically proven inflammatory breast carcinoma axillary lymph node metastases after primary systemic chemotherapy. Cancer 106:1000–1006 Sparano JA, Moulder S, Kazi A, Coppola D, Negassa A, Vahdat L, Li T, Pellegrino C, Fineberg S, Munster P, Malafa M, Lee D, Hoschander S, Hopkins U, Hershman D, Wright JJ, Kleer C, Merajver S, Sebti SM (2009) Phase II trial of tipifarnib plus neoadjuvant doxorubicin-cyclophosphamide in patients with clinical stage IIB-IIIC breast cancer. Clin Cancer Res 15(8):2942–2948 Van Laere SJ, Van den Eynden GG, Van der Auwera I, Vandenberghe M, van Dam P, Van Marck EA, van Golen KL, Vermeulen PB, Dirix LY (2006) Identification of cell-of-origin breast tumor subtypes in inflammatory breast cancer by gene expression profiling. Breast Cancer Res Treat 95(3):243–255

Cross-References ▶ Adriamycin ▶ Amplification ▶ Anthracyclines ▶ Breast Cancer ▶ Cyclophosphamide

See Also (2012) Doxorubicin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1159. doi:10.1007/978-3-642-16483-5_1722 (2012) HER2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1678. doi:10.1007/978-3-642-16483-5_2676

Inflammatory Response and Immunity (2012) Insulin-like Growth Factor Binding Proteins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1877. doi:10.1007/9783-642-16483-5_3079 (2012) Lapatinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1980. doi:10.1007/978-3-642-16483-5_3277 (2012) Magnetic Resonance Imaging. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2136. doi:10.1007/978-3-642-164835_3496 (2012) Mammography. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2159. doi:10.1007/978-3-642-16483-5_6669 (2012) Mastitis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2178. doi:10.1007/978-3-642-16483-5_6668 (2012) Taxanes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3614– 3615. doi:10.1007/978-3-642-16483-5_6648 (2012) Tipifarnib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3703. doi:10.1007/978-3-642-16483-5_6671 (2012) Ultrasound. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3836. doi:10.1007/978-3-642-16483-5_6097 (2012) Wnt. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3953. doi:10.1007/978-3-642-16483-5_6255

Inflammatory Response and Immunity Yaron Meirow, Ilan Vaknin and Michal Baniyash The Lautenberg Center for Immunology and Cancer Research, Israel-Canada Medical, Research Institute Faculty of Medicine, The Hebrew University, Jerusalem, Israel

Definition ▶ Inflammation is the first response of the immune system to infection or irritation and may also be elicited by tissue damage due to an uncontrolled/abnormal growth of self-cells. Various immune system cells and their secreted mediators and products are actively involved in this response. Inflammation is characterized by the following quintet of symptoms: redness, heat,

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swelling, pain, and dysfunction of the organs involved. The inflammatory response could be acute or chronic.

Characteristics An Overview of Inflammation The immune system is composed of a large variety of cells and soluble mediators that interact in a complex and dynamic network to ensure protection against foreign pathogens or abnormally growing autologous tumor cells. The immune system is divided into two arms: ▶ Innate immunity and ▶ adaptive immunity, both participating in the generation of acute and chronic inflammation. Acute inflammation is a short-lasting (several days) immunological process that is mediated by activation of innate and adaptive immune cells. This involves activation and directed migration of innate immune cells (granulocytes, macrophages, and dendritic cells) from the circulation to sites of damage. These cells recruit and activate adaptive immune cells such as T and B cells to increase the efficiency of the immune response. In most cases, this response is beneficial to the host as it results in clearance of the initiating stimulus and resolution of the inflammation. In contrast, chronic inflammation is a response of prolonged duration (weeks, months, or even indefinite) whose extended time course is provoked by persistence of the initiating stimulus. Chronic inflammation may develop either as progression of an acute inflammation, if the original stimulus persists, or after repeated episodes of acute inflammation. Chronic inflammation may be local or systemic, caused by spreading of inflammatory cells and mediators to the entire body via the blood stream and the lymphatic system. This results in disease deterioration and complications such as immunesuppression and subsequent opportunistic infections, as T and NK cells become dysfunctional. This phenomenon is induced by myeloid derived suppressor cells (MDSCs) and is associated with a reduced z chain (CD247) expression in both cell types. The z chain is central for the functionality

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of central immune receptors, the T cell antigen receptor (TCR), and NK killing receptors (NKP30, NKP46, and CD16).

An Overview of Cancer The multiple factors leading to malignant transformation (oncogenic process) involve mutations in somatic cells and subsequent alterations of morphology and growth characteristics, ultimately resulting in transformation, local invasion, and metastasis. Genes controlling cell growth, DNA repair, and programmed cell death (apoptosis) are prime targets for the oncogenic process. DNA alterations are irreversible and can persist almost indefinitely in otherwise normal tissue until the exposure of cells to chemical irritants such as phorbol esters, factors released at the site of wounding, partial organ resection, hormones, or chronic irritation provide the “final hit” leading to abnormal cell growth. A prerequisite for the survival and expansion of cancer cells is the development of strategies for escaping immune system surveillance. The term “cancer immuno-editing” represents a dynamic process comprised of three phases: elimination, equilibrium, and escape. Elimination represents the classical concept of immuno-surveillance against cancer, in which the immune system recognizes and eliminates tumor cells. Equilibrium describes the period of immune-mediated latency after incomplete tumor destruction during the elimination phase, and escape refers to the final outgrowth of tumor cells following their release from the immunological restraints of the equilibrium phase. Tumor growth, characterized by the abnormal expansion of modified self-cells within a normal tissue site, leads to destruction of surrounding tissue. In addition, the inner part of an overgrowing tumor often becomes necrotic due to inefficient ▶ angiogenesis, resulting in poor tumor perfusion and cell death. In the course of cell death and tissue necrosis, a sterile inflammatory response ensues, aimed at clearing the dead cells and injured tissue. However, if the tumor persists, with continuing tissue destruction, chronic inflammation develops.

Inflammatory Response and Immunity

Interrelationship Between Inflammation and Cancer The link between inflammation and cancer promotion was first observed in the nineteenth century, but only in recent years it has become a generally accepted phenomenon. Tumors characterized by mild inflammation invoke a beneficial immune response, which clears the tumor cells and leads to tumor regression. Highly inflammatory tumors display more aggressive characteristics as enhanced growth and invasiveness, in combination with loss of the beneficial immune response. The interrelation between cancer and inflammation is bidirectional: The tumor itself provokes a chronic inflammatory response in its microenvironment, which eventually supports tumor growth and metastatic spread. Necrosis within the tumor releases intracellular components, which are normally concealed from the immune system. These components are recognized by innate immune cells as “danger” signals and invoke an inflammatory response. Moreover, some tumors have the ability to produce inflammatory mediators by themselves, further enriching the pro-inflammatory environment. Initially, the inflammatory response is beneficial as cytotoxic cells are recruited to eradicate the developing tumor. However, when the inflammatory response is prolonged to the inability to clear the tumor cells, it becomes harmful. The tumor microenvironment becomes rich in pro-inflammatory cytokines such as interleukin 1 beta (IL1b), interleukin 6 (IL6), tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF). The pro-inflammatory compounds produced by immune cells in the tumors can directly support tumor growth by providing growth signals to the cancer cells, preventing programmed cell death (apoptosis), disrupting the surrounding tissue and facilitating vasculature in the tumor. Furthermore, the excess of these pro-inflammatory compounds shifts the immune response from a beneficial to a harmful process by the activation and recruitment of MDSCs into the tumor. MDSCs indirectly support cancer, suppressing the antitumor response by inactivating cytotoxic T and NK cells. This

Inflammatory Response and Immunity

suppression is mediated by a myriad of mechanisms including production of nitric oxide (NO), reactive oxygen species (ROS), and depravation of arginine and cysteine from their environment. Moreover, MDSCs skew the activity of other immune cells: induction of regulatory T cells (Treg), polarization of M2 macrophages, and accumulation of immature dendritic cells in the tumor microenvironment, which further suppress the beneficial immune response and directly support tumor progression. MDSCs can directly support tumor growth and invasiveness by expression of matrix metalloproteinases (MMPs), which degrade the surrounding tissue and enhance invasion and metastatic spread. MMPs were also shown to augment the production of VEGF and facilitate vascularization in the cancer microenvironment. MDSCs produce TNF-a and S100A8/9 proteins, creating a positive autocrine feedback loop, which further perpetuates chronic inflammation, MDSC accumulation, immune-suppression, and support of tumor growth. Chronic inflammation in noncancerous tissue increases the risk factor for cancerous transformation. Inflammatory mediators generally contribute significantly to the process of cell transformation by inducing DNA damage, activating oncogenes, or inducing loss of antioncogenic activity. For example, ROS and NO produced in inflamed tissues (▶ Oxidative Stress), whose main role is to attack invading infectious agents and foreign elements could cause injury to host cells and induce ▶ DNA damage and mutations, when produced at excessive levels. These effects are enhanced when chronic inflammation develops and increasing numbers of NO/ROS producing MDSCs accumulate. The skewed inflammatory environment generated by MDSCs leads to an uncontrolled cellular and factors milieu, ensuing in the establishment of an optimal niche for cancer initiation, tumor microenvironment enrichment, and support of a malignant progression (Fig. 1). Examples of Inflammation-Associated Cancers Over the past 20 years, numerous cancer types have been shown to be associated with local chronic inflammation. Studies on liver cancer

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suggested that patients with persistent ▶ hepatitis B virus infections experience inflammation and scarring of liver tissue and thus are at increased risk of liver cancer (Hepatocellular Carcinoma). Other disease characterized by chronic inflammation including ulcers caused by the bacterium, ▶ Helicobacter pylori, and an immune disorder known as ulcerative colitis, predispose patients to stomach cancer and ▶ colon cancer. ▶ Cervical carcinoma, which is caused by infection with human papilloma virus (HPV), has also been suggested to have an inflammatory component. Other examples include the associations between chronic bronchitis and ▶ lung cancer, schistosomiasis (schistosomas, gematobition) and ▶ bladder cancer, chronic pancreatitis and pancreatic cancer, and chronic cholecystitis and gall bladder cancer. In addition, epidemiologic studies showed that regular intake of ▶ non-steroidal antiinflammatory drugs (NSAIDs) decreases the risk of developing several types of cancers. However, in the absence of specific risk factors, NSAIDs are not recommended at present, since many of these drugs, such as aspirin, can cause severe gastrointestinal complications. Based on the knowledge accumulated during the last two decade, it has been estimated that inflammation contributes to the development of at least 15% of all cancers. Therapeutic Strategies Against Cancer As the role of the immune system in combating many types of tumors was revived, immunotherapy has dramatically changed the view of cancer treatment, and numerous novel therapies have been developed and approved. Cancer ▶ immunotherapy is aimed at buttressing the immune system, augmenting its ability to destroy growing tumors. Identification of tumor-associated antigens recognized by the effector arm of the adaptive immune system and check-point inhibitory molecules expressed on T cells that attenuate their function have opened new approaches towards cancer immunotherapy. However, chronic inflammation, which predisposes individuals to cancer, and promoted by developing tumors, ensues in chronic inflammation-induced immunosuppression. Therefore, to attain an

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Cancer progression Stress (UV/IR, carcinogens) I

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Inflammatory Response and Immunity, Fig. 1 Inflammatory Response and Immunity. The interrelationship between cancer and inflammation. (I) Tumors can emerge as a result of carcinogenesis, stress or chronic inflammatory responses during an infection or autoimmune response, leading to tumor initiation. (II) Tumor/pathogen or self-antigens presented by DCs in secondary lymphoid organs activate adaptive and innate immune responses eliciting anti-tumor responses through T and NK-cell-mediated toxicity, antibody-dependent cellmediated cytotoxicity, and antibody-induced complementmediated lysis, resulting in a beneficial acute immune response, which eliminates initially developing malignant

cells. (III) Chronic activation of the innate and adaptive immune cells ensues due to the inability to resolve primary developing tumor cells, resulting in chronic inflammation characterized by the recruitment of MDSCs, M2 macrophages and Treg, generating an immunosuppressive environment. The release of potent pro-survival molecules and/or DNA damaging ROS are involved in the initiation and development of neoplastic cells. The inflammatory cells induce/support tissue remodeling and angiogenesis. Thus, the generated harmful immune milieu that supports tumor development and spreading. The delicate balance between the types of the immune responses will dictate whether tumor will develop or be rejected

effective therapy, anti-inflammatory/anti-MDSC and immunotherapeutic approaches must be combined, taking in consideration the immune status and tumor parameters. Currently, several forms of anticancer immunotherapy are being tested. These approaches fall

into two categories, which are not mutually exclusive: The first involves the administration of ▶ monoclonal antibodies (Mabs) that are directed against check-point molecules as PD1 and CTLA4 or various tumor associated antigens (when the tumor is immunogenic). The check-

Inflammatory Response and Immunity

point molecule PD1 and CTLA-4 play an important role in down regulating the immune system by preventing the activation of T cells. Inhibitory effects of PD-1 are accomplished through a dual mechanism of promoting ▶ apoptosis (programmed cell death) in antigen specific T-cells in lymph nodes while simultaneously, reducing apoptosis in ▶ regulatory T cells (suppressor T cells). The CTLA-4 molecule, which is constitutively expressed in Tregs but only upregulated in conventional T cells after activation, acts as an “off” switch when bound to CD80 or CD86 on the surface of antigenpresenting cells. Thus, antibodies directed against these two molecules are used today in the clinic to “turn off” PD1 and CTLA-4 inhibitory mechanisms, allowing CTLs to destroy cancer cells. Another example for antibody-mediated therapy is directed against tumor antigens, where the Mabs could be conjugated to anticancer drugs, toxins, radioisotopes, or other biologic response modifiers (▶ Cytokine Receptor as Target for Immunotherapy and Immunotoxin Therapy). When the antibodies bind to the antigen-bearing cells, they deliver their cytotoxic load and destroy the tumor. Mabs could also be directed against specific inflammatory compounds that support tumor growth, leading to the inhibition of tumor expansion and recovery of the immune response (▶ Monoclonal Antibody Therapy). In addition, immune response modifiers such as interferons could potentially stimulate immune system cells to act against the tumor. Other strategies that use antiangiogenic agents, MMP-inhibitors, and antiinflammatory drugs have also met with some success. The second category is based on administration of immune cells inducing an active attack against the tumor by several approaches: (i) ▶ Dendritic cell (DCs)-dependent immunotherapy that uses the patient’s own DCs, which initiate the adaptive immune response through their role in antigen presentation. DCs isolated from the patient’s blood are loaded with tumor proteins (antigens) and injected back into the patient to initiate a potent specific immune response directed against the cancer cells. This

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approach is currently being investigated in phase IIb/III clinical trials for ▶ brain cancer and ▶ Prostate Cancer. It has been reported that success rates are in the 20% range for heavily pre-treated otherwise untreatable late stage patients. A larger percentage may not experience remission as such but remain stable with treatment. (ii) T-cell immunotherapy that utilizes tumor-infiltrating T cells (TILs), activated against defined tumor antigens, or genetically modified to express TCRs that recognize a specific tumor antigen. Alternatively, CAR-T cells are CD8 T cells expressing a specificity of a ▶ monoclonal antibody; the classic constructs being a fusion of single-chain variable fragments (scFv) derived from monoclonal antibodies directed against tumor antigen, fused to CD3-z transmembrane and endodomain. These molecules are expressed on cytotoxic T cells and upon tumor antigen recognition (without the presentation requirements) signals are transmitted, leading to the activation of the T-cell killing activity that efficiently destroys the tumor. In both cases, T cells are taken from the cancer patient, selected or modified, and then injected back to the same patient where they are expected to mediate their cytotoxic function. (iii) Vaccine-based immunotherapy depends upon administration of a tumor specific component (antigen), or inactivated cancer cells to elicit an immune response (▶ Cancer Vaccines). While each of these immunotherapeutic approaches has shown some promise, the general success rates are limited due to some critical obstacles. One major obstacle to an effective active tumor immunotherapy is the chronic inflammation and associated MDSC-mediated immunosuppression that often characterizes developing tumors. Moreover, the increased Treg activity in tumor-bearing host may be associated with poor immune responses to tumor antigens and contribute to immune dysfunction. It is expected that the immunosuppressive environment generated in tumor-bearing hosts due to chronic inflammatory response and the increased Treg activity will limit the response to an active immunotherapy, affecting the endogenous host immune system, as well as suppressing any

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newly administered immune modulators. Moreover, it is possible that adjuvants (▶ Adjuvant Therapy) used to enhance a patient’s immune response to weakly immunogenic tumors or pathogenic antigens might result in activation of chronic inflammation, thus negatively affect the specific anti-tumor response. With this knowledge in mind, the functional status of the patient’s immune system must be monitored prior to a given immune-based therapy. Currently, it is already established that expression levels of several immune biomarkers such as CD247 and MDSC subpopulations, and the suppressive function of the latter in the blood and in some cases in biopsies, can provide information about the immune status; low levels of CD247 and elevated levels of MDSCs with suppressive features indicate an immunosuppressed immune system. Accordingly, while a normal immune status encourages immune-based therapy, detected immunosuppression will indicate a higher probability that the patients will respond poorly. Thus, a combined treatment is recommended: neutralization of the chronic inflammation and MDSCs prior to or in conjunction with the given immune-based treatment. Anti-inflammatory and/or anti-MDSC drugs are expected to neutralize the immunosuppressive environment and thus serve a dual purpose; they could be used in cancer prevention or as a treatment to optimize the patient’s response to immunotherapy. In addition, strategies that deplete Tregs, inhibit their function or block their migration, rather than enhance or restore their function, are likely to be advantageous for cancer immunotherapy. Monitoring the immune status could also have a critical impact when using chemotherapy as anticancer treatments. Chemotherapies were shown to have adverse immunoregulatory effects, enhancing or decreasing MDSC levels and activity, thus affecting treatment success. Therefore, it is critical to merge monitoring immune system biomarkers into the traditional patient’s categorization and treatment regiments. This will provide new tools to clinical practice, allowing appropriate management of cancer patients towards a betterpersonalized medicine.

Inflammosome

References Baniyash M (2006) Chronic inflammation, immunosuppression and cancer: new insights and outlook. Semin Cancer Biol 16:80–88 Chen R, Alvero AB, Silasi DA et al (2007) Inflammation, cancer and chemoresistance: taking advantage of the toll-like receptor signaling pathway. Am J Reprod Immunol 2:93–107 Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860–867 DeVisser KE, Eichten A, Coussens LM (2006) Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 6:24–37 Dunn GP, Old LJ, Schreiber RD (2004) The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21:137–148 Meirow Y, Kanterman J, Baniyash M (2015) Paving the road to tumor development and spreading: myeloidderived suppressor cells are ruling the fate. Front Immunol 6:523–535

Inflammosome Fong-Fong Chu Department of Cancer Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA

Definition An inflammasome is an intracellular multiprotein complex that responds to exogenous or endogenous danger or damage signals to activate caspase-1, which promotes secretion of proinflammatory cytokines interleukin (IL)-1b, IL-18, and pyroptosis (a form of phagocyte cell death induced by bacterial pathogens). Different inflammasomes are named after the core protein in the complex; they are NLRP1 inflammasome, NLRP3 inflammasome, NLRP6 inflammasome, NLRC4/IPAF inflammasome, and AIM2 inflammasome. Inflammasomes play major roles in innate immunity by activating an inflammation in response to pathogens but also to self-danger signals.

Inflammosome

Characteristics List of Abbreviations AIM2 ASC CIITA CARD Caspase DAMPs HET-E

HIN200

IL-1 IPAF LRR NACHT

MAMPs MDP NAIP

NLR

NLRC

NLRP NOD PAMPs

Absent in melanoma-2 Apoptosis-associated speck-like protein containing a CARD Major histocompatibility class II transcription activator Caspase activation and recruitment domain Cysteinyl aspartate protease Damage-associated molecular patterns A predicted NTPase and incompatibility locus protein from Podospora anserina bacteria Hematopoietic interferon-inducible nuclear antigens with 200-amino-acid repeats Interleukin Ice protease-activating factor Leucine-rich repeats A domain with predicted NTPases implicated in apoptosis and MHC transcription activation, an acronym of NAIP, CIIA, HET-E, and TP1, and it refers to the same domain as NOD Microbe-associated molecular patterns Muramyl dipeptide, a building block of bacterial wall Neural apoptosis inhibitor protein, a member of IPAF subfamily, which belongs to NLR family NOD-like receptor, the NLR family has three subfamilies, NOD, NLRP, and IPAF NLR family CARD-domaincontaining protein, NLRC proteins belong to either NOD or IPAF subfamilies of NLR family A subfamily of NLR family with 14 members in humans Nucleotide-binding and oligomerization domain Pathogen-associated molecular patterns

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PYD ROS TP1

Pyrin domain Reactive oxygen species Telomerase-associated protein 1

Inflammasome Assembly An inflammasome is assembled after sensing a variety of both exogenous and endogenous signals. The core component of an inflammasome is a multi-domain protein, such as NLRP1 (a.k.a. NARP1, NAC, CARD7, DEFCAP, and CLR17.1) (Fig. 1) (Latz 2010; Horvath et al. 2011). NLRP1 is the first NOD-like receptor (NLR) characterized for inflammasome assembly and caspases-1 activation. NLRP1 has a leucine-rich repeats (LRR) near its C-terminus, which senses the bacterial components muramyl dipeptide (MDP) or lethal toxins to initiate inflammasome assembly. Upon receiving an activating signal, the nucleotidebinding and oligomerization domain (NBD, NOD, or NACHT) of the core protein regulates homo-oligomerization or hetero-oligomerization as the first step of inflammasome assembly. The other two components of an inflammasome are an adaptor protein, ASC (apoptosis-associated speck-like protein containing a CARD), and a cysteinyl aspartate protease, such as caspase-1. Once activated, the core oligomers recruit pro-caspase-1, which binds to a caspase activation and recruitment domain (CARD) of ASC adaptor. The ASC also contains a pyrin domain (PYD), which mediates protein-protein interaction to form a complex with the PYD at the N-terminus of the core protein. In some cases, the core protein binds and activates pro-caspase directly without the adaptor protein. After binding to CARD, the pro-caspase-1 is activated by self-proteolytic processing to become an active protease, which cleaves the pro-IL-1b to form active IL-1b to be secreted. Inflammasome Core Proteins and Their Activation Signals The core components of inflammasomes are intracellular proteins belonging to either NLR family or the PYHIN (pyrin and HIN200 domaincontaining protein) family. HIN200 is

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Inflammosome, Fig. 1 Diagrams of four types of inflammasome. The NLRP1 inflammasome consists of NACHT and LRR domains and can bind to caspase-1 via the C-terminal CARD domain and to the adapter molecule ASC via the N-terminal PYD domain. ASC interacts with caspase-1 via its CARD domain. Muramyl dipeptide (MDP) and lethal factor of Bacillus anthracis send signal to the LRRs of NLRP1. FIIND domain has unknown function. NLRP3 has LRRs sensing an intermediate molecule that is generated by cathepsin or ROS activity.

NLRP3 binds to ASC via its N-terminal PYD domain. ASC, in turn, associates with caspase-1. The NLRC4 inflammasome consists of LRR domains that could interact with bacterial flagellin and a NACHT domain followed by an N-terminal CARD domain. The CARD domain can either directly interact with caspase-1 or via the CARD domain of ASC. The AIM2 inflammasome directly interacts with double-stranded DNA through its C-terminal HIN200 domain. AIM2 activates caspase-1 via ASC at the N-terminus of the protein (Latz 2010)

hematopoietic interferon-inducible nuclear antigens with 200-amino-acid repeats. NLRP1, NLRP3, NLRP6, and NLRC4/IPAF are among the 22 members of human NLR family found in inflammasomes. The NLR proteins typically have three domains: the N-terminal has either PYD or CARD or both; the middle has NOD or NACHT, which has ATPase activity and is necessary for inflammasome activation; and the C-terminal has leucine-rich repeats (LRRs). The LRR domain is involved in auto-inhibition, which is disabled when senses an activation signal. Based on the genetic analysis of domain structures and NACHT sequence, the NLR family can be divided into three subfamilies: the NODs (NOD1–2, NOD3/NLRC3, NOD4/NLRC5, NOD5/NLRX1, CIITA), the NLRPs (NLRP/

NALP1-14), and IPAF. Only three and one members from NLRP and IPAF subfamilies, respectively, have been found in inflammasomes. NLRP1 is the only NLR protein that has an additional CARD at the C-terminus, which allows it to form multimolecular complex with the ASC adaptor when activated by ligand such as MDP, which is a building block of major bacterial cell wall component, peptidoglycan. Because of the large size of this NLRP1-containing complex, this caspases-1 activating complex was coined inflammasome. While human NLRP1 has one gene, mice have three genes encoding Nlrp1a, Nlrp1b, and Nlrp1c. Mouse Nlpr1b senses the lethal toxin expressed by Bacillus anthracis. Mouse NLRP1 inflammasome activation is central to the initiation of the antimicrobial response

Inflammosome

to Bacillus anthracis infection through caspases1-induced pyroptosis of infected macrophages. NLRP3 activates caspases-1 via ASC adaptor and has most diverse activators among all inflammasomes. NLRP3 (NALP3, CIAS1, cryopyrin, CLR1.1, PYPAF1) is activated by a large variety of signals, including pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) that are found in microbes including viruses, bacteria, protozoans, and funguses and damage-associated molecular patterns (DAMPs) that are host-derived, bacterial toxins, reactive oxygen species (ROS), and aggregated substances (asbestos, silica, uric acid, etc.). Because the NLRP3 inflammasome can be activated by a wide spectrum of activators, multiple mechanisms for its activation have been proposed (see below). NLRP6 has similar structure as NLRP3. Nlrp6 mRNA is highly expressed in the intestine but much lower in hematopoietic cells. However, in hematopoietic cells, Nlrp6 is expressed at higher levels in granulocytes and lymphocytes compared with macrophages and dendritic cells. The presence of NLRP6 inflammasome is suggested from the study of Nlrp6-deficient mice that have impaired levels of IL-18 production in the serum and in colon explants cultures compared with wild-type mice. Nlrp6 deficiency leads to dysbiosis (changes in luminal microbiota) (Chen and Nunez 2011; Strowig et al. 2012). NLRC4/IPAF is activated by bacterial flagellin and gram-negative bacteria possessing type III and IV secretion systems, such as Salmonella typhimurium, Shigella flexneri, Legionella pneumophila, and Pseudomonas aeruginosa. NLRC4 activation by Legionella is dependent on NAIP5, another NLR protein, but only partially dependent on NAIP5 during Pseudomonas and Salmonella infection. Since NLRC4 has a CARD domain, it can interact directly with pro-caspase-1 without ASC adaptor. NLRC4-dependent pyroptosis (cell death) does not require ASC upon macrophage infection with Shigella flexneri and Pseudomonas aeruginosa. However, maximal caspases-1 activation by the IPAF inflammasome requires ASC. Among the PYHIN family, AIM2 (absent in melanoma-2) is first identified in an inflammasome in 2009 outside of NLR family.

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AIM2 recognizes double-stranded DNA. Since AIM2 is lack of the NACHT oligomerization domain, protein oligomerization is mediated by the multiple binding sites in the DNA ligand. The C-terminal HIN200 domain of AIM2 interacts with DNA, when the N-terminal pyrin domain recruits ASC adaptor to activate caspases-1. AIM2 may play an essential role in the control of viral and bacterial infections as well as autoimmune disease, since the PYHIN locus is associated with lupus erythematosus susceptibility. Other members of PYHIN family, IFI16, IFIX, and MNDA, are present in the nucleus and have not been found in the inflammasomes. Inflammatory Caspases and ASC Adaptor Both ASC and caspase-1 are found in many tissues and cell types, whereas the inflammasome sensors have tissue-specific expression pattern. Besides caspase-1, caspase-4, caspase-5, and caspase-12 are found in human inflammasomes, caspase-11 and caspase-12 are also found in mouse inflammasomes (Strowig et al. 2012). Possible Mechanisms for NLRP3 Co-activation Because NLRP3 can be activated by many molecules without structural similarity, it is likely that additional priming is involved to activate NLRP3 inflammasome. One such primer is reactive oxygen species (ROS), since ROS scavengers (e.g., N-acetyl cysteine, NAC), an inhibitor of ROS-generating enzymes (diphenyleneiodonium, DPI), or knockdown of p22phox (a subunit of NOX family of ROS-generating enzyme) greatly abolished NLRP3 activation by asbestos, uric acid, silica, or lipopolysaccharide (present in bacterial cell wall). However, since ROS alone is not sufficient to activate NLRP3 inflammasome, it suggests that ROS primes NLRP3 to make it susceptible to other stimulants. Another possible co-activator is rupture of lysosomes. Because lysosomes containing the crystalline or particulate structures, such as uric acid, silica, asbestos, amyloid-b, and alum, may be ruptured in the phagocytes such as macrophages, the lysosomal contents may be sensed by the NLRP3 inflammasome as damaged signals.

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Expression of Inflammasomes in Many Cell Types Although most inflammasomes are studied in macrophages, NLRP3, NLRP6, and NLRC4 inflammasomes are formed in many lineages of hematopoietic cells, and they are also found in non-hematopoietic cells such as epithelial cell, myofibroblasts, and intestinal epithelial stem cells to respond to the invasion of microorganisms. NLRP3 inflammasomes are found in pancreatic b-cells, which along with the infiltrating macrophages can drive b-cell death due to chronic hyperglycemia and leading to type 2 diabetes. NLRP3 inflammasome is detected in cardiomyocytes, fibroblasts, endothelial cells, and leukocytes. Inflammasome-Associated Diseases Mutations in NLRP3 are responsible for a wide range of diseases, named cryopyrin-associated periodic syndromes (CAPS). CAPS are characterized by recurrent fever and inflammation and are comprised of three autoinflammatory disorders. They are familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile neurological, cutaneous, and articular (CINCA) syndrome, a.k.a. neonatal-onset multisystem inflammatory disease (NOMID). NLRP3 inflammasome is also believed to cause gout, an autoinflammatory disease due to elevated blood uric acid levels, and type 2 diabetes.

Infusion of T Cells

Infusion of T Cells ▶ Adoptive T-Cell Transfer

INHBA ▶ Activin

INHBB ▶ Activin

INHBC ▶ Activin

INHBE ▶ Activin

Inherited Human Polycystic Kidney Disease ▶ Polycystic Kidney Disease

References Chen GY, Nunez G (2011) Inflammasomes in intestinal inflammation and cancer. Gastroenterology 141(6):1986–1999 Horvath GL, Schrum JE, De Nardo CM, Latz E (2011) Intracellular sensing of microbes and danger signals by the inflammasomes. Immunol Rev 243(1):119–135 Latz E (2010) The inflammasomes: mechanisms of activation and function. Curr Opin Immunol 22(1):28–33, PMCID: 2844336 Strowig T, Henao-Mejia J, Elinav E, Flavell R (2012) Inflammasomes in health and disease. Nature 481(7381):278–286

Inhibin-b Chain ▶ Activin

Inhibitor of FLICE ▶ FLICE-Inhibitory Protein

INK4A

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INK4A Takehiko Kamijo Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama, Japan

Definition Inhibitor of cyclin-dependent kinase 4; proteins are cyclin-dependent kinase inhibitors that block the action of cyclin-dependent kinase to induce cell cycle arrest. The four INK4 family proteins (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) contain four tandemly repeated ankyrin motifs and have similar biochemical phenotypes. The p16INK4a is an alternative reading frame product (ARF) of p16INK4a and p15INK4b, which are coded at the human chromosome 9p21 region. The significance of the p16INK4a, ARF, and p15INK4b in tumorigenesis is confirmed by the high frequency of the genetic and epigenetic modifications of these genes in tumor samples. Keyword Definitions ARF

Cyclin

The ARF tumor suppressor is encoded by the ARF/p16INK4a genomic region and transcribed as an alternative reading frame product of p16INK4a. By antagonizing Mdm2, a negative regulator of the p53 tumor suppressor, ARF triggers a p53-dependent transcriptional response that diverts incipient cancer cells to undergo growth arrest or apoptosis. Furthermore, ARF physically associates with proteins other than MDM2 to have p53-independent activities. Cyclins are a family of proteins involved in the cell cycle progression, co-operating with its catalytic partner cyclin-dependent kinase (Cdk), which activates the latter’s protein kinase function. The

Polycomb

most important substrate of cyclin/ Cdk complex is retinoblastoma protein (Rb). Polycomb complex modulates histone H3 by methylation of lysine residues, ubiquitination of histone H2A, and deacetylation of histones and binds to the modified histones to inactivate the expression of the adjacent genes. Polycomb protein complex works as a molecular lock to suppress the gene expression to maintain the cellular homeostasis in adult cells.

Characteristics Structure of INK4b/ARF/INK4a Locus and Molecular Functions of INK4s/ARF The INK4b/ARF/INK4a locus is located at the human chromosome 9p21 region. Chromosome region 9p21 is involved in chromosomal inversions, translocations, heterozygous deletions, and homozygous deletions in a variety of malignant cell lines and primary tumor samples, including those from melanoma, pancreatic adenocarcinoma, nonsmall cell lung cancer, leukemia, and glioma. These findings indicate that 9p21 contains a tumor suppressor locus that may be involved in the tumorigenesis of several tumor types. In a region of less than 40 kb of the human genome, three related genes – p15INK4b, ARF (p14ARF in human and p19ARF in mouse), and p16IN4a – are encoded (Fig. 1). The INK4 class of cell cycle inhibitors, p15INK4b and p16IN4a, are homologous inhibitors of the cyclin-dependent kinases, CDK4 and CDK6, which inactivate the tumor suppressor retinoblastoma protein via phosphorylation of its c-terminal region. The association of the INK4 proteins to CDK4/CDK6 induces an allosteric modification that abrogates the binding of these kinases to D-type cyclins, resulting in inhibition of CDK4/6-mediated phosphorylation of retinoblastoma family member proteins. Hence, the existence of p15INK4b and p16INK4a maintains retinoblastoma family member proteins in a hypo-

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INK4A p16INK4a p15INK4b ARF (p19/p14) promoter

p16 promoter

Exon1 beta

Exon 1 Exon 2

Exon 1 alpha

Exon 2 Exon 3

p19/p14ARF

INK4A, Fig. 1 Genomic structure of INK4b/ARF/INK4a locus. Residing on chromosome 9p21 in humans and chromosome 4 in mice, the INK4b/ARF/INK4a locus includes 2 different genes. INK4b and INK4a/ARF. INK4a and ARF, which have the independent exon1 alpha and exon1 beta,

respectively, and common exon2/exon3, encode different proteins via an alternative splicing mechanism. In this commentary, p14ARF (murine p19ARF) will be referred to as ARF

E2F Mitogenic signals

Cyclins

release

P

CyclinD

E2F Rb

CDK4/6

CyclinA/E INK4b

CDK2

INK4a

INK4 locus p21

ARF

MDM2

Constitutive oncogenic signals

p53 Bax Puma

Cell death

Noxa

INK4A, Fig. 2 Physiological roles of INK4s/ARF. Mitogenic signals activated cyclin D-dependent kinases, which phosphorylate RB and RB family proteins to facilitate entry into S phase. Constitutive oncogenic signals can activate the INK4a/ARF locus. By antagonizing the activity of cyclin D-dependent kinases, p16INK4a and

p15INK4b prevent entry into S phase. MDM2 is a p53-inducible gene that normally acts to terminate the p53 response. The ARF protein inhibits MDM2 to induce p53, leading either to p53-dependent apoptosis or to induction of the CDK inhibitor p21Cip1, inhibition of cyclin E/Cdk2, and RB-dependent cell cycle arrest

phosphorylation state, which facilitates binding of E2Fs to induce a cell cycle arrest in the G1 phase (Fig. 2). ARF and p16INK4a have different first exons, exon 1 beta and exon 1 alpha, respectively. These first exons are spliced to a common second exon

and third exon. Although exons 2 and 3 are shared by p16INK4a and ARF, these proteins are encoded in alternative reading frames. The predicted 132-amino acid p14(ARF) is shorter than the corresponding mouse protein, p19(ARF), and the 2 proteins share only 50%

INK4A

identity. However, both proteins have the ability to elicit a p53 response, manifest in the increased expression of both p21Cip1/Waf1 and several p53-downstream proapoptotic molecules, resulting in a distinctive cell cycle arrest in the G1/G2M phases and apoptotic cell death, respectively. Previous reports showed that ARF binds to MDM2 and promotes the segregation of MDM2 in the nucleolus. This interaction is mediated by the exon 1-beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. Roles in Tumorigenesis As human cancers frequently harbor homozygous deletions of the INK4b/ARF/INK4a locus that abrogate expression of all p15INK4b/ARF/ p16IN4a, it has been worth noting which protein or proteins of the locus represents the principal tumor suppressor activity located at human chromosome 9p21. In a large number of human cancers, specific somatic loss of p16INK4a, through point mutation or small deletion, has been reported. Furthermore, silencing of p16INK4a through promoter methylation is reported at high frequency in numerous types of human malignancies. Therefore, p16INK4a is an important tumor suppressor in human malignancies. In the case of p15INK4b, specific epigenetic silencing by hypermethylation of the p15INK4b promoter has been described in hematologic malignancies including leukemia and myelodysplastic syndrome and rare cases of glial tumors. In myelodysplastic syndrome, hypermethylation of p15INK4b has been reported in the absence of p16INK4a hypermethylation. p15INK4b seems to be an important tumor suppressor in specific lineages of human malignancies, e.g., hematological malignancies. To identify the significance of the proteins in tumorigenesis, genetically modified mouse experiments were performed. Serrano et al. reported the phenotype of mice carrying a targeted deletion of the INK4b/ARF/INK4a locus that eliminates both p16INK4a and p19ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate

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rapidly and have high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-Ras into INK4a-deficient fibroblasts can result in neoplastic transformation. Next, we reported that mice lacking p19ARF, but expressing functional p16INK4a develop tumors early in life. Their MEFs do not senesce and are transformed by oncogenic Ha-Ras alone. Conversion of ARF+/+ or ARF+/ MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. These results suggest that ARF is a bona fide tumor suppressor in murine malignancies. However, selective inactivation of ARF, in the absence of additive loss of p16INK4a and p15INK4b, has only been reported in a small number of human malignancies, e.g., familial melanoma/astrocytoma patients and somatic ARF-specific mutations and promoter methylations in colon carcinoma patients. Therefore, it seems that ARF cooperates with p16INK4a and p15INK4b in tumorigenesis of human malignancies, and their relative and combinational importance in any given tumor types remains to be elucidated. Roles in Senescence Cellular senescence is a fundamental cellular program that is activated in various situations of stress and acts to prevent further cell proliferation. Senescence induced by extrinsic stress, such as DNA damage or oncogene activation, occurs relatively rapidly, in a matter of days. As a population of cells is propagated in culture, cells are exposed to various extrinsic and intrinsic stresses and the population gradually stops dividing. These findings have led to a distinction between “stress-induced premature senescence,” a term referring to rapid senescence triggered by extrinsic stress, and “replicative senescence,” a term referring to senescence that occurs following extended proliferation, presumably triggered by various stresses. Cellular senescence is thought to play an important role in tumor suppression and contribute to organismal aging. In fact, the two definitive tumor suppressor pathways, ARF/MDM2/p53 and p16INK4a/Rb, have been shown to play critical roles in the induction of cellular senescence. In tissue culture of primary

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cells, the accumulation of one or more INK4b/ ARF/INK4a locus genes can eventually lead to cell cycle arrest through the mechanisms presented in Fig. 2. The cellular senescence is canceled by downregulation of expression caused by gene deletion or epigenetic regulation, by inactivation of the gene products by mutation, or by cellular resistance to those cell cycle inhibitors. Considering the significance of the INK4b/ARF/ INK4a locus genes in the cellular life span, p16INK4a plays a central role in senescence in human cells, whereas ARF assumes a prominent role in mouse cells. For mice, p19ARF and p16INK4a both accumulate significantly after passage, but spontaneous escape from senescence occurs through deletion of INK4/ARF or p53 mutation in wild-type MEFs. Consistent with this, ARF-null MEFs do not have proliferation failure, but p16INK4a-null and p15INK4b-null MEFs indicate limited proliferation. Whereas senescence generally occurs in the setting of increased expression of p16INK4a, but not ARF, and enforced Ras-Raf pathway activation also appears to induce only p16INK4a, along with senescence in cultured human cells. Furthermore, only p16INK4a induction has been reported with human aging, although ARF expression studies in human aging have not been reported.

INK4A

mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Whereas oncogenic Ras induces ARF transcription in MEFs, similar effects have not been detected in human cells. One of the key molecules activated by Ras signaling pathway is DMP1, which binds directly to the p19ARF promoter region. Although there are putative binding sites in the human ARF promoter region, the effects of DMP1 have not been demonstrated in human cells. E2F

The E2F transcriptional factors are divided into two groups: 1. transcriptional activating E2F1, E2F2, and E2F3; and 2. transcriptional repressing E2F4, E2F5, and E2F6. Several E2Fs have been detected at the endogenous ARF promoter by chromatin immunoprecipitation assay and directly activate ARF transcription without association with DP-1. Since ARF transcription is not regulated by a cell cycle-dependent manner, the unphysiological level of E2F seems to be required to induce ARF. Some studies described the inverse correlation between pRb status and p16INK4a expression in tumor cells. However, the physiological relation remains to be elucidated in terms of the relationship between the pRb/E2Fs and INK4 pathway.

Activators of the p15INK4b/ARF/p16INK4a c-Myc Ras-Raf-p38MAPK Pathway

Involvement of the Ras signaling pathway into the regulation of the INK4b/ARF/INK4a locusencoded genes has been reported in detail. Expression of oncogenic Ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by Ras is accompanied by accumulation of p53 and p16 and is phenotypically indistinguishable from cellular senescence. Constitutive activation of MAPKK induces both p53 and p16 and is required for Ras-induced senescence of normal human fibroblasts. Ras signaling pathway activation can result in phosphorylation and enhance binding of Ets2 transcription factor to the p16INK4a promoter. These results imply that premature senescence via INK4b/ARF/ INK4a locus activation acts as a fail-safe

Studies addressed that c-Myc has the ability to control p16INK4a transcription in human cells. In detail, c-Myc binds to the promoter and first intron of human p16INK4a, which is in line with the reports that c-Myc upregulates p16INK4a transcription in human cells. However, c-Myc has little effect on p16INK4a expression in mouse cells. In mouse cells, establishment of MEFs as continuously growing cell lines is normally accompanied by loss of the p53 or p19ARF, which act in a common biochemical pathway. c-Myc rapidly activates ARF and p53 transcription in MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive c-Myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. These

INK4A

observations are consistent with the co-operation of c-Myc and Bmi1 in mouse lymphomagenesis, suggesting that the ARF/p53 pathway is a physiological safeguard system against c-Myc-induced oncogenic stresses. Suppressors of the p15INK4b/ARF/p16INK4a AML1/ETO

AML1/ETO chimeric protein results from the t (8;21) translocation in human acute myelogenous leukemia. AML1/ETO can bind directly to the ARF promoter regions as well as the POZ/BTB domain protein, ZBT7B. The translocation seems to convert the wild-type AML1 from being an inducer of ARF to a repressor. p53

One of the most important tumor suppressors p53 seems to have a significant role in ARF transcriptional suppression because ARF is generally transcriptionally upregulated in p53-inactivated cells. However, the mechanism of the transcriptional repression of ARF by re-introduction of wildtype p53 into p53-null cells remains to be elucidated. Polycomb Proteins

Traditionally, cancer has been viewed as a genetic disease that is driven by the sequential acquisition of mutations, leading to the constitutive activation of proto-oncogenes and loss of function of tumorsuppressor genes. However, it has become increasingly evident that tumor development also involves “epigenetic changes” patterns of altered gene expression that are mediated by mechanisms that do not affect the primary DNA sequences. The INK4b/ARF/INK4a region is known to be regulated by not only the genetic alteration but also by epigenetic modifications. Polycomb group (PcG) genes were first identified in Drosophila as a group of genes required for maintenance of stable repression of Hox cluster genes during development. There are increasing lines of evidence that PcG proteins themselves affect cellular proliferation and replicative senescence. Targeted disruption of Bmi1, Mel18, rae28, and M33, which are members of the class

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II PcG complex, leads to proliferation defects in hematopoietic stem cells and mouse embryo fibroblasts, indicating that inactivation of these PcGs results in cell proliferation failure. Replicative senescence of MEFs derived from Bmi1-, M33-, and Phc2-null mice has been shown to be mediated by de-repression of the central mediators of senescence signals, p19ARF and p16INK4a, which are encoded by the INK4b/ARF/INK4a region. The molecular mechanism underlying the transcriptional regulation of these genes by mammalian PcG complexes, however, has not yet been appropriately addressed except for physical interactions of Bmi1 and Phc2 gene products to p19ARF and p16INK4a genomic regions. Long Noncoding RNA in INK4b-ARF-INK4a Regulation

Long noncoding RNAs (lncRNAs), with size larger than 200 nucleotides and lack proteincoding capacity, have gained widespread attention as potentially new and important molecules of biological regulation. lncRNA is a new class of the noncoding RNA that regulates cancer development and progression. Roles for several lncRNAs in cancers have been addressed, and strategies targeting them have inhibitory effects to malignant cells. As described above, expression of the INK4b-ARF-INK4a locus is tightly controlled, and PcG complexes are required to initiate and maintain the silenced state of these tumor suppressors. Reports indicated that ANRIL, the lncRNA transcript antisense noncoding RNA in the INK4 locus, is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases including cancers. Both polycomb repressive complex-1 (PRC1) and 2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression.

Cross-References ▶ Acute Myeloid Leukemia ▶ CDKN2A

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▶ Chromosomal Translocation t(8;21) ▶ Cyclin D ▶ Cyclins ▶ Epigenetic ▶ Epigenetic Gene Silencing ▶ Ets Transcription Factors ▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ MDM2 ▶ Myc Oncogene ▶ Myelodysplastic Syndromes ▶ Non-Small-Cell Lung Cancer ▶ Polycomb Group ▶ RAS Genes

References Aguilo F, Zhou M-M, Walsh MJ (2011) Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARFINK4a expression. Cancer Res 71:5365–5369 Gil J, Peters G (2006) Regulation of the INK4b-ARFINK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677 Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4aArf: progress and puzzles. Curr Opin Genet Dev 13:77–83 Ruas M, Peters G (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378:F115–F177 Sherr CJ (2001) The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2:731–737 Sparmann A, van Lohuizen M (2006) Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6:846–856

See Also (2012) ARF. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 265. doi:10.1007/978-3-642-16483-5_383 (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) Deletion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1080. doi:10.1007/978-3-642-16483-5_1553 (2012) Glioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1557. doi:10.1007/978-3-642-16483-5_2423 (2012) Homozygous Deletion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1729. doi:10.1007/978-3-642-16483-5_2807 (2012) Hypermethylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1784. doi:10.1007/978-3-642-16483-5_2910

Innate Immunity (2012) Inversion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1907. doi:10.1007/978-3-642-16483-5_3136 (2012) Leukemia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2005. doi:10.1007/978-3-642-16483-5_3322 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Point Mutation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2934. doi:10.1007/978-3-642-16483-5_4653 (2012) Polycomb. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2940. doi:10.1007/978-3-642-16483-5_4663 (2012) Senescence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3370. doi:10.1007/978-3-642-16483-5_5236 (2012) Translocation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5942

Innate Immunity Gonghua Huang Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

Synonyms Natural immunity

Definition Innate immunity represents the first line of defense against a wide range of pathogens or damage signals and plays key roles in generating a protective inflammatory response as well as activating and instructing the adaptive immune response. It is present at birth and can pass down genetically from parents to children.

Characteristics Previously, innate immunity was characterized by a rapid, nonspecific immune response to

Innate Immunity

pathogens. However, studies demonstrate that innate immune responses show a greater degree of specificity to discriminate self from pathogens than previously appreciated. Furthermore, innate immunity is the master regulator of all immune responses and plays a key role in the activation of adaptive immunity. As the first line of defense, the innate immune system has three main components to allow for rapid and efficient removal of invading pathogens: barriers and cellular and humoral effector components. Barriers When pathogens enter into the body, they are quickly attacked, blocked, or killed by the barrier of the innate immune system. Innate immunity consists of many kinds of barriers. Anatomical Barriers

Among the most important anatomical barriers are the skin and mucous membranes. These barriers are tight and tough connections that block the entry of pathogens into the body. Hair follicles and sweat glands in the skin produce lysosome and toxic lipids to directly kill the invading bacteria. Epithelial cells in mucous membranes can produce antimicrobial peptides (AMPs), such as defensins and cathelicidins, to kill the microbes. In addition, dead keratinized cells are continuously being sloughed off, thereby regularly removing surface microbes. Cilia on the surface of epithelial cells propel mucus and prevent microbes from penetrating the host. Physiological and Chemical Barriers

These barriers include body temperature, pH, and other body secretions. The temperature of the skin is normally below body temperature (37  C) and is not favorable for bacterial growth. Once the body temperature is higher than 37  C, it can inhibit bacterial growth. The most common example of a pH barrier is the acidic environment in the stomach. Due to the acidic condition, most bacteria cannot survive and are directly killed. Moreover, bodily secretions contain lysozymes, which also possess potent antimicrobial properties and thus deter infection.

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Biological Barriers

The skin and other mucous membranes harbor approximately 100 trillion commensal bacteria and other microorganisms. These floras are harmless to the host under normal circumstances and help to keep potentially harmful opportunistic pathogens under check. Moreover, commensal flora can inhibit the colonization of pathogens by either producing metabolic products to inhibit the growth of other bacteria or consuming essential nutrients needed for the growth of pathogens. Destruction of these normal bacterial floras either by mechanic damage or by the use of broad spectrum antibiotics can result in serious infections or overgrowth by other opportunistic commensal bacteria which are resistant to the antibiotics. Cellular Components of Innate Immunity Although the barriers of the innate immune system play a vital role in protection from invading pathogens at the interface between the host and outside world, some microbes can still pass through these barriers and invade the host. To protect the host from infection, phagocytes under the skin and mucous or in the circulation will take over the task to ingest and kill the bacteria immediately. Phagocytes

Phagocytes include hematopoietic-derived macrophages, dendritic cells (DCs), mast cells, neutrophils, basophils, and eosinophils, as well as cells from the skin and the respiratory, gastrointestinal, and genitourinary systems. Among all of these phagocytes, macrophages, DCs, and neutrophils are believed to play essential roles in the killing of bacteria. Macrophages and DCs can also secrete chemotactic cytokines to recruit other phagocytes, such as neutrophils, to the site of infection. Other phagocytes, such as mast cells, basophils, and eosinophils, are involved in the antimicrobial response by releasing inflammatory mediators to activate resting phagocytes. In addition, macrophages and DCs present antigens to CD4+ T cells and secrete T cell-polarizing cytokines to initiate adaptive immune responses. The adaptive immunity will not be effective without the essential antigen-presenting function of innate immune cells.

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Cell-Based Innate Immune Recognition

Recognition of pathogens is the first and most important step to initiate effective immune responses. Unlike adaptive immunity, which harbors the antigen-specific recognition, innate immunity does not have a strict antigen-specific recognition. Instead, at the site of infection, innate immune cells (i.e., phagocytes) can immediately recognize invading pathogens by using surface pattern recognition receptors (PRRs), which are germline-encoded receptors. PRRs recognize and bind to structurally conserved pathogenassociated molecular patterns (PAMPs) expressed by most bacteria, but not expressed by the host. The best studied and characterized PRRs are the toll-like receptors (TLRs), which are a family of transmembrane receptors with an extracellular leucine-rich repeat binding domain, a transmembrane domain and a conserved cytoplasmic toll/ interleukin-1 receptor homology domain. To date, at least thirteen different TLRs have been identified and characterized in the mammalian system. Each TLR shows specific recognition to unique PAMPs. TLR3 recognizes double-stranded RNA (dsRNA), TLR4 recognizes LPS, TLR5 recognizes flagellin, TLR7 recognizes single-stranded (ssRNA), and TLR9 recognizes dsDNA. Moreover, TLRs are differentially expressed by different cell types, which endow them with unique recognition responsibilities. Other transmembrane PRRs expressed on the host innate immune cells include dectin-1 and dectin-2, C-type lectin family members. These receptors detect b-glucans and mannan which are expressed on fungal cell walls. In addition to transmembrane receptors, phagocytes also possess cytosolic PRRs to recognize intracellular pathogens. These include the RIG-I-like receptors (RLRs) and the Nod-like receptors (NLRs). Three RLRs have been identified to date: RIG-I, melanoma differentiation factor 5 (MDA5), and LGP2. Each of these RLRs plays essential roles in detecting the viral RNA in the cytoplasm. Like RLRs, NLRs are another large family of cytosolic sensors that recognize intracellular PAMPs as well as stress signals. According to the different ligand specificity, NLRs include NOD1 and NOD2. NOD1 recognize the dipeptide g-D-glutamyl-

Innate Immunity

diaminopimelic acid (iE-DAP), which is derived from most Gram-negative and some Grampositive bacteria. NOD2 detects muramyl dipeptide (MDP), which is a component of peptidoglycan. Moreover, a number of NLR family members can form multiprotein complexes, called inflammasomes. Studies have identified several inflammasomes, including IPAF (ICE proteaseactivating factor) inflammasome which is triggered by bacterial flagellin, NALP1b (NACHT domain-, leucine-rich repeat-, and PYDcontaining protein 1b) inflammasome which is induced by anthrax lethal toxin, and NALP3/ cryopyrin/NLRP3 inflammasome which assembles in response to a variety of PAMPs and “danger” signals (i.e., uric acid crystals). AIM2 (absent in melanoma 2), a newly identified inflammasome, recognizes cytoplasmic dsDNA. Notably, unlike the antigen-specific receptors which are only expressed on T and B cells, PRRs are widely expressed in a variety of cells, which enables the innate immune response to rapidly response to invading microorganisms. Dysregulation of PRRs can lead to uncontrolled inflammation and is thus believed to contribute to numerous autoinflammatory diseases. Cell-Based Innate Immune Effectors

The main function of innate immunity is not solely to sense the invading microbes but also to kill the microbes. After activation by inflammatory mediators such as bacterial products, phagocytes can be attracted to the site of infection, where they recognize and bind to the pathogens. This recognition and binding will lead to a series of intracellular processes, which include the recruitment of adaptors to the intracellular domains of PRRs and the activation of their downstream kinases, such as the IKK complex and MAP kinase or transcription factors such as the NF-kB and AP-1. These processes lead to the expression of inflammatory cytokines, chemokines, and other mediators. Some cytokines and chemokines can directly destroy bacteria, promote an inflammatory response, and/or further activate resting phagocytes. In addition, certain PRRs, such as TLRs, have been found to act as adjuvant receptors that create a bridge between

Innate Immunity

innate and adaptive immunity and to have important roles in the induction of adaptive immunity, which serves as a second line of defense. Aside from the receptor-mediated innate immune responses, activated phagocytes can also ingest and kill the microbes in an oxygen-dependent or oxygen-independent system. In the oxygendependent killing manner, the cytoplasmic membrane of phagocytes contains the enzyme oxidase which converts oxygen into superoxide anion. Combined with water, the superoxide anion is converted into hydrogen peroxide and hydroxyl radicals, which further catalyze chloride ions and nitric oxide to form the hypochlorous acid and peroxynitrite radicals, respectively. All of these compounds are powerful microbicidal agents. However, in the oxygen-independent killing system, phagocytes harbor lysosomes, which are produced from the Golgi apparatus and contain various digestive enzymes, antimicrobial peptides, proteases, and lipases. These components can alter or damage the bacterial membrane or break down microbial proteins, RNAs, and lipids. Humoral Components of Innate Immunity To augment the cellular defense functions of phagocytes, innate immunity also has humoral effector components to kill the microbes which have not been cleared by the phagocytes. Like the cellular components, humoral components also act through the recognition of the microbes and then produce an effective innate immune response to kill the invading microbes. Humoral-Based Innate Immune Recognition

Aside from recognition of PAMPs by PRRs on the phagocytes, some extracellular molecules can also directly recognize and bind to microbes. The most well-known molecules are mannosebinding proteins. These proteins can recognize microbial surface terminal mannosyl residues or other determinants to activate the complement pathway. LPS, which is recognized by TLR4 of phagocytes, can be also recognized by the LPS binding protein and CD14. Moreover, C-reactive protein has also been found to bind to microbes to induce a lectin-like innate immune response.

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Humoral-Based Innate Immune Effectors

The abovementioned extracellular molecules can only bind to the microbes. They cannot kill the microbes. To induce effective bacterial clearance, other proteins are required. Among the most important humoral components involved in the defense against the microbial infection is the complement. The complement system is a complicated enzymatic cascade, which comprises more than 30 proteins in the blood plasma or on cell surfaces. There are three distinct pathways to activate the complement system: classical, mannosebinding lectin, and alternative pathway. Although the components involved in the activation pathways are different, they all start with binding to specific ligands and subsequent activation of the recognition unit of each pathway. Through a series of downstream activation events, all three complement pathways ultimately lead to the activation of the complement component C3, which then activates the membrane-attack complex and subsequently leads to lysis of the bacteria or infected apoptotic cells. Complement pathway also plays an important role in clearing the immune complex and apoptotic cells. Moreover, the complement pathway has also been shown to bridge innate and adaptive immunity. However, under certain conditions, such as genetic variation or autoantibodies, the functions of the complement pathway can be altered and involved in pathology by contributing to tissue damage, induction of certain autoimmune diseases, and chronic inflammation. Thus, the functions of the complement system, especially the activation of complement cascade, must be tightly regulated to provide the host essential protection against infection while maintaining homeostasis. Aside from complement, AMPs are another important component that defends against microbial infection. Initially, AMPs were found in bacteria and fungi and regarded as unique defense molecules in unicellular organisms. Subsequently, more and more AMPs were isolated, characterized, and discovered in all kinds of life, from plants to animals. Although there is great sequence diversity in the AMPs, they share certain common structural features: AMPs have a length around 30 residues and are cationic in nature (+2 to +9). Each AMP

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consists of about 40–50% hydrophobic residues. These residues can adapt an amphipathic structure to directly recognize and bind to microbial structures and lyse the bacteria membrane, thereby killing the microbes immediately. Studies show that there are multiple AMPs in mammals. The most thoroughly characterized are the defensins and cathelicidins, which play a role in human immunity, health, as well as disease. Although the use of AMPs as a single antibiotic agent has received lots of attention and data from in vitro antibiotic potential are encouraging, none of them have obtained FDA approval for clinical applications. Thus, the use of AMPs as a new class of antibiotic drugs and their value as external therapeutic agents still needs to be further established. In addition, cytokines also play essential roles in the defense against infection. Cytokines are small molecular weight, soluble protein molecules and can be produced by numerous cells in response to infections and function as regulatory molecules to instruct the innate and adaptive immunity as well as directly destroy the microbes. The acute phase response is also dependent on cytokines. However, dysregulation of cytokine productions can be harmful to the host. To date, large amounts of cytokines can be produced by recombinant gene and protein expression technology, providing a very promising method to further investigate the mechanisms and clinical applications of cytokines. Conclusion Defense against microbial infection is essential for all living organisms. Vertebrates, invertebrates, and even plants have evolved a set of strategies to protect the host from infection. Although the precise characteristics of the innate immunity differ between the various species, they all share several central features, including the utilization of PRRs to rapidly response to a large spectrum of pathogens and serving as a first line of defense to rapidly and effectively clear most invading pathogens through the cellular- or humoral-based innate immune responses. Advancements in cellular and molecular biology should foster in a new wave of discoveries in innate biology.

Inositol Lipids

For more reading about the complement, please see http://www.faqs.org/health/bios/44/ Jules-Bordet.html) and http://www.ncbi.nlm.nih. gov/pubmed/21704094. For more reading about the antimicrobial peptides, please see http://aps.unmc.edu/AP/. For more reading about the roles of cytokine in the innate immune system, please see http:// pathmicro.med.sc.edu/bowers/imm-reg-ver2.htm.

References Beutler B (2004) Innate immunity: an overview. Mol Immunol 40:845–859 Imler JL, Hoffmann JA (2000) Toll and Toll-like proteins: an ancient family of receptors signaling infection. Rev Immunogenet 2:294–304 Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216 Schenten D, Medzhitov R (2011) The control of adaptive immune responses by the innate immune system. Adv Immunol 109:87–124 Steinman RM (2006) Linking innate to adaptive immunity through dendritic cells. Novartis Found Symp 279:101–109; discussion 109–113, 216–219

Inositol Lipids Matilda Katan CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, London, UK

Definition Are a class of phospholipids where inositol is the polar headgroup. The simplest inositol phospholipid is phosphatidylinositol (PtdIns). The inositol moiety can be phosphorylated at several different positions giving rise to a number of other molecular species.

Characteristics Among different inositol lipids, the importance in transmembrane signaling and regulation of cell

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Inositol Lipids, Fig. 1 Structure of phosphatidylinositol (4,5) bisphosphate (PtdIns (4,5)P2) shows a typical phospholipid containing an inositol ring as a headgroup. The positions on the inositol ring are designated 1–6 and two phosphate groups are present at positions 4 and 5. Phosphatidylinositol (3,4,5) trisphosphate (PtdIns

(3,4,5)P3) is generated in a phosphorylation reaction; the third phosphate group is added at position 3 of the inositol ring. Hydrolysis of PtdIns(4,5)P2 at the C-bond separates the hydrophobic part that contains two lipid chains, from water-soluble inositol that contains phosphates at the positions 1, 4, and 5

functions are best documented for PtdIns(4,5)P2 and PtdIns(3,4,5)P3. There are several ways in which these low-abundance inositol lipids (less than 1% of membrane phospholipids) could provide a signaling link or fulfill other roles in different cellular processes.

and, in turn, cause their activation. Ins(1,4,5)P3 binds to specific receptors in the endoplasmic reticulum causing a release of calcium from this intracellular store into the cytoplasm. Membrane-resident diacylglycerol (DAG) is required for activation of several isoforms of protein kinase C (PKC). These second messengers act as a common component in different signaling pathways, contributing to diverse cellular responses. Specificity of the pathways is provided at the level of a receptor and downstream components (e.g., calcium-regulated proteins and PKC substrates) present in a specific cell type or state.

Hydrolysis of PtdIns(4,5)P2 to Generate Second Messenger Molecules Hydrolysis of PtdIns(4,5)P2 occurs in response to a large number of extracellular signals and generates two second messenger molecules, inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) molecules. The reaction is catalyzed by phosphoinositide-specific phospholipase C (PI-PLC) (Fig. 1). There are several isoforms of this enzyme (PLCb, PLCg, PLCd, PLCe and PLCz) linked to and activated by different cellular receptors. For example, PLCg is regulated through tyrosine kinase receptors such as receptors for ▶ Epidermal Growth Factor Inhibitors, ▶ fibroblast growth factor, and ▶ platelet-derived growth factor, while PLCe could be a novel target for ▶ RAS Genes proteins. The second messengers generated from PtdIns (4,5)P2 interact with specific intracellular targets

Binding of PtdIns(4,5)P2 to Specific Proteins In addition to its role as a precursor of Ins(1,4,5) P3 and DAG, PtdIns(4,5)P2 has emerged as a highly versatile signaling molecule in its own right. These other functions are mediated through direct binding of PtdIns(4,5)P2 to specific protein targets and include fundamental processes in membrane trafficking and plasma membranecytoskeleton linkages. Many proteins that regulate actin cytoskeleton (e.g., gelsolin and profilin) and proteins involved in ▶ endocytosis

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(e.g., dynamin and AP-2 adaptor) bind PtdIns(4,5) P2. The binding involves different positively charged protein surfaces that in some proteins are present within the modular pleckstrin homology domain (PH domain). The result of the binding could be a direct change in the protein function or a regulated membrane targeting. For example, the PH domain of phospholipase Cd1 associates with membranes of many cell types, but after PLC stimulation and the reduction in PtdIns(4,5)P2 concentrations, it translocates to the cytoplasm. Concentration of PtdIns(4,5)P2 is not only regulated at the level of hydrolysis by PLC but also through regulation of several types of inositol lipid kinases and phosphatases. Generation of PtdIns(3,4,5)P3 and Other 3-Phosphorylated Inositol Lipids and Their Binding to Specific Intracellular Targets Inositol lipids phosphorylated at the 3-position of the inositol ring (PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3) are generated by phosphoinositide 3-kinase (PI3-K). PI3-Ks are grouped into three classes on the bases of their structure, and according to the inositol lipid, they preferentially utilize as a substrate. For example, the class I PI3-Ks are receptor-regulated signaltransducer proteins that preferentially phosphorylate PtdIns(4,5)P2 in vivo and generate PtdIns (3,4,5)P3 (Fig. 1). Several target proteins for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been described, and they include protein kinases such as PKB/Akt, PDK1, and Btk. In the case of PKB/Akt, the direct binding to the PH domain (with high affinity and specificity toward 3-phosphorylated inositol lipids compared with more abundant PtdIns(4,5)P2) results in both membrane targeting and conformational changes that lead to phosphorylation and activation of this protein kinase. Activated kinases, in turn, phosphorylate and regulate downstream targets and thus propagate the signal. The 3-phosphorylated inositol lipids also participate in diverse cellular functions including cell survival, proliferation, migration, and vesicle budding. In addition to regulation of PI3-K, the levels of PtdIns(3,4,5) P3 are also controlled by a 3-phosphatase.

Inositol Lipids

Clinical Relevance There is considerable experimental evidence that the key enzymes involved in the control of inositol lipids, PI-PLC and PI3-K, play an important role in processes critical for tumor development and spreading, including cell proliferation, survival, and ▶ migration. The involvement of the PI3-K in human cancer has been supported by both genetic and functional studies, including the discovery of frequent, activating missense mutations in a wide variety of common human tumor types. Important components downstream of PI3K, also relevant in the context of cancer, include kinase PKB/Akt and the protein kinase complex mTORC. Inhibitors targeting PI3-K and PI-3K pathway are entering clinical trials and treatments. Furthermore, the importance of the control of inositol lipid levels in human cancers have been emphasized by the findings that the tumor suppressor protein PTEN is a 3-phosphatase that dephosphorylates PtdIns(3,4,5)P3. The PTEN gene is deleted or mutated in a wide variety of human cancers. Activating or inactivating, disease-linked mutations can also occur in other inositol lipid kinases and phosphatases. Among PIPLC enzymes, PLCg gamma activating mutations have been reported in several cancer types and in specific immune disorders. Many human tumors have also been found to express increased levels of PI-PLC or PI3-K.

Cross-References ▶ Endocytosis ▶ Epidermal Growth Factor Inhibitors ▶ Fibroblast Growth Factors ▶ Migration ▶ PI3K Signaling ▶ Platelet-Derived Growth Factor ▶ RAS Genes

References Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93(3):1019– 1137. doi:10.1152/physrev.00028.2012. PMCID: PMC3962547

Insulin Receptor Bunney TD, Katan M (2010) Phosphoinositide signalling in cancer: beyond PI3K and PTEN. Nat Rev Cancer 10 (5):342–352. doi:10.1038/nrc2842. Review. PMID: 20414202 Fruman DA, Rommel C (2014) PI3K and cancer: lessons, challenges and opportunities. Nat Rev Drug Discov 13 (2):140–156. doi:10.1038/nrd4204. Review. PMID: 24481312 Koss H, Bunney TD, Behjati S, Katan M (2014) Dysfunction of phospholipase C γ in immune disorders and cancer. Trends Biochem Sci 39(12):603–611. doi:10.1016/j.tibs.2014.09.004. Epub 2014 Oct 30. Review. PMID: 25456276 Vanhaesebroeck B, Stephens L, Hawkins P (2012) PI3K signalling: the path to discovery and understanding. Nat Rev Mol Cell Biol 13(3):195–203. doi:10.1038/ nrm3290. PMID: 22358332

See Also (2012) Hemangiosarcoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 1640. doi:10.1007/978-3-642-164835_2614 (2012) Isoform. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1920– 1921. doi:10.1007/978-3-642-16483-5_3158 (2012) Phosphatidyl inositol 3-kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2865. doi:10.1007/978-3-642-164835_4527 (2012) Phospholipids. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2869– 2870. doi:10.1007/978-3-642-16483-5_4542 (2012) Phospholipase C. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2869. doi:10.1007/978-3-642-16483-5_4539 (2012) Pleckstrin homology domains. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2919. doi:10.1007/978-3-642-164835_4620 (2012) Posttranslational modification. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2966. doi:10.1007/978-3-642-164835_4696 (2012) Retrovirus. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, pp 3296–3297. doi:10.1007/978-3-64216483-5_5084 (2012) Second messenger. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3343. doi:10.1007/978-3-642-16483-5_5192 (2012) Signal-transducer proteins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3411. doi:10.1007/978-3-642-164835_5299 (2012) Transmembrane signaling. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3774. doi:10.1007/978-3-642-164835_5952

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Insulin Receptor Antonio Brunetti Department of Health Sciences, University of Catanzaro “Magna Græcia”, Catanzaro, Italy

Definition IR is a phylogenetically ancient ▶ receptor tyrosine kinase protein embedded in the plasma membrane of virtually all cells. When the peptide hormone insulin binds to the IR, the receptor becomes activated and induces a cascade of intracellular events that will lead to several metabolic and growth-promoting effects.

Characteristics The IR belongs to the tyrosine kinase growth factor receptor family and functions as an enzyme that transfers phosphate groups from ATP to tyrosine residues on intracellular target proteins. The IR consists of two identical extracellular alpha subunits (130 kDa) that house insulin binding domains and two transmembrane beta subunits (95 kDa) that contain ligand-activated tyrosine kinase activity in their intracellular domains (Fig. 1). When insulin binds to the alpha subunits, the receptor is first activated by tyrosine autophosphorylation, and then the IR tyrosine kinase phosphorylates various intracellular effector molecules (e.g., IRS proteins and Shc) which in turn alters their activity, thereby generating a biological response. The IR exists as two splice variant isoforms: the IR-B isoform that is responsible for signaling metabolic responses involved mainly in the regulation of glucose uptake and metabolism by increasing glucose transporter (Glut4) molecules on the plasma membrane of the insulin-responsive tissues – muscle, liver, and fat – and the IR-A isoform that is capable of binding IGF-2 with high affinity and signals predominantly mitogenic responses. As a consequence of these cellular activities, abnormalities of IR expression and/or function can

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Insulin Receptor, Fig. 1 In the absence of insulin, most of the IRs in the plasma membrane are in a non-tyrosine phosphorylated inactive state, and only a very small proportion of receptors are lightly phosphorylated and subjected to constitutive ▶ endocytosis and recycling. Upon binding of insulin, the IR undergoes autophosphorylation which enables the receptor to have a

Insulin Receptor

kinase activity and phosphorylates various cytoplasmic substrates, such as IRSs. From this point, signaling proceeds via a variety of signaling pathways (i.e., PI3K signaling pathway, Ras and MAP kinase cascade) that are responsible for the metabolic, growth-promoting, and mitogenic effects of insulin

Insulin Receptor

facilitate the development of several metabolic and neoplastic disorders in humans as well as in animal models. Regulation Gene expression in eukaryotic cells is controlled by nuclear regulatory proteins (trans-acting factors) that modulate the transcription of genes by binding to specific cis-acting transcriptional elements in the promoter of target genes. The IR gene promoter extends over 1,800 bases 50 upstream from the IR gene ATG codon, contains a series of GGGCGG repeats that are putative binding sites for the mammalian transcription factor Sp1, and has neither a TATA box nor a CAAT box, reflecting the common features for the promoters of constitutively expressed genes (so-called housekeeping genes). Like other housekeeping promoters, the IR gene promoter confers a basal level of transcriptional activity common to all cells, whereas significantly higher transcriptional activity is induced in the muscle, liver, and fat, at which levels the IR has been shown to be under the regulation of hormones, metabolites, and differentiation. Promoters of genes that are activated in a tissue-specific manner are often regulated by a combination of tissue-specific and ubiquitous transcription factors, where the ubiquitous element facilitates or enhances the action of one or more tissue-specific transcription factors. The molecular mechanisms regulating IR gene expression are being elucidated and evidence has been provided showing that the architectural transcription factor HMGA1 is required for proper transcription of the IR gene in cells expressing IRs. HMGA1 acts on the IR gene promoter as an element necessary for the formation of a transcriptionally active multiprotein–DNA complex involving, in addition to the HMGA1 protein, the ubiquitously expressed transcription factor Sp1 and the CCAATenhancer binding protein beta (C/EBP-beta). By potentiating the recruitment and binding of Sp1 and C/EBP-beta to the IR promoter, HMGA1 greatly enhances the transcriptional activities of these factors in the context of the IR gene.

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Conversely, repression of HMGA1 function in cells and tissues adversely affects transactivation of the IR gene promoter by Sp1 and C/EBP-beta and considerably reduces IR protein expression. Clinical Relevance The IR is of major importance in certain states of insulin resistance in humans, in which abnormalities of the receptor may lead to defective transmembrane signaling. In this respect, dysfunctional IR signaling is implicated in certain common dysmetabolic disorders, including obesity, type 1 and type 2 diabetes, the dysmetabolic syndrome X, and the polycystic ovary syndrome (PCOS). Also, clinical syndromes due to mutations in the IR gene have been identified in patients with genetic forms of severe insulin resistance (i.e., leprechaunism, type A insulin resistance, and the Rabson–Mendenhall syndrome). Many of these patients have point mutations in the coding sequence of the IR gene, leading to reduced or absent IR expression in target tissues. Previously, defects in IR gene regulation have been reported in individuals with insulin resistance and type 2 diabetes, in which the generation of IR mRNA was considerably impaired, although the IR genes were normal. In these individuals, cell-surface IRs were decreased and the expression of HMGA1 was markedly reduced. These observations have been confirmed and mechanistically underpinned in studies. Even though it is an open question whether IR plays a critical role in aging and longevity in mammals, disturbance of the neuronal IR seems to be of pathogenetic relevance in human Alzheimer disease and depressive disorders, suggesting a neurotrophic role of IR in the brain. According to studies, IR in the brain begins to disappear early in Alzheimer and continue to decline as the disease progresses. It has been shown that stimulation in the brain of a receptor that mediates insulin responses can halt or diminish the neurodegeneration of Alzheimer disease. Also, a high prevalence of insulin resistance has

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been reported in patients with depression, and an increased risk of cognitive decline has been found in women with insulin-resistant PCOS. A relation between IR and cancer has been established following the observation that overexpression of functional IRs can occur in human ▶ breast cancer and other epithelial tumors including ovarian and colon cancer, in which the IR may exert its oncogenic potential via abnormal stimulation of multiple cellular signaling cascades, enhancing growth factor-dependent proliferation, and/or by directly affecting cell metabolism. An explanation for increased IR expression in epithelial tumors has been provided before for several human breast cancers, in which overexpression of the transcription factor AP2alpha accounts for increased IR expression in neoplastic breast tissue. In these cases, it has been demonstrated that transactivation of the IR gene by AP-2 alpha needs direct physical association of AP-2 with HMGA1 and Sp1, which represents a fundamental prerequisite for AP-2 alpha to activate IR gene transcription in neoplastic breast tissue. Epidemiological and clinical evidence points to a link between insulinresistant syndromes such as obesity and type 2 diabetes and cancer of the colon, liver, pancreas, breast, and endometrium. The mechanistic link between insulin resistance and cancer is unknown, but constitutive activation of the tyrosine kinase activity of IR and related downstream signaling pathways by chronic sustained hyperinsulinemia (a hallmark of insulin resistance) in these clinical syndromes appears to have a role in the neoplastic transformation process. Also, insulin and the related insulin-like growth factor I (IGF-I) stimulate the ovarian production of sex steroids, whose effects in breast epithelium and endometrium can promote cell proliferation and inhibit apoptosis. Furthermore, an increased risk of cancer among insulinresistant subjects can be due to overproduction of reactive oxygen species (ROS) that by damaging DNA can contribute to mutagenesis and carcinogenesis. In addition to that, it is also important to remark that the accumulation of inflammatory cells in adipose tissue of obese and diabetic patients may promote systemic chronic

Insulin Receptor

inflammation, which in turn can result in a protumorigenic conditions. Thus, the IR plays a major role in the pathophysiology of a wide range of metabolic, neurodegenerative, and proliferative disorders in humans. Selective modulation of IR expression and/or function may represent a useful therapeutic strategy for these diseases. Also, measures to decrease insulin resistance in insulin-resistant patients with obesity and type 2 diabetes may offer a general approach to prevention of cancer in these predisposed individuals.

Cross-References ▶ Breast Cancer ▶ Endocytosis ▶ Receptor Tyrosine Kinases

References Arcidiacono B, Iiritano S, Nocera A et al (2012) Insulin resistance and cancer risk: an overview of the pathogenetic mechanisms. Exp Diabetes Res 2012:789174. doi:10.1155/2012/789174 Chiefari E, Iiritano S, Paonessa F et al (2010) Pseudogenemediated posttranscriptional silencing of HMGA1 can result in insulin resistance and type 2 diabetes. Nat Commun 1:40. doi:10.1038/ncomms1040 Chiefari E, Tanyolaç S, Paonessa F et al (2011) Functional variants of the HMGA1 gene and type 2 diabetes mellitus. JAMA 305:903–912 Foti D, Chiefari E, Fedele M et al (2005) Lack of the architectural factor HMGA1 causes insulin resistance and diabetes in humans and mice. Nat Med 11:765–773 Goldfine ID (1987) The insulin receptor: molecular biology and transmembrane signalling. Endocr Rev 8:235–255 Paonessa F, Foti D, Costa Vet al (2006) Activator protein-2 overexpression accounts for increased insulin receptor expression in human breast cancer. Cancer Res 66:5085–5093 White MF, Khan CR (1994) The insulin signaling system. J Biol Chem 261:1–4 Yarden Y, Ullrich A (1998) Growth factor receptor tyrosine kinases. Annu Rev Biochem 57:443–478

See Also (2012) AP2-alpha. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 230. doi:10.1007/978-3-642-16483-5_346

Insulin Signaling (2012) ATP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 302. doi:10.1007/978-3-642-16483-5_440 (2012) Diabetes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1105. doi:10.1007/978-3-642-16483-5_1601 (2012) Glut4. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1559. doi:10.1007/978-3-642-16483-5_2435 (2012) HMGA1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1710. doi:10.1007/978-3-642-16483-5_2772 (2012) Insulin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1873–1874. doi:10.1007/978-3-642-16483-5_3075 (2012) Insulin resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1877. doi:10.1007/978-3-642-16483-5_3078 (2012) IRS. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1917. doi:10.1007/978-3-642-16483-5_3150 (2012) Obesity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2595. doi:10.1007/978-3-642-16483-5_4185 (2012) Plasma membrane. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2900. doi:10.1007/978-3-642-16483-5_4599 (2012) SHC. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3401. doi:10.1007/978-3-642-16483-5_5284 (2012) TATA box. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3613–3614. doi:10.1007/978-3-642-16483-5_5682

Insulin Signaling Yong Zhang Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Synonyms Insulin-like growth factor (IGF) signaling

Definition Insulin/insulin-like growth factor signaling pathway is one of the most important signaling pathways in controlling cell and organ size. It also

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plays key roles in metabolism, stress resistance, reproduction, and longevity. Insulin binding to insulin receptors triggers an intracellular signaling cascade consisting of PI3K and Akt (PKB). Akt in turn deactivates glycogen synthase kinase 3 (GSK-3), leading to activation of glycogen synthase (GYS) and thus glycogen synthesis. Activation of Akt also results in the translocation of GLUT4 vesicles from their intracellular pool to the plasma membrane, where they allow uptake of glucose into the cell. Akt also leads to mTORmediated activation of protein synthesis by eIF4 and p70S6K.

Characteristics Insulin Signaling and Cell Growth In metazoans, tissue growth relies on the availability of cell-intrinsic and cell-extrinsic factors. Hormones and growth factors are known to play a predominant role in regulating organ growth by controlling cell growth, cell proliferation, and cell survival. Insulin signaling pathway is one of the most important pathways in growth control. Biochemical studies have shown that on binding of insulin or IGFs, insulin receptor (InR) recruits phosphoinositide 3-kinase (PI3K) to the membrane, either directly or through insulin receptor substrates (IRS) as intermediates. Phosphorylation of the membrane lipid phosphatidylinositol 4,5-biphosphate (PIP2) by PI3K produces the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3), which activates Akt, a Ser/Thr kinase. Akt phosphorates and activates target of rapamycin (TOR), another Ser/Thr kinase which ultimately controls the phosphorylation of two downstream effectors, p70 S6 kinase (S6K) and 4E-binding protein (4E-BP), which are involved in translational control. Mutations in any one of these components lead to change in cell size and in cell number as well. Overexpression of Dp110 (catalytic subunit of PI3K) or Akt results in increased cell size. Loss of function of PTEN, a negative regulator of the insulin pathway, also leads to increase in cell and organ size.

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Genetic studies in Drosophila have made important contributions in deciphering the insulin signaling pathway. The Drosophila homolog of the insulin/IGF1 receptor, InR, is essential for normal development and is required for the formation of the epidermis and the central and peripheral nervous systems during embryogenesis. Only weak heteroallelic combinations of InR alleles were found to be viable and yield adults with a severe developmental delay, small body size, and female sterility. It has also been shown that InR functions cell autonomously in a tissuespecific manner. PI3K phosphorylates membrane lipid phosphatidylinositol 4,5-biphosphate (PIP2) to produce second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3); the binding of PIP3 to the PH domain anchors Akt to the plasma membrane and allows its phosphorylation and activation by PDK1 (phosphoinositide-dependent kinase-1). Ectopic expression of Dp110, the catalytic subunit of PI3K, affects wing and eye growth in Drosophila. Phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (also known as protein kinase B or PKB) affects cell and organ size in Drosophila in a cell autonomous manner. Akt has a PH domain that binds PIP3, and the translocation of Akt to the plasma membrane leads to its activation by PDK1. Overexpression of Akt in the Drosophila wing imaginal disk leads to marked enlargement. In Drosophila, the overexpression of Akt affects organ size by altering cell growth without modulating the cell number. However, Akt may also positively regulate cell cycle progression in mammalian system. Genetic and biochemical studies have shown that TOR impinges on the insulin signaling pathway by autonomously affecting growth through regulating the activity of S6 kinase (S6K). TOR has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. TOR forms two structurally and functionally distinct complexes termed TOR complex 1 (TORC1) and TORC2. In mammals, TOR specifically associates with raptor to form TORC1, whereas TORC2 contains rictor, SIN1, and PRR5

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(also known as protor-1). Both complexes contain the shared component LST8. Acute rapamycin treatment inhibits TORC1 but not TORC2, although prolonged treatment can also indirectly inhibit TORC2. TORC1 is activated by nutrients (amino acids), growth factors (insulin, IGF, etc.), and cellular energy status (AMP/ATP ratio) and regulates several growth-related process including protein synthesis, ribosome biogenesis, lipid synthesis, nutrient import, and autophagy. The best-characterized TORC1 substrates are S6K and 4E-BP1 via which TORC1 controls protein synthesis. TORC2 regulates the actin cytoskeleton, cell survival, and other processes via phosphorylation of AGC kinase family members including Akt, SGK1, and PKC. Since we are discussing insulin signaling, we will be focusing on TORC1 in the following discussion. As a major cell growth regulator, TOR integrates a wide range of intracellular and extracellular signals to regulate translation and cell growth. PKB/Akt can relay the positive input on TOR by modulating PRAS40 function. Studies have shown that PRAS40 interacts with the TOR complex (either TOR kinase or Raptor), and in vitro it prevents TOR activation. PKB/Akt directly phosphorylates PRAS40 on T246 and diminishes the inhibitory activity of PRAS40 on TOR. A key downstream target of TOR function is protein synthesis. TOR positively regulates protein synthesis by modulating the activities of two essential translational components: translation initiation factor 4E-binding protein (4E-BP) and the ribosomal protein S6 kinase (S6K). Rapamycin acts by forming an inhibitory complex with its intracellular receptor FKBP12; the complex binds the FRB domain in the C-terminus region of the TOR protein and inhibits TOR kinase activity. In Drosophila, TOR is required cell autonomously for normal growth and proliferation during larval development; loss of TOR function leads to reduced nucleolar size and cell-typespecific pattern of cell cycle arrest. The phenotypes associated with the complete loss of TOR function are remarkably similar to phenotypes associated with mutations in the insulin-like receptor (Inr) pathway. Mutation of components

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in both pathways arrests development at similar early developmental stage. TOR and insulin signaling pathway component mutant clones both have significant proliferative disadvantage. TOR is also activated by amino acids and nutrients; a major discovery in understanding TOR regulation by nutrient was the identification of the tuberous sclerosis complex (TSC) that negatively regulates TOR activity. Mutation of TSC1 or TSC2 gene in Drosophila significantly increases cell size, consistent with their negative role in TOR regulation. The human syndrome TSC is caused by mutations in TSC1 or TSC2. The discovery of a connection between the TSC and TOR pathway provides the first molecular link between TOR and tumor formation. TSC2 encodes a putative GTPase-activating protein (GAP), whereas TSC1 encodes a protein that contains two coiled-coil domains. Biochemical and genetic experiments have shown that small GTPase Rheb is a direct downstream target of the TSC2 GAP activity, which promotes GTP hydrolysis of Rheb, thereby inactivating Rheb. Loss of Rheb decreases S6K activity and inhibits cell growth, whereas overexpression of Rheb increases S6K activity and promotes cell growth. Furthermore, overexpression of Rheb confers resistance to amino acid starvation, similar to that caused by loss of Tsc1 or Tsc2 in the S2 cells of Drosophila. Genetic analyses place Rheb downstream of the TSC complex and strongly suggest that Rheb is a target of the GAP activity of TSC2. Consistent with this hypothesis, co-overexpression of TSC1 and TSC2 dramatically decreases the relative ratio of GTP-to GDP-bound Rheb, whereas RNAi inhibition of TSC2 results in increased GTP loading on Rheb. In vitro, purified GAP domain of TSC2 displays specific GAP activity toward Rheb but not toward the closely related small GTPase Ras, demonstrating the specificity of the GAP activity of TSC2. Together, these studies suggest an evolutionarily conserved role for Rheb as direct target of TSC1–TSC2 in regulating TOR signaling. Insulin/IGF signaling and amino acid signaling converge on TOR in regulating cell growth and onset and progression of diabetes, cancer, and aging.

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Insulin Signaling and Glucose Metabolism Like the receptors for other protein hormones, the receptor for insulin is embedded in the plasma membrane. The insulin receptor is composed of two alpha subunits and two beta subunits linked by disulfide bonds. The alpha chains are entirely extracellular and house insulin-binding domains, while the linked beta chains penetrate through the plasma membrane. The insulin receptor is a tyrosine kinase. It functions as an enzyme that transfers phosphate groups from ATP to tyrosine residues on intracellular target proteins. Binding of insulin to the alpha subunits causes the autophosphorylation of beta subunits, thus activating the receptor. The activated receptor then alters the activity of intracellular molecules in a signaling cascade, thereby generating a biological response. Insulin acts on cells throughout the body to stimulate uptake, utilization, and storage of glucose. The major transporter used for uptake of glucose (called GLUT4) is made available in the plasma membrane through the action of insulin. Insulin regulates membrane trafficking and whole body glucose homeostasis. Insulin promotes glucose uptake and its utilization via glycolysis. One of the main acute effects of insulin is to regulate the disposal and storage of dietary glucose by stimulating the uptake of glucose into muscle and fat. Insulin regulates glucose uptake into these cells by recruiting membrane vesicles containing the GLUT4 glucose transporters from the interior of cells to the cell surface, where it allows glucose to enter cells by facultative diffusion. Once in the cytoplasm, the glucose is phosphorylated and thereby trapped inside the cells. The effect of insulin on GLUT4 distribution is reversible. Within an hour of insulin removal, GLUT4 is removed from the membrane and restored intracellular in vesicles ready to be re-recruited to the surface by insulin. Insulin release from pancreas oscillates with a period of 3–6 min. The release of insulin from beta cells is rapidly triggered in response to increased blood glucose levels. Increased blood insulin content causes cells in the liver, muscle, and fat tissue to take up glucose from the blood,

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storing it as glycogen in the liver and muscle. When the glucose level comes down to its physiological range, insulin release from the b-cells slows or stops. Failure to control insulin levels results in diabetes mellitus; in patients with type 1 diabetes, the pancreas no longer makes insulin. The beta cells have been destroyed, and they need insulin shots to use glucose as their energy source. Patients with type 2 diabetes make insulin, but they are often insulin resistant. Some patients with type 2 diabetes may eventually require insulin shots if other medications fail to control blood glucose levels adequately. Insulin Signaling and Aging Decreased signaling through the insulin/IGF1 pathway can increase the lifespan of worms by up to 250% and that of flies by up to 85%. The insulin/IGF1 signaling pathway is highly conserved between worms, flies, and mammals. In aging, the most important AKT/PKB substrates are the class O of forkhead box transcription factors (FoxOs). Upon insulin activation, AKT/PKBmediated phosphorylation of FoxOs results in sequestration of FoxO in the cytoplasm. Mutations that decrease the insulin/IGF1 signaling pathway reduce the phosphorylation of FoxOs, resulting in their nuclear translocation. In the nucleus, FoxOs modulate the expression of genes that increase lifespan. In flies, transgenic overexpression of FoxO in the fat body is sufficient to prolong lifespan. Tissue-specific FoxO overexpression in the cerebral fat body also increases the levels of FoxO in the nuclei of peripheral fat body cells. The relationship between insulin signaling and aging is more complicated in mammals than in lower metazoans. Due to the essential role of insulin in the regulation of carbohydrate and lipid metabolism in mammals, dramatic reductions in insulin signaling lead to metabolic disarray and shortened lifespan. Female mice that lack one functional copy of the IGF-1 receptor are long lived, have a normal size, and show normal fertility and feeding behavior. Caloric restriction in mammals is associated with decreased insulin levels and normal or low glucose levels, which also suggests a positive

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correlation between insulin sensitivity and longevity.

References Pan D, Dong J, Zhang Y, Gao X (2004) Tuberous sclerosis complex: from Drosophila to human disease. Trends Cell Biol 14(2):78–85 The Interactive Fly. http://www.sdbonline.org/fly/aimain/ 1aahome.htm White MF (2003) Insulin signaling in health and disease. Science 302(5651):1710–1711 Yang Q, Guan KL (2007) Expanding mTOR signaling. Cell Res 7(8):666–681

Insulin-Like Growth Factor (IGF) Signaling ▶ Insulin Signaling

Insulin-Like Growth Factors Ruslan Novosiadly1 and Derek LeRoith2 1 Department of Cancer Immunobiology, Eli Lilly and Company, New York, NY, USA 2 Division of Endocrinology, Diabetes and Bone Diseases, Mount Sinai School of Medicine, New York, NY, USA

Definition The insulin-like growth factors (IGF-I and IGF-II) are structurally related molecules that play essential roles in the regulation of cell survival, growth, proliferation, differentiation, and metabolism. The IGF family is comprised of (i) ligands (IGF-I, IGF-II, and insulin), (ii) six wellcharacterized high-affinity binding proteins (IGFBP-16), (iii) IGFBP proteases, and (iv) cell surface receptors that mediate the biological functions of IGFs. These transmembrane receptors include the IGF-I receptor (IGF-IR), IGF-II/mannose 6-phosphate receptor (IGF-II/

Insulin-Like Growth Factors

M6PR), ▶ insulin receptor (IR), and insulin receptor-related receptor (IRR). In many tumor cells, the IGF-IR is often upregulated and/or hyperactivated. Furthermore, increased circulating IGF-I levels are considered a significant risk factor for the development of various types of cancers. Although the oncogenic role of IGFs (i.e., their ability to initiate ▶ carcinogenesis) is still under the debate, numerous lines of evidence suggest that these powerful growth factors enhance tumor growth and ▶ progression.

Characteristics IGFs The insulin-like growth factors (IGF-I and IGF-II) are ubiquitously expressed growth factors that are structurally related to insulin. However, in contrast to insulin and other peptide hormones, they are not stored within cells of a specific tissue but are produced by numerous cell types in the body and circulate in approximately 1000-fold higher concentrations than most other known peptide hormones. These properties suggest more universal functions of the IGFs in the body compared to the more specific metabolic role of insulin. The IGFs are critical in a broad range of functions during pre- and postnatal life. In adult tissues, IGFs are important trophic factors that support normal differentiated functions of various tissues and prevent programmed cell death (▶ apoptosis). IGF-I is known as a major regulator of postnatal growth. Most of the circulating IGF-I is produced by the liver, although other tissues are capable of synthesizing this peptide locally. Thus, IGF-I has characteristics of both a circulating hormone and a tissue growth factor. In contrast to IGF-I, IGF-II plays a fundamental role in embryonic and fetal growth, whereas due to ▶ imprinting of the Igf2 gene, its role in postnatal period of life is less important, especially in rodents. IGF Receptors Most of the actions of both IGF-I and IGF-II are mediated via the IGF-IR, which is expressed by virtually all cell types except adult hepatocytes. The IGF-IR and IR belong to the large family of

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▶ receptor tyrosine kinases. The two receptors are structurally related and are composed of two a-subunits localized entirely extracellularly and two b-subunits spanning the membrane and localized primarily intracellularly. Both subunits are linked together by disulfide bonds. They assemble a a2b2 configuration with ligand binding primarily mediated by the a-subunits, which form a binding pocket. Binding of the ligand to the a-subunit leads to conformational changes resulting in stimulation of the b-subunit intrinsic tyrosine kinase activity with subsequent multisite phosphorylation of the b-subunit. The current concept is that insulin and the IGFs act as bivalent ligands; both IGF-IR and IR are capable of binding insulin and IGF-I or -II, though each receptor binds its own ligand with a 100–1000-fold higher affinity than the heterologous peptide. In cells expressing both receptor genes, hybrid insulin/IGF-I receptors can form. The hybrid receptors have ligand specificity profiles more comparable to the IGF-IR than to the IR since they bind IGF-I with an affinity similar to the IGF-IR but insulin with a much lower affinity. Moreover, the IR is also responsible for some of the mitogenic actions of IGF-II. IGF-II is an agonist of the A isoform of the IR. This splice variant of the IR is expressed at high levels in fetal and neoplastic tissues. IRR and hybrid IR/IRR have not yet been extensively studied, and their ability to bind all the different insulin-like peptides as well as their biological significance remains unclear. The IGF-II/M6PR is structurally distinct from the IGF-IR and is actually identical to the cationindependent mannose 6-phosphate receptor, which lacks tyrosine kinase activity and is not considered to have any role in IGF ▶ signal transduction. The IGF-II/M6PR functions as a scavenger receptor and is involved in uptake and degradation of IGF-II. The IGF-II/M6PR is strongly expressed during tissue differentiation and organogenesis, and high levels of the IGF-II/ M6PR were found in fetal tissue, which decline in the late gestation and in the early postnatal period due to genomic imprinting. IGFBPs Unlike insulin, the IGFs are present in the circulation and throughout the extracellular

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compartments almost entirely bound to a family of multifunctional, structurally related, highaffinity IGF-binding proteins (IGFBPs), which can modulate biological effects of the IGFs. To date, six IGFBPs with high affinity have been cloned and sequenced. All share structural homology with each other and specifically bind the IGFs. They differ in molecular mass, binding affinities for the IGFs, posttranslational modifications such as phosphorylation and glycosylation, as well as susceptibility to proteolysis. The IGFBPs act as carrier proteins in plasma, control the efflux of the IGFs from the vascular space, and prolong half-lives of the IGFs. They regulate metabolic clearance and tissue- and cell-specific localization of the IGFs thereby modulating their biological actions in a negative or a positive manner. Finally, some IGFBPs also have intrinsic bioactivities that are independent of the IGFs (Fig. 1). Mechanisms The biological effects of IGFs are mainly mediated by the IGF-IR and two principal signaling pathways including mitogen-activated protein kinase (MAPK) pathway that plays a pivotal role in cell growth and proliferation and phosphatidylinositol-triphosphate kinase (PI3K) pathway, which is mainly involved in mediating the metabolic, antiapoptotic, and other more differentiated effects of IGF-I. Upon ligand binding and receptor autophosphorylation, the IGF-IR recruits and phosphorylates several adaptor proteins including Shc and members of the insulin receptor substrate (IRS) family of proteins. They bring together and coordinate the activity of other signaling intermediates, finally resulting in the activation of the MAPK and PI3K pathways. Typically, the MAPK pathway is initiated by the recruitment of growth factor receptor-bound 2 protein (Grb2) that via the guanine nucleotide exchange factor Son of Sevenless (SOS) stimulates the activity of the GTPases Ras and Rac, which, in turn, through the sequential phosphorylation of certain kinases finally lead to activation of terminal MAP kinases ERK1/ERK2, JNK, and p38 kinase. Although JNK and p38 kinase are primarily activated by environmental stressors, several lines of evidence suggest that they can

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also be activated in response to IGFs. The activated MAP kinases phosphorylate several important cytoplasmic substrates and also translocate to the nucleus where they phosphorylate transcription factors leading to immediate early gene induction followed by progression of the cell cycle. Alternatively, signal transduction through the PI3K pathway results in the activation of protein kinase B also known as Akt, which is known to block apoptosis by phosphorylating numerous cellular substrates such as Bad, GSK-3, Foxo, Mdm2, CREB, IKK, caspase-9, p21, and p27. The PI3K pathway also activates p70S6 kinase involved in the regulation of ribosome biogenesis as well as some isoforms of protein kinase C, which are capable of potentiating signal transduction through the MAPK pathway. Thus, IGFs induce cell proliferation both by enhancing cell cycle progression and by inhibiting apoptosis. Furthermore, evidence for both direct and indirect interaction between the IGF-IR and other growth regulatory signals has been demonstrated, thereby expanding the traditional view of highly specific IGF-IR/IGF interactions and rendering the IGF-IR central in cellular response. For instance, in many cell types IGF-IR and ▶ epidermal growth factor receptor, family members physically interact and transphosphorylate each other. ▶ Estrogen receptor activation augments the IGF-I response in estrogen receptor-positive MCF-7 mammary carcinoma cells at multiple levels. Moreover, estrogens enhance the tyrosine phosphorylation of the IGF-IR and IRS-1 and eventually increase expression of cyclins and reduce the level of cdk inhibitors (Fig. 2). Altered Expression of the IGF System Components in Tumor Cells The expression of the components of the IGF system is often altered in malignant cells. In certain tumors, the ▶ imprinting of the Igf2 gene is lost, and this results in increased IGF-II gene expression. In general, IGF-II is more commonly expressed by tumors than IGF-I, although increased IGF-I expression has been found in numerous tumors as well. The IGF-IR is often upregulated or hyperactivated in tumor cells. The expression of

Insulin-Like Growth Factors

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Insulin-Like Growth Factors, Fig. 1 The IGF family is comprised of (i) ligands (IGF-I, IGF-II, and insulin), (ii) IGF-binding proteins (IGFBPs), (iii) IGFBP proteases, and (iv) cell surface receptors. IGFBPs act as carrier proteins in plasma, control the efflux of the IGFs from the vascular space, prolong half-lives of the IGFs, regulate their metabolic clearance, provide tissue- and cell-specific localization of the IGFs, and modulate biological actions of the IGFs, and some also have intrinsic bioactivities that are IGF independent. The IGFs can be released from the IGF/IGFBP complexes by the action of IGFBP proteases. The insulin receptor (IR), insulin-like growth factor I receptor (IGF-IR), and insulin receptor-related receptor (IRR) are heterotetrameric complexes composed of

extracellular a-subunits that bind the ligands and b-subunits that anchor the receptor in the membrane and contain tyrosine kinase activity in their cytoplasmic domains. Hybrids consist of a hemireceptor from both IR and IGF-IR. The IGF-II/M6PR is not structurally related to the IGF-IR and IR or the IRR, having a short cytoplasmic tail and no tyrosine kinase activity. IR is responsible for metabolic effects, whereas IGF-IR and hybrid IR/IGF-IR are responsible for cell survival, growth, proliferation, and differentiation. The insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/M6PR) functions as scavenger receptor and is responsible for uptake and degradation of the IGFs. This receptor is not considered to have any role in IGF signaling

the IGF-IR is regulated by tumor suppressors and growth factors in a negative and positive manner, respectively. Tumor suppressors such as ▶ Wilms tumor 1 (WT1) and p53 bind to the Igf1r gene promoter and inhibit receptor gene expression. Mutations of these genes occur in various tumors and paradoxically enhance the activity of the Igf1r gene promoter. This explains the upregulation of

the IGF-IR gene expression in Wilms tumor (a pediatric kidney tumor) and colon cancer, which is often accompanied by p53 mutations. Both WT1 and p53 also inhibit IGF-II gene expression. By analogy with the IGF-I receptor, IGF-II gene expression is increased when WT1 and TP53 genes are mutated. Thus, the autocrine IGF-IR/IGF-II loop is turned on under these

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Insulin-Like Growth Factors, Fig. 2 Signal transduction cascades initiated by the IGF-IR. Activation of the IGF-IR kinase results in receptor autophosphorylation and tyrosine phosphorylation of several docking proteins such as Shc and insulin receptor substrate (IRS) proteins. Once activated, IRS molecules recruit Src homology 2 (SH2) domain containing molecules such as Grb2 and the p85 subunit of phosphatidylinositol-30 -kinase (PI3-K). Grb2 via SOS stimulates the activity of the GTPases Ras and Rac, which through the phosphorylation of numerous kinases finally lead to the activation of terminal MAP

kinases ERK, JNK, and p38. Activated MAP kinases are translocated to the nucleus where they activate a variety of transcription factors. Alternatively, the binding of the p85 and p110 subunit of PI3-K to IRS proteins generates phospholipids that participate in the activation of 3-phosphoinositide-dependent kinases (PDKs) 1 and 2. In turn, they phosphorylate several targets involved in the regulation of different biological processes including glucose transport, protein synthesis, glycogen synthesis, cell proliferation, and cell survival

circumstances. Basic ▶ fibroblast growth factor and ▶ platelet-derived growth factor are capable of enhancing the IGF-IR gene expression. Since tumors often express these and other growth factors, this could also upregulate the IGF-IR gene expression. Certain ▶ oncogenes can also regulate the IGF-IR at posttranslational level. For instance, ▶ Src augments the phosphorylation state of the IGF-IR, thereby increasing its kinase activity. Neoplastic growth can be also enhanced

by injections of recombinant human IGF-I into mice. In this case, the latency period is shortened and tumor growth is accelerated. This response is particularly enhanced in tumors with higher levels of IGF-IR expression. In contrast to the IGF-IR, the IGF-II/M6PR expression is often decreased or lost in tumor cells. The IGF-II/M6PR possesses properties of a ▶ tumor suppressor gene. Tumor cell growth is inhibited when the IGF-II/M6PR expression is restored and is increased when its

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expression is reduced. In addition to the IGFs and their receptors, the IGFBP expression is also altered in tumor cells. Various IGFBPs are expressed by numerous tumors and often in different combinations. For example, estrogen receptor-positive ▶ breast cancer cells release IGFBP-2, whereas estrogen receptor-negative breast tumors release IGFBP-1 and IGFBP-3. The IGFBP production can be altered by growth factors, steroid hormones, and ▶ cytokines, and these changes in IGFBP levels may alter biological effects of the IGFs on tumor growth and progression. Interestingly, wild-type p53 induces the expression of IGFBP-3 that seems to be critical for the inhibitory function of p53 on cell growth. Furthermore, enhanced IGFBP proteolysis is thought to contribute to carcinogenesis. Numerous IGFBP proteases produced by tumor cells mediate the release of the IGFs from the IGFBP/ IGF complexes that eventually leads to increased IGF-IR stimulation. For instance, prostate cancer cells secrete ▶ prostate-specific antigen (PSA), which exerts IGFBP-3 proteolytic activity thereby enhancing the local bioavailability of free IGFs. Syndrome of Hypoglycemia An emerging clinical syndrome is tumor-induced hypoglycemia. This phenomenon is often seen in terminally ill, poorly nourished patients. In addition, it can be also observed in patients with large mesenchymal tumors in the abdomen or thorax that secrete large quantities of IGF-II. In these patients, IGF-II is not fully processed, and therefore it is poorly bound to the circulating IGFBPs. This allows IGF-II to interact more readily with insulin receptors thereby causing hypoglycemia. Clinically, tumor-induced hypoglycemia can be diagnosed in cancer patients that have normal or elevated circulating IGF-II levels, whereas their insulin, growth hormone (GH), IGF-I, and IGFBP-3 levels are suppressed. These patients usually have a poor prognosis. Surgical excision and ▶ chemotherapy- or radiation therapyinduced reduction of tumor size is palliative, and GH and corticosteroid therapy provides symptomatic relief.

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Clinical Aspects An increasing body of evidence suggests that the IGF system is a promising target for adjuvant anticancer therapy. If chemotherapy inadequately ablates tumor cells, then blockade of the proliferative and antiapoptotic effects of the IGFs may be helpful. The IGF system could potentially be targeted at various levels. Reduction of circulating IGF-I levels can be achieved by GHRH antagonists or somatostatins as well as GH receptor antagonists. In the application of inactive IGF molecules, small-molecule competitive binding antagonists or soluble IGF-I receptors may inhibit binding of endogenous IGFs to the receptor, thereby abrogating their tumor-promoting effects. The IGF-IR represents another attractive therapeutic target. Approaches that have been tested in preclinical and clinical studies include the use of blocking monoclonal antibodies directed against the extracellular portion of the IGF-IR and ▶ small-molecule drugs that inhibit the tyrosine kinase activity of the IGF-IR. Application of ▶ RNA interference and ▶ antisense therapy to reduce the IGF-IR expression, as well as overexpression of altered or truncated IGF-IR that acts in a dominant-negative manner, represents additional approaches that have been effective in laboratory studies.

Cross-References ▶ Antisense DNA Therapy ▶ Apoptosis ▶ Breast Cancer ▶ Carcinogenesis ▶ Chemotherapy ▶ Cytokine ▶ Epidermal Growth Factor Receptor ▶ Estrogen Receptor ▶ Fibroblast Growth Factors ▶ Imprinting ▶ Insulin Receptor ▶ Oncogene ▶ Platelet-Derived Growth Factor ▶ Progression

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▶ Prostate-Specific Antigen ▶ RAS Genes ▶ Receptor Tyrosine Kinases ▶ RNA Interference ▶ Signal Transduction ▶ Small Molecule Drugs ▶ Src ▶ Tumor Suppressor Genes ▶ Wilms Tumor

References Khandwala HM, McCutcheon IE, Flyvbjerg A et al (2000) The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocrine Rev 21:215–244 LeRoith D, Werner H, Beitner-Johnson D et al (1995) Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine Rev 16:143–163 Pollak MN, Schernhammer ES, Hankinson SE (2004) Insulin-like growth factors and neoplasia. Nat Rev Cancer 4:505–518 Rubin R, Baserga R (1995) Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest 73:311–331 Samani AA, Yakar S, LeRoith D et al (2007) The role of the IGF system in cancer growth and metastasis; overview and recent insights. Endocrine Rev 28:20–47

See Also (2012) AKT. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163 (2012) Dominant Negative. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1150. doi:10.1007/978-3-642-16483-5_1705 (2012) MAPK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) Mitogen-Activated Protein Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2336. doi:10.1007/978-3-642-16483-5_3770 (2012) Monoclonal Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Palliative. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2759. doi:10.1007/978-3-642-16483-5_4350 (2012) Radiation Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3144. doi:10.1007/978-3-642-16483-5_4907 (2012) Shc. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3401. doi:10.1007/978-3-642-16483-5_5284

Insulinoma

Insulinoma Boaz Hirshberg Cardiovascular and Metabolic Diseases, Pfizer Inc, Groton, CT, USA

Synonyms b-cell tumor of the islets

Definition Insulinomas are functioning insulin-producing tumors of the pancreatic islets of Langerhans, resulting in hypoglycemia.

Characteristics Insulinomas arise from the b-cell of the pancreatic islets. The nonregulated secretion of insulin from these tumor cells into the blood stream results in fasting hypoglycemia. Insulinomas are relatively rare, with approximately four cases per million person-years. However, they are the most common tumor of the pancreatic islets. Insulinomas may appear at any age, but the majority appears in the fifth decade. Insulinomas are evenly distributed along the pancreas. Insulinomas, associated with ▶ multiple endocrine neoplasia type 1 (MEN1), tend to appear one decade earlier. Insulinomas are usually solitary and their localization is even along the pancreas, exceptions are those associated with MEN1, where multiple tumors are the rule. Diagnosis The presence of Whipple Triad (hypoglycemia and neuroglycopenic symptoms that are corrected by the administration of carbohydrate) is the hallmark of the diagnosis of insulinoma. Fasting, therefore, is the major maneuver used in the diagnosis of insulinoma and has two important purposes. The first goal is to document hypoglycemia and its relationship to the patient’s symptoms, and

Insulinoma

the second is to demonstrate inappropriate insulin concentrations in the face of hypoglycemia. The prolonged (48–72 h) fast is the gold standard for the diagnosis of hypoglycemia. This study should be conducted under supervised conditions (i.e., while hospitalized). Diagnosis of insulinoma has been centered on the 72-h fast that was introduced long before to measure insulin or insulin-related components. Now the insulin and proinsulin measurements are widely available, all of the necessary information from a fast can be derived in the first 48 h. Thus, the 48-h fast has become the new standard. The diagnosis is based on detectable insulin levels (6 mU/ml), detectable C-peptide, and elevated proinsulin levels, when the patient has symptoms of hypoglycemia and glucose levels 45 years, male subjects, alcohol use, and drinking water from a well were significantly associated with IM progression.

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Etiology Related to Gastric Cancer

Gastric adenocarcinomas can be classified into two: intestinal (well differentiated) and diffuse (poorly differentiated). IM is involved in the genesis of intestinal type gastric cancer, but not in that of diffuse type. Intestinal type tumors are causally related to H. pylori infection. Chronic gastritis is induced in the normal gastric mucosa by infection with H. pylori. At this stage, the gastric mucosa can be reversed to normal by H. pylori eradication. When inflammation is continued, the gastric mucosa undergoes a sequence of changes that leads to atrophic gastritis and IM. Once IM is induced in the gastric mucosa, it does not appear to regress to normal even when H. pylori is eradicated. A Chinese study showed that eradication of H. pylori significantly decreased the development of gastric cancer in H. pylori carriers without IM but that it failed to prevent gastric carcinogenesis in patients harboring IM at entry, indicating that IM may be the breakpoint of gastric carcinogenesis. The ratio of gastric carcinogenesis in patients with IM is small. In a Japanese study for 8 years, gastric cancer developed in 2.9% of the H. pylori-infected and none of the uninfected subjects. In a Dutch nationwide histopathology registry, the annual incidence of gastric cancer was 0.25% for patients with IM, 0.6% for those with mild-to-moderate dysplasia, and 6% for those with severe dysplasia within 5 years after diagnosis. Thus other genetic and epigenetic changes are necessary for IM to progress to gastric cancer, including APC mutations, TGFbRII mutations, promoter methylation of MLH1 and RUNX3 genes, mutation and/or loss of TP53. Molecular Biology

CDX2 ParaHox gene is expressed only in the intestinal area in the gut endoderm in normal development but is expressed in IM and Barrett esophagus. Ectopic expression of Cdx2 in gastric epithelial cells is sufficient to induce IM in the stomach in transgenic mice. Moreover, loss of Cdx2 function or its conditional deletion in the intestine results in replacement of intestinal epithelial cells with stratified squamous cells. These results suggest that expression of CDX2 is

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essential for the development of IM in gastric epithelial cells. What induces expression of CDX2 in the gastric epithelial cells? RUNX3 is a tumor suppressor originally identified in gastric cancer, and it is inactivated by promoter hypermethylation or protein mislocalization in most gastric cancer cases. RUNX3 not only regulates TGF-b/BMP signaling pathway by directly binding to SMADs but also attenuates oncogenic Wnt signaling through ternary complex formation with b-catenin/TCFs. CDX2 is upregulated by b-catenin/TCFs, and RUNX3 indirectly represses the CDX2 promoter activity by attenuating b-catenin/TCFs. Therefore, RUNX3 deficiency, coupled with a consequent increase of b-catenin/TCF transactivation, leads to activation of the CDX2 promoter to induce IM. Actually, RUNX3 expression is significantly decreased in IM compared with the surrounding normal gastric mucosa. Epigenetic analysis showed that inflammation induced by H. pylori infection causes aberrant DNA methylation of tumor suppressor genes, including RUNX3, CDKN2A, CDH1, and MLH1 in the gastric mucosa, and this connection between infection-inflammation-epigenetic alterations is deeply involved in gastric carcinogenesis. IM may be a sign where RUNX3 tumor suppressor gene is inactivated by promoter hypermethylation induced by H. pylori infection in the gastric mucosa.

Intestinal Metaplasia

be good to reduce the risk for gastric carcinogenesis, but it may increase the risk for esophageal carcinogenesis. Barrett Esophagus Barrett esophagus (BE; synonyms: Barrett metaplasia, columnar-lined esophagus) is a condition where the stratified squamous epithelium lining the esophagus is replaced by a simple columnar one with goblet cells that is usually found in the intestines. In BE, junctional-cardiac type and gastric-fundic type mucosae are sometimes found in addition of intestinal type mucosa, which exhibits histological features of incomplete IM of the gastric mucosa, and it is rare that matured absorptive cells with well-defined brush border are found there. BE is considered a premalignant condition that predisposes to the development of esophageal adenocarcinoma. BE is classified into short segment (10% mast cells in peripheral blood, or >20% mast cells in bone marrow aspirate smears. Very rare Local destructive (sarcoma-like) growth of a tumor consisting of highly atypical immature mast cells, similar in morphology to those seen in MCL. Very rare Benign extramedullary mast cell tumor in extracutaneous tissue such as the lungs. Mast cells are mature with nonaggressive growth pattern. Very rare

may accelerate osteoporosis. Other tests such as gastrointestinal endoscopies, skeletal X-rays, and imaging of the chest and abdomen may be necessary depending on the presenting symptoms. Classification Mastocytosis is a hematopoietic neoplasm classified according to the World Health Organization into seven categories on the basis of the site of involvement and aggressiveness of the disease (Table 2). Therapy Treatments for mastocytosis can be divided into two broad categories: (i) those intended to control

Prognosis Good. Resolves or improves by adolescence in most children Good. Inadequate control of mast cell mediator symptoms may lead to poor quality of life

Poor. Determined by the progression of non-mast cell hematologic disorder Poor. Average survival 3–5 years

Poor. Average survival 3–9 months

Poor. Average survival 3–9 months

Good

symptoms due to mediators released from mast cells and (ii) those intended to reduce the mast cell burden. Treatment of mastocytosis aimed at controlling mast cell mediator symptoms is offered in all categories of mastocytosis. Mast cell cytoreductive therapies are generally reserved for patients with aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and mast cell sarcoma (MCS). However, none of the current cytoreductive approaches has resulted in the cure of mastocytosis. Because there is no therapy proven to cure mast cell disease, symptom control is an important aspect of the treatment strategy. Symptom control may be accomplished through the use of

Mastocytosis

antihistamines (H1 and H2 receptor blockers), antagonists to ▶ leukotrienes, and mast cell stabilizers such as sodium cromoglycate. Patients with malabsorption, ▶ ascites, or recurrent anaphylactoid (syncopal) episodes are generally managed with glucocorticoids. Calcium and vitamin D supplementation and ▶ bisphosphonates are useful in the treatment of patients with osteoporosis. Self-injectable epinephrine should be prescribed to patients with life-threatening episodes of generalized mast cell mediator release resulting in cardiovascular collapse or respiratory compromise. The avoidance of known triggers of mast cell mediator release is an important nonmedical management strategy. The potential of certain medications to stimulate mast cell mediator release should be considered in the overall medical management of the patient. These medications include, but are not limited to, opioid analgesics, nonsteroidal anti-inflammatory drugs, muscle relaxants, general anesthetics, and medications that were not tolerated by the patient in the past. Despite symptomatic treatment with available anti-mediator drugs, some patients continue to have a poor quality of life, probably because of the drugs’ inability to block all mast cell mediators. Patients with ASM, MCL, and MCS and selected patients with indolent systemic mastocytosis (ISM) who have a high mast cell burden and/or poor quality of life may be candidates for several approaches that aim to control the mast cell burden. ▶ Interferon-alpha (IFN-a) has been the subject of most of the research in this area. Due to rarity of the disease, however, most of the reports on the use of IFN-a (with or without prednisolone) are limited in the number of patients reported. For example, a review of the literature identified 14 well-documented patients with aggressive mast cell disease treated with IFN-a (with or without prednisolone); three (21%) had a major response, with dissolution of signs and symptoms of the disease, and five patients (36%) had a partial response. ▶ Clinical trial experience with IFN-a is less favorable, however. The largest phase II trial of IFN-a in 20 adult patients with systemic mastocytosis resulted in

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seven (35%) partial responses and no major response. Although given primarily to patients with aggressive forms of mastocytosis, IFN-a may also be considered in patients with ISM when they present with severe osteoporosis or when symptomatic therapy fails to control anaphylactic episodes. Patients with ISM who have a high mast cell burden and progressive disease (i.e., involvement of the organs but with preserved organ function) may also benefit from biological cytoreductive therapy with IFN-a. Patients with systemic mastocytosis with associated clonal hematological non-mast cell lineage disease (SM-AHNMD) may have an indolent or aggressive mast cell disease component independent of the AHNMD, and therapy depends on the severity of the individual disorders. In most cases, treatment of the latter usually takes precedence and should follow standard guidelines for the associated hematologic disease. When the SM component is clinically more prominent, therapy with IFN-a (with or without the addition of corticosteroids) or chemotherapy can be beneficial. Patients with ASM have poor quality of life and shortened life expectancy and are therefore treated with cytoreductive therapy (usually IFN-a  corticosteroids as first-line therapy). Experience suggests that there exists a subpopulation of patients with ASM who respond to 2-chlorodeoxyadenosine (▶ cladribine; 2-CDA). One report of four patients with ASM treated with cladribine noted a major response in two patients and a partial response in a third patient. Another report documented improvement in symptoms (partial response) in 10 of 10 patients treated with cladribine, although no major responses were observed. Some patients with ASM may benefit from hematopoietic stem cell transplantation. Patients with MCL traditionally have been treated with polychemotherapy regimens similar to those employed in patients with acute myeloid leukemia. While these modalities can render significant mast cell reduction and symptom control in some patients, responses are transient and the prognosis remains very poor. The discovery of activating point mutations near the activation loop of the KIT tyrosine kinase

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domain has driven investigative efforts to identify suitable drugs that inhibit this target. The therapeutic potential of many target-specific small molecules in systemic mastocytosis, including ▶ imatinib, dasatinib, and ▶ nilotinib, is currently being evaluated in clinical trials. The first such medication to be tried in patients with systemic mastocytosis was imatinib mesylate. Imatinib is a potent competitive inhibitor of Abl, ▶ plateletderived growth factor receptor (PDGFR), and KIT tyrosine kinases and is the standard first-line therapy for chronic myeloid leukemia associated with the ▶ BCR-ABL oncoprotein. Because of its inhibitory effect on wild-type (non-mutated) KIT, some investigators have utilized imatinib mesylate in patients with systemic mastocytosis, and responses were seen in selected patients. Unfortunately, the mutated D816V KIT found in the great majority of systemic mastocytosis patients is not sensitive to imatinib, as shown by in vitro laboratory studies. Therefore, the initial high expectations for imatinib treatment have diminished significantly. A review of all experience with imatinib confirmed, however, its potential to improve signs and symptoms of the disease in some patients. This led the US Food and Drug Administration to approve imatinib in the USA, as therapy for adult patients with ASM without the D816V KIT mutation or with unknown KIT mutational status. Another indication for therapy with imatinib is systemic mastocytosis associated with chronic eosinophilic leukemia characterized by the presence of the FIP1L1-PDGFRa ▶ fusion gene. Approximately 20% of cases of systemic mastocytosis present with persistent peripheral blood eosinophilia, and clonality can be demonstrated in a significant proportion of them. Some of these cases express the imatinib-sensitive FIP1L1-PDGFRa oncogene, which results from an interstitial deletion of chromosome 4q12, leading to the constitutive activation of the PDGFRa tyrosine kinase. For this subset of patients, imatinib must be considered first-line therapy, as it eliminates the disease in the great majority of cases. Other targets for therapy that are involved in neoplastic mast cell survival and activation are currently being investigated.

Mastzellen

References Butterfield JH (2006) Systemic mastocytosis: clinical manifestations and differential diagnosis. Immunol Allergy Clin North Am 26:487–513. http://dx.doi.org/10.1016/ j.iac.2006.05.006 Escribano L, Akin C, Castells M et al (2006) Current options in the treatment of mast cell mediator-related symptoms in mastocytosis. Inflamm Allergy Drug Targets 5:61–77. http://dx.doi.org/10.2174/ 187152806775269303 Pardanani A, Akin C, Valent P (2006) Pathogenesis, clinical features, and treatment advances in mastocytosis. Best Pract Res Clin Haematol 19:595–615. http://dx. doi.org/10.1016/j.beha.2005.07.010 Quintas-Cardama A, Aribi A, Cortes J et al (2006) Novel approaches in the treatment of systemic mastocytosis. Cancer 107:1429–1439. http://dx.doi.org/10.1002/ cncr.22187 Robyn J, Metcalfe DD (2006) Systemic mastocytosis. Adv Immunol 89:169–243. http://dx.doi.org/10.1016/ s0065-2776(05)89005-4

Mastzellen ▶ Mast Cells

Matriptase: Epithin, MT-SP1, Suppression of Tumorigenicity 14 ▶ Serine Proteases (Type II) Spanning the Plasma Membrane

Matriptase-2 (TMPRSS6) ▶ Serine Proteases (Type II) Spanning the Plasma Membrane

Matriptase-3 (TMPRSS7) ▶ Serine Proteases (Type II) Spanning the Plasma Membrane

Matrix Metalloproteinases

Matrix Metalloproteinase Inhibitor ▶ RECK Glycoprotein

Matrix Metalloproteinase-3 ▶ Stromelysin-1

Matrix Metalloproteinases M. Sharon Stack Northwestern University Medical School, Chicago, IL, USA

Synonyms Matrixins; MMPs

Definition The matrix metalloproteinases (MMPs), or matrixins, are a family of zinc-dependent metalloendopeptidases that are widely expressed. They cleave a variety of extracellular substrates including, but not limited to, protein and proteoglycan components of the extracellular matrix (ECM). MMPs belong to the metzincin family of endopeptidases, characterized by the presence of three zinc-binding histidine residues within the active site (HexxHxxGxxH). MMPs are believed to catalyze localized hydrolysis of extracellular matrix proteins including collagens, fibronectin, laminins, and proteoglycans, thereby modifying the integrity of the connective tissue.

Characteristics Domain Structure MMPs are synthesized as prepro-enzymes and, following removal of the signal peptide, are

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generally secreted in proenzyme or zymogen form. The latency of the zymogen is maintained by the presence of an unpaired cysteine residue in the propeptide domain (Fig. 1), contained within the conserved sequence PRCG(V/N)PD. This cysteine residue ligates the catalytically essential zinc atom in the enzyme active site, thereby preventing enzymatic activity. Exceptions exist including stromelysin 3 (MMP-11) and membrane-type MMPs (MT-MMPs), which contain a conserved RX(K/R)R sequence within the propeptide and can be activated intracellularly by proprotein processing enzymes of the PACE family, such as furin. In addition to the catalytic zinc atom described above, an additional zinc and two calcium ions provide structural stability. Inserted into the active site of the gelatinases (MMP-2 [gelatinase A] and MMP-9 [gelatinase B]) are a series of three fibronectin type II repeats that function in substrate recognition. The catalytic domain is followed by a short connecting (hinge) region and, with the exception of MMP-7 (matrilysin), a hemopexin-like domain. The role of the hemopexin-like domain is the subject of current investigation, and studies have demonstrated that this region participates in cell surface association (MMP-2) and collagen recognition and cleavage (MMP-1). The MT-MMPs also contain a hydrophobic transmembrane sequence and a short (20 amino acids) cytoplasmic tail. Like the hemopexin domain, the cytoplasmic tail of MT-MMPs is also currently under active investigation to evaluate the potential for association with other cytoplasmic proteins that may regulate MT-MMP localization or function. The domain structures of the MMPs most widely expressed in tumor tissues are summarized in Fig. 1. Zymogen Activation In addition to regulation of MMP gene expression by a number of growth factors and cytokines, the activity of MMPs is stringently regulated by posttranslational mechanisms, predominantly zymogen activation and enzyme/inhibitor binding. Proteolytic activation of proMMP zymogens functions primarily via a cysteine switch

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Matrix Metalloproteinases

Matrilysin (MMP-7) (28,000/19,000) CIV, G,E,FN,LN,proMMP-1, -2, and -9

SH Sig

Pro

Cat

SH Collagenase-1 (MMP-1) Sig (55,000/45,000) CI,G,A stromelysin-1 (MMP-3) (57,000/45,000) CIII,CIV,G,P,FN, proMMP-1,-7,-9,-13

Gelatinase A, B (MMP-2,-9) (72,000/66,000 & 92,000/86,000) CIV,CV,G,E,FN,LN

Membrane-type MMP (MT1-MMP) (66,000/60,000) proMMP-2, proMMP-13, CI,CII,CIII,G,E,FN,VN,P

Pro

Cat

H

HP

SH Sig

Pro

Cat

FNII Cat H

HP

SH Sig

Pro

F

Cat

H

HP

TM

Matrix Metalloproteinases, Fig. 1 Domain structure of selected tumor-associated MMPs. The common name and MMP number are listed, followed by the approximate molecular weight of the latent and active species, respectively. Reported substrates are summarized as follows: CI, CII, CIII, CIV, CV – collagen types I, II, III, IV, and V, respectively; G gelatin, E elastin, FN fibronectin, LN laminin-1, A aggrecan, P proteoglycan, VN vitronectin.

Structural/functional domains are denoted as follows: Sig signal peptide, pro propeptide domain containing unpaired Cys residue denoted by -SH, Cat catalytic domain containing the zinc-binding consensus sequence that chelates the catalytically essential zinc atom (circle), H hinge region, HP hemopexin-like domain, FNII fibronectin type II-like repeats, F furin cleavage site, TM transmembrane and cytoplasmic domain

mechanism (Fig. 2a) and involves cleavage of the propeptide, thus destabilizing the cysteine-zinc interaction and generating catalytically active enzyme. The initial cleavage in many MMP propeptides can be carried out by proteinases of other mechanistic classes, predominantly serine proteinases such as plasmin. Following the initial proteolytic event, the partially active enzymes often undergo further inter- or intramolecular cleavages, resulting in complete removal of the prodomain and generation of fully active enzyme. Proteinase activity is frequently regulated by zymogen activation cascades, such that an initial event will generate an active proteinase that processes a downstream zymogen (Fig. 3). Many studies suggest that coupling of serine and MMP zymogen activation pathways may function to promote pericellular proteolysis during tumor invasion and metastasis.

proMMP-2 (gelatinase A) is an exception to serine proteinase activation of proMMPs. Detailed biochemical studies from a number of laboratories have demonstrated that proMMP-2 is activated on the surface of many neoplastic cells following formation of a trimeric complex containing the transmembrane proteinase MT1-MMP and a molecule of tissue inhibitor of metalloproteinases 2 (TIMP-2). The MT1-MMP/ TIMP-2 complex forms a binding site for proMMP-2, which is then proteolytically processed by a second MT1-MMP molecule (Fig. 2b). This is an interesting example of a reaction in which a proteinase inhibitor (TIMP-2) is also required for zymogen activation. Activation of proMT1-MMP is believed to occur intracellularly, as the zymogen contains a dibasic recognition motif (Arg108-Arg-Lys-Arg) in the propeptide region that may be cleaved by

Matrix Metalloproteinases

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a

b S

Conformational change

Autolysis Furin

S

Limited proteolysis

Autolysis S

ProMT1-MMP Active MT1-MMP TIMP-2

Pro-MMP-2 Active MMP-2

Matrix Metalloproteinases, Fig. 2 Activation of proMMPs. (a) Cysteine switch mechanism for proMMP activation. The latency of MMP zymogens is maintained by an interaction between an unpaired cysteine residue in the propeptide region (S) and the catalytically essential zinc atom (circle). Following conformational perturbation via the action of agents such as SDS, the Cys-Zn2+ interaction is disrupted, leading to autolytic activation characterized by propeptide cleavage. Alternatively, limited proteolysis of the propeptide initiated by serine or metalloproteinases may result in partial propeptide

processing and disruption of the Cys-Zn2+ interaction, followed by autolytic processing to generate the fully active species. (b) Cell surface activation of proMMP-2 by MT1-MMP. ProMT1-MMP is processed intracellularly by proprotein convertases, such as furin, and active enzyme is inserted in the plasma membrane. A ternary “activation complex” form, comprised of MT1-MMP/ TIMP-2/proMMP-2. ProMMP-2 in the “activation complex” is proteolytically processed by a neighboring MT1-MMP molecule, followed by additional autolytic activation and release of the fully active proteinase

proprotein processing enzymes including the PACE family serine proteinase furin.

the nonspecific plasma proteinase inhibitor a-2macroglobulin. Substantial research efforts have also been directed toward generation of synthetic MMP inhibitors to prevent pathologic proteolysis prevalent in diseases such as cancer and arthritis. These compounds contain a zinc-binding functionality such as a hydroxamic acid group coupled with a peptide or peptidomimetic sequence designed to target the inhibitor to the enzyme active site. Most synthetic inhibitors are broad spectrum, blocking the activity of a wide range of MMPs. However, a current alternative strategy in inhibitor design is the generation of more specific compounds that target only a specific subset of MMPs (for example, gelatinase inhibitors that do not alter collagenase function).

Inhibition Aside from zymogen activation, a predominant mechanism for posttranslational control of MMP enzymatic activity is via interaction with protein inhibitors. Tissue inhibitors of metalloproteinases (TIMPs) inhibit MMP activity via the formation of a tight, noncovalent enzyme/inhibitor complex in a 1:1 molar ratio. Four TIMPs (TIMPs 1–4) have been identified, although the function of TIMP-1 and TIMP-2 are the most well characterized. Studies have demonstrated that the amino terminal cysteine residue in TIMPs is required for inhibitory activity, functioning by coordinating the catalytically essential zinc ion. Additional contact sites have been described that may interfere with substrate binding or other proteinase functions, suggesting that the inhibitor presents an extended contact surface to the enzyme. In addition to TIMPs, MMPs are also inhibited by

Substrate Cleavage Although it is widely believed that MMPs function in vivo to process extracellular matrix macromolecules, the precise substrates for the majority of MMPs remain unclear. In vitro

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Matrix Metalloproteinases

Pg

MMP-3 uPAR u-PA

Pm MMP-3

Pg Pm MMP-3

uPAR u-PA

MMP-1

MMP-1

MMP-9

MMP-9

MMP-9

MMP-13

MMP-13

MMP-13

MMP-1

Matrix Metalloproteinases, Fig. 3 Cell surfaceinitiated zymogen activation cascade. Interaction of the serine proteinase uPA with its cell surface glycosyl phosphatidyl inositol-anchored receptor (uPAR) initiates activation of cell-associated plasminogen (Pg), forming the

broad-spectrum serine proteinase plasmin (Pm). Plasmin can initiate propeptide processing of MMP-1, -3, -9, or -13. This is followed by additional autolytic cleavages or MMP-3 processing events, leading to the generation of fully activated MMPs

experiments have demonstrated a wide range of substrates for MMPs including native and denatured collagens, adhesive glycoproteins, such as fibronectin, laminins, and vitronectin, and proteoglycans. Substrate gel electrophoresis, or zymography, is a commonly utilized method to evaluate relative MMP activity levels in tissue extracts or tumor cell conditioned media. Although not quantitative, this method provides a rapid and sensitive initial evaluation of cellular MMP profiles. However, data obtained using zymography are frequently misinterpreted as:

Preliminary results obtained using zymography should be confirmed by other methods such as those based on solution interaction of the MMP and its potential target substrate such as collagen, gelatin, or other matrix macromolecules. Cleavage may then be assessed by electrophoretic examination of reaction products or other methods. Alternatively, a number of quenched fluorescent peptide MMP substrates have been described that have broad utility for continuous kinetic monitoring of peptidase activity. These synthetic substrates have proven useful for comparative evaluation of synthetic MMP inhibitors. A more complex approach for elucidation of MMP substrates in vivo involves generation of mice genetically engineered for a specific MMP deficiency by targeted gene inactivation (MMP “knockouts”). The majority of these animals display relatively mild phenotypes, with the exception of animals deficient in MT1-MMP

• MMP zymogens attain proteolytic activity in the presence of sodium dodecyl sulfate without propeptide cleavage • MMP/TIMP complexes are noncovalent and are thus dissociated during electrophoresis, leading to the potential for overestimation of enzymatic activity

Mature/Immature

expression. These mice display severe connective tissue abnormalities characterized by dwarfism, osteopenia, and arthritis, suggesting that MT1-MMP is required for pericellular collagenolysis in vivo. Control of MMP Gene Expression MMP gene expression is tightly regulated and transcription of many MMP genes is inducible by a wide variety of effectors including hormones, growth factors, cytokines, and tumor promoters. In addition to soluble mediators, studies have demonstrated changes in MMP expression or activity associated with physical factors such as alterations in cell-cell or cell-matrix contact, or changes in cell shape associated with conditions such as physical stress that induce cytoskeletal rearrangement. Clinical Relevance MMPs have been implicated in a wide variety of pathologic processes including arthritis, cardiovascular disease, periodontal disease, emphysema, and cancer. In tumor tissues, enhanced or de novo expression of MMPs is often correlated with disease progression. Conversely, experimental manipulation of TIMP levels has been shown to decrease tumor invasion and metastasis in many experimental models. MMP production in malignant tissues is not necessarily limited to neoplastic cells, as several studies have demonstrated that tumor cells can “recruit” stromally produced enzymes to promote pericellular proteolysis. Furthermore, MMPs have also been implicated in tumor-associated angiogenesis, suggesting an additional mechanism whereby MMPs can contribute to tumor progression. Therapeutic administration of MMP inhibitors is currently under investigation as a potential strategy to prevent tumor invasion and metastasis, and several synthetic MMP inhibitors have demonstrated efficacy in preclinical models of human cancers including colon, breast, and lung carcinoma and melanoma. The observed inhibition of tumor growth may be due in part to inhibition of tumor angiogenesis. In human trials, a decrease in the rate of rise in cancer antigens was observed in patients with prostate, ovarian, colorectal, and

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pancreatic cancer. However treatment was associated with musculoskeletal pain and inflammation. Additional studies are necessary using MMP inhibitors alone or in combination with cytotoxic chemotherapies to assess the efficacy of these compounds in inhibition of tumor growth, metastasis, and angiogenesis.

Cross-References ▶ Tissue Inhibitors of Metalloproteinases

References Benbow U, Brinckerhoff CE (1997) The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15:519–526 Birkedal-Hansen H (1995) Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 7:728–735 Ellerbroek SM, Stack MS (1999) Membrane associated matrix metalloproteinases in metastasis. Bioessays 21:940–949 Holmbeck K, Bianco P, Caterina J et al (1999) MT1-MMPdeficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99:81–92 Nagase H, Woessner JF (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494

Matrixins ▶ Matrix Metalloproteinases

Maturation-Promoting Factor ▶ Cyclin-Dependent Kinases

Mature/Immature ▶ Ovarian Tumors During Childhood and Adolescence

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MBP1, UPH1, H411, EFEMP2, FBLN-4 ▶ Fibulins

Mch5 ▶ Caspase-8

MCJ ▶ Methylation-Controlled J Protein

MCL ▶ Mantle Cell Lymphoma

Mcl Family Shinichi Kitada Burnham Institute for Medical Research, La Jolla, CA, USA

Synonyms Mcl-1; Myeloid cell leukemia sequence 1

Definition Mcl family is a subfamily of the ▶ BCL-2 (B-cell leukemia/lymphoma 2) family proteins involved in the regulation of ▶ apoptosis (programmed cell death), as represented by Mcl-1 (Homo sapiens myeloid cell leukemia sequence 1).

Characteristics Mcl-1, a member of the Bcl-2 family proteins (where Bcl-2 family proteins include both

MBP1, UPH1, H411, EFEMP2, FBLN-4

antiapoptotic and proapoptotic members), was originally cloned as an early response gene upregulated in the ML-1 human myeloblastic leukemia cell line following addition of cytokines. It is a mitochondrial protein of 350 amino acids (its molecular weight is 42/40 kDa) and the gene map to chromosome 1, q21.2. Mcl-1 contains BH-1 (Bcl-2 homology domain 1), BH-2, and BH-3 (one helix only) but not BH-4. Mcl-1 preferentially heterodimerizes with Bak (a proapoptotic member of Bcl-2 family proteins) via BH-3 binding pocket which represents hydrophobic grooves, in contrast to Bcl-2 which preferentially heterodimerizes with Bax. The BH-3 binding pocket is a crucial domain for functions of Mcl-1 and Bcl-2. Imbalances in the levels or activities of these proteins are commonly associated with malignancy, including lymphocytic malignancies such as non-Hodgkin lymphomas where the founding member of the family, Bcl-2, was first discovered nearly two decades ago by virtue of involvement of its encoding gene in chromosomal translocations, including t(14:18) translocations. Analogously, transgenic mice overexpressing Mcl-1 protein in lymphocytes frequently develop lymphomas, attesting to the in vivo importance of this antiapoptotic protein for B-cell neoplasia. The carboxyl regions of Mcl-1 and Bcl-2 share significant sequence homology, as is the case for other members of the Bcl-2 family. Like Bcl-2, Mcl-1 elicits and enhances cell viability under apoptotic conditions. Unlike Bcl-2, Mcl-1 is very labile (its half-life = 2 h), presumably because of its PEST sequences that target it for rapid degradation. In addition, Mcl-1 resembles Bcl-2 in that both proteins are found in the outer membrane of mitochondria. Differentiated cells lose their proliferative capacity, but remain viable and capable of carrying out normal physiological functions. Physiologically, Mcl-1 is important in keeping cells viable during the early induction of differentiation. Regulation A simplified model of cell survival and cell death pathways, involving Mcl-1, is presented in Fig. 1. Cell numbers in the body are governed not only

Mcl Family

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Simplified model of the apoptosis pathway involving Mcl-1 Chemo Rx Fludarabine

Mcl-1

Chlorambucil Ara C

Fodrin

Mitochondria

Rituximab Steroid

Initiator Caspases

Effector Caspases

PARP Lamin

Radiation Rx IAPs XIAP

Growth factor Deprivation Cytokine withdrawal UV irradiation Death stimuli

Core machinery

Apotosis substrates

Mcl Family, Fig. 1 Simplified model of the apoptosis pathway involving Mcl-1

by cell division, which determines the rate of cell production, but also by cell death, which sets the rate of cell loss. Normally, these two processes of cell division and cell death are tightly coupled so that no net increase in cell numbers occurs. However, alterations in the expression or function of genes that control programmed cell death (PCD) can upset this delicate balance, contributing to the expansion of neoplastic cells. As shown in Fig. 1, Mcl-1 is located in the outer membrane of mitochondria and inhibits apoptotic process in core machinery of PCD, involving mitochondria. CLL (▶ chronic lymphocytic leukemia) (B-CLL) is one of the representative diseases caused by defect in programmed cell death primarily as a result of overexpression of Bcl-2. As shown in Fig. 1, chemotherapeutic agents, such as ▶ fludarabine, chlorambucil, cytosine arabinoside (ara C), ▶ rituximab (chimeric anti-CD20 monoclonal antibody), Campath-1H, human leukocyte antigen, and steroid, as well as radiation therapy are believed to induce programmed cell death via mitochondrial pathway, and Mcl-1 inhibits this

core machinery of programmed cell death. Therefore, overexpression of Mcl-1 accounts for chemoresistance commonly seen in patients with CLL and AML (acute myeloblastic leukemia), at least in part. Under physiological conditions, Mcl-1 is regulated at multiple levels. In many hematopoietic cells, growth factors promote cell survival by triggering Mcl-1 transcription and stabilizing Mcl-1 protein. Conversely, cytokine withdrawal and other stress signals such as UV irradiation initiate cell death by promoting Mcl-1 degradation. Clinical Relevance Previous studies documented high levels of antiapoptotic Bcl-2 relative to proapoptotic Bax protein in CLL, with higher Bcl-2:Bax ratios often correlating with aggressive disease or poor response to therapy. The mechanisms responsible for the high levels of Bcl-2 in CLL are poorly understood, but gene hypomethylation has been reported. Occasionally chromosomal translocations activate the Bcl-2 gene in CLL, but these

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are relevant to only a few percent of cases. Thus, the reasons for elevated Bcl-2 expression in CLL are incompletely resolved. Also, Bcl-2:Bax ratios are insufficient to explain differences among CLL patients with respect to progression and chemoresponses. Searches for other prognostic determinants of outcome and response have uncovered Mcl-1 as a candidate predictor of more aggressive disease. Roughly one-half of CLLs contain higher levels of Mcl-1 protein, as measured by immunoblotting using peripheral blood B cells. Though cohort sizes are small, correlations have been noted for higher Mcl-1 and failure to achieve complete remission (CR) following single-agent chemotherapy (either fludarabine or chlorambucil). In fact, in two independent clinical studies conducted in North America, not a single patient with higher Mcl-1 protein achieved complete remission. Thus, while larger cohorts of patients must be analyzed before firm conclusions are reached, higher Mcl-1 protein may represent an indicator of adverse outcome for patients with CLL. Interestingly, comparisons of matched pairs of untreated and relapsed specimens from acute leukemia patients have revealed elevations in Mcl-1 during progression to chemorefractory disease, further supporting a role for this antiapoptotic protein in chemoresistance. The unique requirement for Mcl-1 for survival of lymphoid cells in vivo suggests it might make a good target for treatment of lymphoid malignancies such as CLL and B lymphoma. Strategies for targeting Mcl-1 include reducing its expression using antisense oligonucleotides recognizing its mRNA and kinase inhibitors such as flavopiridol, an experimental drug, derived from a medicinal plant from India, that has been used for centuries in many indigenous medicines; it is a cyclindependent kinase (cdk) inhibitor that can cause cell cycle arrest, induce apoptosis in cancer cells, and inhibit tumor cell growth in vivo. In use are also therapeutic monoclonal antibodies such as anti-CD20 (rituximab), which have been shown to interfere with signaling events necessary for maintaining expression of Mcl-1 protein in CLL, and small-molecule compounds that mimic the inhibitory BH-3 domain of endogenous Mcl-1

Mcl Family

antagonists. Currently, a number of investigators in academia and industries are making efforts to develop small-molecule inhibitors targeted on the BH-3 binding pocket of Mcl-1 and other antiapoptotic Bcl-2 family proteins, and strategies for development of chemical antagonists of Mcl-1 are illustrated in Fig. 2. As shown in this figure, disruption of protein–protein interactions, in particular disruption of Mcl-1–Bak interactions, has been employed as strategies for development of chemical antagonists of Mcl-1. Mcl-1 consists of three major domains, BH-1, BH-2, and BH-3, and Mcl-1 heterodimerizes preferentially with Bak, a proapoptotic member of Bcl-2 family proteins through the BH-3 binding pocket, thereby neutralizing proapoptotic activity of Bak. Smallmolecule antagonist of Mcl-1 binds to BH-3 binding pocket of Mcl-1 and displaces Bak. Initially, Bak is inactive, but Bid or Bim binds to inactive Bak subsequently and induces conformational changes in Bak protein, resulting in activation of Bak, leading to apoptosis. At present, a number of investigators in academia and industries have been screening natural compounds, semisynthetic compounds, and fully synthetic compounds to identify chemical antagonists of Mcl-1. Among them, GX15–070 (Gemin X Pharmaceuticals Inc., Montreal, Canada) is the first small-molecule antagonist of Mcl-1 that has been under clinical development (▶ small-molecule drugs). At present, GX15–070 has entered phase I clinical trials, using patients with CLL (chronic lymphocytic leukemia) and solid tumors, although no results are yet available. Gossypol (from cotton seeds) has been reported to occupy the BH-3 binding site on Bcl-2 and Bcl-XL and tested clinically and shown to have bioactivities against refractory cancers. An enantiomer with superior affinity for Bcl-2, ()-gossypol, termed “AT-101,” is currently being developed by Ascenta Therapeutics Inc. (San Diego, CA) in collaboration with the DTP (Developmental Therapeutics Programs) at the NCI (National Cancer Institute). AT-101 has been shown to be a broad-spectrum inhibitor of Bcl-2 family proteins, including Mcl-1, Bcl-2, Bcl-xl, and Bcl-w. Clinical trials of AT-101 have been currently conducted in patients with CLL and B lymphoma, and antitumor activities of

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Mcl Family, Fig. 2 Disruption of protein–protein interactions reveals strategy for chemical antagonists of Mcl-1

Disruption of protein-protein interactions reveals strategy for chemical antagonists of Mcl-1

“Inactive” bak

BH1

Loop

“Active” bak Bid / Bim Apoptosis

BH3

BH2 Loop

Mcl-1

BH1

Antagonist BH3

BH2

Mcl-1

AT-101 have been reported. Selective inhibition of Mcl-1 and Bcl-2 may represent a useful strategy for leukemia and cancers (chemosensitization), including CLL (chronic lymphocytic leukemia), AML (acute myeloblastic leukemia), and small cell lung cancer. However, conditional gene ablation studies in mice indicate that Mcl-1 is also required for survival of hematopoietic progenitor cells, raising the specter of toxicities that might limit utility.

leukemia: correlations with in vitro and in vivo chemoresponses. Blood 91:3379–3389 Saxena A, Viswanathan S, Moshynska O et al (2004) Mcl-1 and Bcl-2/Bax ratio are associated with treatment response but not with Rai stage in B-cell chronic lymphocytic leukemia. Am J Hematol 75:22–33 Thallinger C, Wolschek MF, Wacheck Vet al (2003) Mcl-1 antisense therapy chemosensitizes human melanoma in a SCID mouse xenotransplantation model. J Invest Dermatol 120:1081–1086 Zhou P, Levy NB, Xie H et al (2001) MCL1 transgenic mice exhibit a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes. Blood 97:3902–3909

Cross-References

See Also

▶ Apoptosis ▶ Bcl2 ▶ Chronic Lymphocytic Leukemia ▶ Fludarabine ▶ Rituximab ▶ Small Molecule Drugs

References Kaufmann S, Karp J, Svingen P et al (1998) Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 91:991–1000 Kitada S, Andersen J, Akar S et al (1998) Expression of apoptosis-regulating proteins in chronic lymphocytic

(2012) BH3 Binding Pocket. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 389. doi:10.1007/978-3-642-16483-5_600 (2012) Campath-1H. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 603. doi:10.1007/978-3-642-16483-5_790 (2012) Heterodimer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1688. doi:10.1007/978-3-642-16483-5_2694 (2012) HLA. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1706. doi:10.1007/978-3-642-16483-5_2765 (2012) PEST Sequence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2828. doi:10.1007/978-3-642-16483-5_4478 (2012) Translocation t(14;18). In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3773. doi:10.1007/978-3-642-16483-5_5946

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Mcl-1 Klaus Podar Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Heidelberg, Germany

Definition Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family and plays an integral role in cell survival and apoptosis. Mcl-1 is characterized by its short half-life, its wide intracellular localization, and the tight regulation of its transcription, translation, and degradation.

Characteristics Structurally, Mcl-1 is unique among the Bcl-2 family. As other antiapoptotic Bcl-2 family proteins, Mcl-1 contains Bcl-2 homology regions (BH1-4 domains) and a transmembrane domain, which confer the ability to heterodimerize with other family members (e.g., proapoptotic proteins Bak and Bax) or to bind to membranes (e.g., cell membrane, mitochondrial membrane, nuclear envelope). Mcl-1 differs from its pro-survival family members in its larger size of 350 residues due to the presence of two polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST). PEST regions are made responsible for Mcl-1 degradation via the proteasome pathway, localization, and phosphorylation, thus providing the mechanistic base for the fine-tuned Mcl-1 protein functions in response to environmental stimuli and the cellular origin. The pro-survival function of Mcl-1 is predominantly mediated by its binding to the proapoptotic effector Bak and the proapoptotic BH3-only molecule Bim. Conversely, release of Bak or Bim from their interaction with Mcl-1 induces apoptosis. Importantly, the BH3-only family members BIM, BID, and PUMA have the ability to bind to all antiapoptotic molecules with similar affinities. In contrast, the BH3-only protein NOXA

Mcl-1

only binds Mcl-1 and BFL-1. A new technology, entitled BH3 profiling, which defines the dependency of tumor cells to Mcl-1 and other antiapoptotic Bcl-2 family members, is under investigation for diagnostic and therapeutic use. Physiologically, Mcl-1 is essential in the survival of hematopoietic stem cells, T and B lymphoid development, the survival of mature granulocytes, the maintenance of long-lived plasma cells, the development of neuronal progenitors, and the survival of mature neurons, as well as the survival of mature cardiomyocytes. Clinical Aspects Mcl-1 is frequently amplified in a variety of solid and hematologic malignancies including cancers of the breast, the ovary, the kidney, the pancreas, the colon, and the lung, as well as melanoma, leukemia, lymphoma, and multiple myeloma. Elevated Mcl-1 levels have been correlated with increased tumor cell survival, growth, and poor prognosis. Importantly, Mcl-1 additionally mediates resistance against widely used anticancer therapies and radiotherapy, as well as against early clinical BH3-mimetic drugs such as ABT-199/GDC-0199/venetoclax that bind into the hydrophobic pocket of the antiapoptotic Bcl-2 proteins (e.g., Bcl-2 or Bcl-xL) and thereby block their ability to interact with proapoptotic proteins. Based on these data, Mcl-1 holds great promise as a high-priority therapeutic target. Indeed, Mcl-1 is the current focus of worldwide cancer-drug development efforts, and a number of Mcl-1 inhibitors are in the cancer-drug development pipeline. Approaches to target Mcl-1 include strategies that block Mcl-1 mRNA synthesis or protein translation, that induce its proteasomal degradation, and that trigger upregulation of NOXA. Moreover, BH3 mimetics, small molecules, which have been designed to inhibit the binding of pro-survival Bcl-2 family members to the BH3 domain of proapoptotic proteins, are the currently most promising agents to target Mcl-1 and other Bcl-2 antiapoptotic proteins. Importantly, due to their functional redundancy, pan-active Bcl-2 inhibitors with at least some activity against Mcl-1 or the combination of Mcl-1 with other Bcl-2/Bcl-xL inhibitors are likely to achieve higher

MCT-1 Oncogene

response rates than targeting one individual member of the Bcl-2 subfamily.

Cross-References ▶ Apoptosis ▶ Bcl2 ▶ Breast Cancer ▶ Lung Cancer ▶ Mcl Family ▶ Multiple Myeloma ▶ Pancreatic Cancer

References Del Gaizo Moore V, Letai A (2013) BH3 profiling – measuring integrated function of the mitochondrial apoptotic pathway to predict cell fate decisions. Cancer Lett 332(2):202 Opferman JT (2015) Attacking cancer’s Achilles heel: antagonism of anti-apoptotic BCL-2 family members. FEBS J Perciavalle RM, Opferman JT (2013) Delving deeper: MCL-1’s contributions to normal and cancer biology. Trends Cell Biol 23(1):22–29 Quinn BA, Dash R, Azab B et al (2011) Targeting Mcl-1 for the therapy of cancer. Exp Opin Investig Drugs 20(10):1397–1411 Thomas LW, Lam C, Edwards SW (2010) Mcl-1; the molecular regulation of protein function. FEBS Lett 584(14):2981–2989

MCPH1 ▶ BRIT1 Gene

MCT-1 Oncogene Ronald B. Gartenhaus and Ari L. Landon The University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD, USA

Definition MCT-1/MCTS1 is an oncogene initially identified in a human T-cell lymphoma and has been shown

2683

to induce cell proliferation, activate survivalrelated pathways, and preferentially translate mRNA with an upstream open reading frame. The MCT-1 protein contains a PUA domain, which acts as an RNA binding domain. MCT-1 interacts with the eIF4F cap-binding complex and modulates the translation profiles of a subset of mRNAs.

Characteristics The MCT-1 oncogene is mapped to chromosome Xq22–24. MCT-1 was shown to interact with the density-regulated protein (DENR/DRP), a protein whose expression is increased in cultured cells grown at high density. DENR has a SUI1 domain, which is involved in recognition of the initiation codon by the eIF2-GTP-Met-tRNAi ternary complex. MCT-1 has been shown to directly bind density-regulated protein (DENR) to form an RNA-binding complex. This heterodimer is analogous to Ligatin (eIF2D) by combining the MCT-1 PUA RNA-binding domain with the DENR containing SUI-1 domain, thus binding to potential start codons within the 5’-UTR of mRNA. Regulation Expression analysis of a variety of normal human tissues revealed low-level ubiquitous expression of MCT-1 message. Levels of MCT-1 protein were shown to be increased after exposure to DNA damaging agents; this increase did not require new protein synthesis. Phosphorylation of MCT-1 protein by p44/p42 ERK1/2 MAPK is critical for stabilization of MCT-1 protein and for its ability to promote cell proliferation. Interestingly MCT-1 was shown to regulate upstream open reading frames, which were able to cause drastic phenotypic changes in drosophila demonstrating a conserved requirement. The knockdown of MCT-1 results in apoptotic cell death of lymphatic and lung cancer. Clinical Relevance A subset of primary diffuse large B-cell lymphomas exhibited significantly elevated levels of MCT-1 protein compared with normal lymphoid

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tissue. The region of chromosome Xq22–24 has been shown to contain amplified DNA in a variety of primary B-cell lymphoid neoplasms using comparative genomic hybridization, suggesting that increased copy number of genes located in this region confers a growth advantage. The signaling pathways that regulate the translational machinery are abnormally active in many human cancers and are causally related to tumor formation in mouse models. The MCT-1 protein interacts with the cap-binding complex and modulates mRNA translational profiles through its binding to DENR. Using a comparative genomics approach, a homologue of MCT-1 was identified in the archaea Pyrococcus abyssi. MCT-1 is the only known oncogene homologue in archaea and highlights that MCT-1 is a highly conserved gene with critical conserved biological function. Further underscoring the functional relevance of MCT-1 is the observation that the human MCT-1 gene can complement in yeast the translation defects observed by the loss of the yeast gene TMA20, having significant homology (48%) to MCT-1. MCT-1 is overexpressed in a majority of primary diffuse large B-cell lymphomas (DLBCLs). Many human malignancies gain a selective advantage through the abnormal activation of this pathway, and its targeting offers a potential selective therapeutic approach in malignancies that exhibit function through MCT-1-mediated protein translation. Ongoing studies have demonstrated DLBCL is reliant upon MCT-1 for survival.

mda-6 uORFs to control tissue growth. Nature 512 (7513):208–212 Shi B, Hsu HL, Evens AM et al (2003) Expression of the candidate MCT-1 oncogene in B- and T-cell lymphoid malignancies. Blood 102(1):297–302 Sinnathamby L, Nandi S, Helen Hui BS et al (2006) MCT1protein interacts with the cap complex and modulates mRNA translational profiles. Cancer Res 66(18):8994– 9001

See Also (2012) Archaea. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 265. doi:10.1007/978-3-642-16483-5_381 (2012) Cap-binding complex. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 640. doi:10.1007/978-3-642-16483-5_826 (2012) Comparative genomics. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 961. doi:10.1007/978-3-642-16483-5_1282 (2012) Comparative genomic hybridization. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 960. doi:10.1007/978-3-642-16483-5_1281 (2012) Initiation codon. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1865. doi:10.1007/978-3-642-16483-5_3059 (2012) SUI1 domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3555. doi:10.1007/978-3-642-16483-5_5556

mda-6 ▶ p21

MDC References Fleischer TC, Weaver CM, McAfee KJ et al (2006) Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. Genes Dev 20(10):1294–1307 Nandi S, Reinert LS, Hachem A et al (2006) Phosphorylation of MCT-1 by p42/44 MAPK is required for its stabilization in response to DNA damage. Oncogene 26(16):2283–2289 Prosniak M, Dierov J, Okami K et al (1998) A novel candidate oncogene, MCT-1, is involved in cell cycle progression. Cancer Res 58(19):4233–4237 Schleich S, Strassburger K, Janiesch PC, Koledachkina T, Miller KK, Haneke K, Cheng YS, Küchler K, Stoecklin G, Duncan KE, Teleman AA (2014). DENR-MCT-1 promotes translation re-initiation downstream of

▶ ADAM Molecules

MDM Genes Fabiola Moretti Institute of Cell Biology and Neurobiology, National Council Research of Italy, Rome, Italy

Synonyms Mouse double minute gene

MDM Genes

2685 p53-binding domain

NLS NES

Zinc finger Caspase cleavage domain signal

Ring finger domain

MDM2

MDM4

MDM Genes, Fig. 1 Scheme of MDM proteins. The main regions are indicated. NLS indicates nuclear localization signal; NES indicates nuclear export signal; caspase

cleavage signal indicates a tetrapeptide that mediates cleavage of the entire protein at this site

Definition

depicted in Fig. 1, is composed of a p53-binding domain at their amino-terminus, with a structure similarity of 54% between the two proteins: a zinc finger motif with 42% of structural similarity whose activity has not been completely defined, and a RING finger domain at their carboxyterminus, with a structure similarity of 53%. Through this last domain, MDM2 and MDM4 interact with each other forming a functional heterodimer. Increasing in vitro and in vivo data demonstrate the relevance of MDM2/MDM4 heterodimerization for proper control of p53 at least during embryo development. Additionally, data suggest the presence of different heterodimer complexes in subcellular sites. The central part of the two proteins is the most divergent region. Despite their similarity, the two proteins are not redundant and play different roles in the cell and during mouse development. Indeed, their loss in knockout mice causes a different lethal phenotype, regardless of the presence of the other gene. Notably, both mice are viable when p53 is simultaneously deleted, indicating that MDM2 and MDM4 lethality is closely related to the deregulation of p53 activity.

The MDM are two genes, MDM2 and MDM4, found amplified in different human tumors. Their main activity is the binding and regulation of the oncosuppressor p53.

Characteristics The MDM gene family comprises MDM2 and MDM4 (also called MDMX), identified in 1992 and 1997, respectively. The first identified gene, MDM2, derives its name from having been isolated from small, extrachromosomal bodies present in a mouse cell line and called mouse double minute. On the same double minute, MDM1 and MDM3 were also isolated, but they resulted completely unrelated to MDM2. MDM2 and MDM4 show high similarity at the level of gene sequence and structure and may be considered paralogs. Orthologs of MDM2 and MDM4 have been described in different animal species across evolution, from Homo sapiens to the zebra fish, Danio Rerio, to the frog Xenopus laevis and to the lampreys, suggesting an ancient origin of these genes. MDM2 and MDM4 are indeed essential genes for mouse development and viability, as demonstrated by the embryonic lethal phenotype of the mice lacking both alleles of each gene (Mdm2 / or Mdm4 / knockoutKO-mouse). MDM2 and MDM4 have been mainly studied for their ability to bind and regulate the oncosuppressor p53 protein. Their structure,

MDM2 The human ortholog of the MDM2 gene (also indicated as Hdm2 or hMDM2) is located on chromosome 12q13–14. It codes for a protein composed of 491 amino acids, indicated also as MDM2p90. In addition, a short protein, called MDM2p76 (or MDM2p75), derived from the initiation of translation at two internal ATG, has been detected. MDM2p90 is present in all tissues with

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levels varying in response to cellular growth conditions. MDM2 is an E3-type ubiquitin ligase, able to covalently bind ubiquitin moieties to specific targets through its RING finger domain. Multiubiquitin chains are a signal that brings proteins to degradation through the proteasome system. The main target of MDM2 is p53 but other targets have been described, many of which are proapoptotic molecules. Degradative activity of MDM2 independent of its ubiquitination function has been also reported (i.e., toward the oncosuppressor Rb). Biologically, the MDM2 most relevant activities are those exerted toward p53. To date, all studies targeting inactivation of MDM2 in different mouse tissues yielded a severe phenotype, recovered by simultaneous knocking down of p53, thus pointing to an essential role of MDM2 in the regulation of p53. MDM2 function on p53 can be summarized in two kinds of actions: 1. MDM2 controls p53 protein levels through its degradative function. 2. MDM2 inhibits p53 transcriptional activity. This function takes place through different mechanisms: by binding p53 in the same region through which p53 binds basal transcription factors, thereby preventing such interaction; by promoting a modification of p53 (NEDDylation) that inhibits its transcriptional activity; and by ubiquitinating histones surrounding the DNA p53-binding regions and inducing gene silencing. It is still a matter of debate if and which activity prevails over the other. Mouse models have provided evidence that the two activities are differently involved during animal development or cell stress conditions. MDM2 activities not related to p53 regulation have also been described. Many of them confer to MDM2 a further prooncogenic role: degradation of the oncosuppressor Rb and of ▶ E-cadherins (a component of cell–cell junction) and the activation of the E2F1 transcription factor are representatives of these activities. Although these MDM2 activities do not seem to be essential for mouse development and viability,

MDM Genes

they might play a relevant role in tumor promotion. Notably, two independent transgenic studies targeting MDM overexpression in all tissues or specifically in mammary gland have evidenced MDM2 tumorigenic properties independent of p53. On the other hand, MDM2 growth inhibitory activity in normal cells has also been described. In agreement with this function, MDM2 is expressed at high levels in terminally differentiated “growtharrested” tissues such as the brain, muscle, and skin. Several studies have investigated the regulation of MDM2 function. Of particular interest it is its transcriptional up-regulation by p53, that establishes a negative feedback loop between MDM2 and its target p53. When p53 activity raises, levels of MDM2 rise too, and this in turn ensures shutdown of p53 activity, thus limiting the duration of the ▶ stress response. Other proteins that regulate MDM2 activity include (i) kinases and ▶ phosphatases that modify its interactions by phosphorylating or dephosphorylating different MDM2 residues, (ii) proteins that delocalize MDM2 in different cell compartments, and (iii) proteins that inhibit MDM2 degradative activity. Interestingly, some posttranslational modifications trigger MDM2 degradative activity toward itself, thereby favoring an autocontrol of its levels. MDM2 and Human Tumors Direct mutation of the p53 gene that results in abrogation of the p53 oncosuppressive function is present in ~50% of human cancers. Deregulation of p53 is supposed to exist in the remaining 50% of cancers. Alterations of p53 regulators, particularly of the MDM genes, represent a good way to achieve it. Indeed, MDM2 is amplified in many different human tumors that harbor wild-type p53, with the highest frequency in human sarcomas. Analysis of data obtained from 3,889 tumor samples from 28 tumor histotypes revealed an overall MDM2 ▶ amplification frequency of 7%. In addition to gene amplification, overexpression of the protein caused by different molecular mechanisms has also been described in human tumors, indicating that the previous frequency underestimates the incidence of MDM2 deregulation in human

MDM Genes

cancer. Moreover, a polymorphism in the promoter region of MDM2 gene (SNP309) has been identified. This polymorphism causes an increased MDM2 expression that in turn should lead to attenuation of p53 oncosuppressive function. Indeed, a survey of the presence of this polymorphism in different human tumors has shown a significant earlier age of onset of several tumors, both hereditary and sporadic. The genetic background is a strong determinant for MDM2SNP309 activity, as it accelerates tumor formation in a gender-specific and hormonal-dependent manner. Additionally, the MDM2-SNP285 modulates the effect of SNP309, furtehr increasing the complexity of MDM2 in human cancer. Some studies have pointed to the worse prognosis of the tumors harboring p53 mutation and MDM2 overexpression, in respect to the tumors harboring only one of these alterations. These data confirm that MDM2 possesses tumorigenic properties also independently of p53. Despite wide characterization of MDM2 molecular functions, the predictive value of MDM2 overexpression on cancer features is still controversial. It probably relies on additional factors as tumor type and genetic alterations that need to be comparatively evaluated to obtain significant predictive biomarkers. The relevance of the genetic background in the activity of MDM2SNP309 is a good example in this respect. In addition to full-length protein, different forms of MDM2 have been found in human tumors. Some of these forms arise from alternative splicing, others from aberrant splicing of fulllength mRNA. Some of these forms are considered oncogenic, and their activity has been evaluated by in vitro studies. However, the genetic background in which they work seems to strongly affect the biological outcome they cause. Therefore, further studies are necessary to define their function and tumorigenic potential. In agreement with its proto-oncogenic function, different strategies aimed at inhibiting MDM2 have been set up. They can be essentially summarized in two types: 1. Downregulation of its levels by antisense nucleotides and ▶ siRNA.

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2. Molecules that prevent its activity, mainly the binding to p53. To this group belong synthetic peptides and “low-molecular-weight compound” that displace/impair MDM2 binding to p53. This new group includes (i) Nutlin-3a (a cis-imidazoline analog) that disrupts p53–Mdm2 interaction by binding to the p53-binding pocket of MDM2 and (ii) RITA (a furan-derived compound) that binds to p53 and alleviates its inhibition by MDM2. Many of these approaches have shown antitumor activity in in vitro and in vivo human cancer models, used alone or in combination with chemotherapy and radiation therapy and have entered clinical trials. MDM4 Human MDM4 (also called HDMX) is located on chromosome 1q32 and codes for a protein of 490 amino acids. Its mRNA is expressed in all human tissues, with particularly high levels in the brain and thymus. Despite structure similarity with MDM2, MDM4 molecular activities are different from those of MDM2. MDM4 does not function as an E3 ubiquitin ligase in vivo, although, contribution of MDM4 in the MDM2mediated control of p53 is important. Indeed, mouse models have demonstrated that genetic mutation/s that impairs MDM2/MDM4 heterodimerization cause p53-dependent embryonic lethality, indicating that the heterodimer rather than the the monomeric proteins is necessary for proper control of p53 activity at least during embryo development. Conversely, these mutants are compatible with adult life indicating that the requirement of heterodimer activity for p53 proper control is different depending on life stages. Additionally, MDM4 per se inhibits p53-transcriptional function especially the activation of growth arrest genes. Interaction of MDM4 with other proteins has been described too, but the biological relevance is not completely defined. Biologically, the recovery of MDM4 KO mouse lethality by simultaneous p53 deletion clearly indicates an essential function of MDM4 in the regulation of p53 during mouse development. In contrast to MDM2, loss of MDM4 in

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different cell types leads to different phenotypes, in some cases with no apparent defects, indicating that its function is more cell-type specific than that of MDM2. Different studies have concurred to characterize regulation of MDM4. In contrast to MDM2, MDM4 transcriptional levels are not modulated by p53. At present, mainly posttranslational modifications (such as phosphorylation, ubiquitination, and SUMOylation) have been identified that regulate MDM4 stability and activity. Interestingly, MDM4 levels are also controlled by MDM2 during cell stress response, highlighting the complexity of the p53–MDM2–MDM4 interplay. MDM4 and Human Tumors In analogy to MDM2, amplification of MDM4 gene in human tumors characterized by wildtype p53 has been observed. Particullarly, its overexpression has been reported in gliomas, in breast carcinomas and in pediatric retinoblastoma with different percentages. Overexpression has been observed in melanoma, lung and colon tumors too. These data concur to define MDM4 as a proto-oncogene, similar to MDM2. However, some studies reported downregulation of MDM4 associated with advanced stage or therapy-resistant tumors, leading to the hypothesis that the presence of MDM4 might be detrimental to tumor progression. In analogy to MDM2, splicing forms of MDM4 deriving from alternative or aberrant splicing events have been described, and the oncogenic properties of these spliced forms have been shown. Their presence coexists with that of the full-length protein, raising the question of the extent to which gene amplification contributes to their expression and of their relevance with respect to full-length protein. Similarly to MDM2, strategies aimed at counteracting MDM4 inhibitory function and reactivating p53 oncosuppression have been proposed. However, despite structural similarity between the MDM2 and MDM4 p53-binding domain, majority of the strategies that work with MDM2 appear to be not effective with MDM4, indicating the need to search for optimal MDM4

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antagonists. Some works have reported the finding of double MDM2/MDM4 inhibitors acting on the p53 bindng domain or on the MDM2/MDM4 interaction interface, thus impairing the heterodimer function.

Cross-References ▶ Amplification ▶ Clinical Trial ▶ E-Cadherin ▶ Phosphatase ▶ SiRNA ▶ Stress Response

References Burgess A, Chia KM, Haupt S, Thomas D, Haupt Y, Lim E (2016) Clinical overview of MDM2/X-targeted therapies. Front Oncol 6:1–7 Khoo KH, Verma CS, Lane DP (2014) Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov 13:217–236 Marine JC, Francoz S, Martens M et al (2006) Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ 13:927–934 Moretti F (2016) Novel insights about the MDM2/MDM4 heterodimer. Mol Cell Oncol 3:e1066923 Tollini LA, Zhang Y (2012) p53 Regulation Goes LiveMdm2 and MdmX Co-Star: Lessons Learned from Mouse Modeling Genes Cancer 3:219–225 Onel K, Cordon-Cardo C (2004) MDM2 and prognosis. Mol Cancer Res 2:1–8.

See Also (2012) Alternative RNA Splicing. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 148. doi:10.1007/978-3-642-16483-5_212 (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 408409. doi:10.1007/978-3-642-16483-5_6601 (2012) Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1943. doi:10.1007/978-3-642-16483-5_3217 (2012) Neddylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2469. doi:10.1007/978-3-642-16483-5_3995 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Polymorphism. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 29542955. doi:10.1007/978-3-642-16483-5_4673

MDM2 (2012) RING Finger Domain. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3309. doi:10.1007/978-3-642-16483-5_5108 (2012) Sumoylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3562. doi:10.1007/978-3-642-16483-5_5572 (2012) Transgenic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3763. doi:10.1007/978-3-642-16483-5_5919 (2012) Ubiquitin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3825. doi:10.1007/978-3-642-16483-5_6083 (2012) Zinc-Finger Proteins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3977. doi:10.1007/978-3-642-16483-5_6299

MDM2 David W. Meek Division of Cancer Research, Jacqui Wood Cancer Centre/CRC, University of Dundee, Dundee, UK

Definition The protein human homologue of mouse double minute 2 (MDM2) was first identified as the product of the MDM2 gene amplified in transformed murine cells. It is a p53-binding nuclear protein that antagonizes and downregulates p53 activity. The gene maps to 12q14–q15 and is amplified in certain human tumors, including sarcomas, glioblastomas, and astrocytomas.

Characteristics Discovery and Significance MDM2 was discovered by Donna George’s laboratory as a gene that is amplified within doubleminute chromosomes in 3T3DM cells and encodes a cellular transforming activity that promotes tumorigenicity in xenografts. Its association with p53 was first observed in immunoprecipitation analyses of p53 as a coprecipitating protein migrating with an apparent molecular weight of 95 kDa. Subsequently, it was shown that overexpression of MDM2 leads to increased

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degradation of p53 via the proteasome, thereby establishing its role as a crucial regulator of p53 levels. Function MDM2 is a ubiquitin E3 ligase of the RING (really interesting new gene) finger type. It has several established biological functions and targets in the cell. Its principal function is the regulation (inhibition) of the p53 tumor suppressor protein which it controls through several coordinated activities. These are: • Mediating monoand subsequently polyubiquitylation of p53, leading to degradation of p53 by the 26S proteasome • Exporting p53 to the cytoplasm • Targeting of p53 to the proteasome • Inducing a conformational change in p53 that blocks the site-specific DNA-binding function of p53 • Targeting histone deacetylase activity toward p53 leading to deacetylation of C-terminal lysine residues in p53 that are the major targets of ubiquitylation • Mediating the modification of p53 by NEDD8 and by SUMO-1, SUMO-2, and SUMO-3 In contrast to these inhibitory activities, there are also reports that under conditions of cellular stress, MDM2 can bind to and stimulate translation of p53 mRNA. The central importance of MDM2 in regulating p53 function in vivo is underpinned by two mouse models. Firstly, Mdm2-null mice die in utero at E5–E6 owing to widespread unregulated p53-dependent apoptosis. Significantly, this lethality can be rescued in a p53-null background, underscoring the critical level of regulation that MDM2 provides. In contrast to the knockout model, overexpression of Mdm2 in mice inhibits p53 function in vivo and increases susceptibility to various cancers including lymphomas, softtissue sarcomas, and osteosarcomas. Structure Full-length human MDM2 (apparent molecular weight 90 kDa) comprises 491 amino acids and

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MDM2

MDM2, Fig. 1 MDM2 interacting partners. MDM2 is shown schematically, highlighting the various important functional domains. The interacting partners shown are those for which the binding site/sites in MDM2 has/have been mapped (in each case represented by the bar/bars)

contains several structured and unstructured regions (there are a number of truncated MDM2 isoforms of 85, 76, and 57 kDa). A schematic representation of its structure is given in Fig. 1 which highlights a number of its salient features. These include: • An N-terminal p53-binding pocket (amino acids 25–110: see below for 3D structure and the development of inhibitors) • Nuclear import (amino acids 161–163 and 179–185) and export (amino acids 191–202) sequences that permit nucleocytoplasmic shuttling • An unstructured central acidic domain (amino acids 215–300) • A zinc finger domain (amino acids 290–335) that mediates interaction with the inhibitory partners, ribosomal proteins L5, L11, and S7 • A C-terminal regulatory region (amino acids 386–429) that is targeted for phosphorylation through DNA damage pathways (see below)

• A RING finger domain (amino acids 436–491) that participates in the transfer of ubiquitin from E2 ligases (e.g., UBCH5) to p53 Three of the structured regions of MDM2 have been resolved: the N-terminal p53-binding pocket (Fig. 2), the zinc finger, and the RING finger domain. The coordinates for these structures are available from the NCBI web site. MDM4 (Also Known as MDMX) The MDM4 protein is structurally related to MDM2. It is a defective ubiquitin ligase and is therefore unable to mediate transfer of ubiquitin to p53. However, it is thought to interact with p53 via its N-terminal p53-binding pocket (which closely resembles that of MDM2) leading to an inhibition of p53-mediated transcription. Importantly, MDM2 is, itself, a relatively weak E3 ligase for p53 but is stimulated by MDM4 through hetero-oligomerization mediated by their respective RING fingers. This hetero-oligomerization

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MDM2, Fig. 2 X-ray crystal structure of the MDM2 N-terminus in association with an N-terminal peptide of p53. MDM2 is shown in blue/ gray. The p53 peptide is shown in salmon with the three important interacting amino acids shown in red. Two separate views are shown. The images were generated using PyMOL from the coordinates in protein database (ID: 1YCR)

also stabilizes MDM2 by blocking its autoubiquitylation. The level of MDM4 is not directly controlled by p53-dependent gene expression (as is the case for MDM2: see below). However, it is regulated by MDM2 via ubiquitylation and degradation. Like MDM2, MDM4 is an essential regulator of p53, and its involvement in controlling p53 activity is strongly supported from experiments with genetically altered mice. Structure/Function Relationships The N-Terminal p53-Binding Pocket

This region of MDM2 (amino acids 25–110) provides a hydrophobic cleft that forms a tight complex with the N-terminus of p53 by accommodating p53 amino acids F19, W23, and L26, respectively (see Fig. 2). Interaction of p53 within this domain of MDM2 leads to ensuing contacts between the two molecules that permit subsequent ubiquitylation of p53 (discussed in detail below). It was previously thought that the binding of p53 to this domain of MDM2 could sterically block to the interaction of p53 with key transcription factors. However, studies with mouse models that express a mutant Mdm2 protein (C462A) that can still bind through its N-terminus to p53, but cannot mediate ubiquitylation, have challenged this idea. Research has found that the C462A Mdm2 mutant can actually stimulate p53dependent transcription but the mechanism for this has not yet been established.

The N-terminal p53-binding domain of MDM2 is also the target of novel drugs aimed at disrupting the p53/MDM2 interaction (see below). There are several published structures of the N-terminal p53-binding domain of MDM2 (e.g., see protein database ID: 1YCR). Nuclear Import and Export Sequences

MDM2 contains two nuclear import sequences and one nuclear export sequence that mediate the ability of MDM2 to shuttle between the nucleus and the cytoplasm. The nuclear export function is essential for the ability of MDM2 to downregulate p53 levels, and its discovery led to a model whereby MDM2 binds to p53 in the nucleus and mediates its export to the cytoplasm where it is degraded. The Acidic Domain

The acidic domain is a largely unstructured region in the central part of the protein (amino acids 215–300) and is thought to play a number of key functions. Firstly, several groups identified the acidic domain as mediating a second point of contact with p53 that is required for the ubiquitylation and degradation of p53 (the so-called p53 ubiquitylation signal). In this model, the binding of the N-terminus of p53 to the cleft in the N-terminal domain of MDM2 (the principal p53-binding site) leads to subsequent association of the acidic domain with the p53 core (site-specific DNA-binding domain). This,

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in turn, permits ubiquitylation of p53 as mediated by the MDM2 RING finger. It was shown that two segments of the acidic domain are required to mediate p53 ubiquitylation and destruction: Thus, amino acids 247–258 are required for ubiquitylation of p53, while these amino acids, together with amino acids 270–274, are required to mediate p53 destruction. These new data support previous findings that the acidic domain comprises distinct but possibly overlapping biochemical functions that are required for p53 turnover. However, it has not yet been demonstrated whether or how these regions within the acidic domain contact p53 and mediate their overlapping effects. The acidic domain is also the interaction site for numerous regulators of MDM2 (see below) including the important ARF (alternative reading frame) tumor suppressor that inhibits MDM2 in response to hyperproliferative signaling (see below). Importantly, it is a focal point for most (if not all) signals that promote the induction of p53.

p53 target activation and apoptosis in response to whole body irradiation that are indistinguishable from wild-type mice. However, agents that induce ribosomal stress (such as low levels of actinomycin D) fail to induce a robust p53 response in these mice. L5/L11 binding therefore constitutes an MDM2-targeted p53 activation pathway that is essentially independent of the DNA damage and ARF pathways. Notably, L11 is a transcriptional target of the Myc oncoprotein. Mice expressing Myc under control of the immunoglobulin enhancer (E-mu-Myc mice) develop aggressive B-cell lymphomas. When crossed with Mdm2C305F/C305F mice lymphoma, development and death are accelerated. The zinc finger therefore offers crucial protection against Myc-induced lymphomagenesis in vivo. It does not, however, accelerate prostatic tumorigenesis induced by inactivation of Rb family members and therefore has a selective role in its responsiveness to tumorassociated stimuli. The structure of the zinc finger has been solved (protein database ID: 2C6B).

The Zinc Finger

The RING Finger

The zinc finger domain plays an important role in regulating MDM2 function specifically in response to changes in ribosome integrity and/or availability. Upon reduction in ribosome biogenesis (e.g., through reduced rRNA expression), excess ribosomal proteins L5 and/or L11 bind directly to the MDM2 zinc finger, inhibit MDM2 function, and thereby induce a p53 response. In this manner, MDM2 regulation is linked to cellular growth (via ribosome number) and is sensitive to “ribosomal stress.” The physiological role of the zinc finger was underscored through the development and analysis of Mdm2C305F/C305F homozygous mutant mice. The C305F mutation within the zinc finger had previously been shown to prevent ribosomal protein binding. Under normal conditions, Mdm2C305F/C305F mice show no obvious phenotypical or developmental differences from wild-type mice nor do they show any altered susceptibility to the development of spontaneous tumors. Fibroblasts from these mice show a normal DNA damage response to various genotoxic agents, and the mice themselves show levels of

The RING finger of MDM2 contains a Cys3His2-Cys3 consensus which coordinates two ions of zinc leading to proper folding of the domain. It is required to mediate the transfer of ubiquitin to p53 from ubiquitin E2 ligases; to mediate the binding of MDM2 to its homologue, MDM4; and to bind to specific RNA sequences. A cysteine to alanine substitution at residue 464 completely abolishes the ability of MDM2 to ubiquitylate p53 in vitro and in transfected cultured cells. The RING finger is also required to mediate p53 degradation. Consistent with this idea, a mouse model expressing Mdm2C462A/C464A fails to suppress p53. Additionally, the RING finger is required for the export of p53 to the cytoplasm. Regulation Autoregulatory Feedback Loop

MDM2 is regulated at several levels. Firstly, MDM2 expression is controlled by p53 as part of a negative feedback loop in which p53 stimulates

MDM2

expression from one of two MDM2 promoters, the “P2” promoter that lies upstream of MDM2 exon 2. The subsequent increased level of MDM2 protein stimulates the ubiquitylation and degradation of its activator, p53, thus maintaining p53 protein levels. In addition to promoting the maintenance of p53 levels homeostatically (there are several other p53 ubiquitin ligases that contribute to the maintenance of its levels), increased MDM2 expression following induction of p53 is thought to be a major player in resetting p53 to preinduction levels. The MDM2 gene has an alternative promoter, “P1,” that is upstream of exon 1. P1 is constitutively active, is independent of p53, and can maintain a low level of MDM2 expression in the absence of p53. Protein Turnover

MDM2 protein levels are also regulated by autoubiquitylation (i.e., where one molecule of MDM2 ubiquitylates another). There is also evidence that MDM2 ubiquitylation and turnover can be mediated by additional ubiquitin ligases such as PCAF (p300/CBP-associated factor).

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compartment where it is physically separated from p53). • Ribosomal proteins L5, L11, L23, and S7: Reduced growth rate or ribosomal stress leads to increased levels of these proteins which then interact with the acidic domain and/or zinc finger of MDM2, with the outcome that they block MDM2-mediated ubiquitylation and degradation of p53. Regulation by Posttranslational Modification

In addition to ubiquitylation, MDM2 is subject to other regulatory posttranslational modifications including SUMOylation mediated by UBC9 and acetylation of K466 and K467 within the RING domain by CREB-binding protein (CBP). Importantly, MDM2 is regulated by multisite phosphorylation mediated by several independent but cooperating pathways. The protein kinases within these pathways that phosphorylate MDM2, together with the target sites and the biochemical/biological outcomes of the phosphorylation events, are summarized in Fig. 3. The key features of MDM2 regulation by phosphorylation are as follows:

Regulation by Protein-Protein Interactions

MDM2 participates in a wide variety of proteinprotein interactions, many of which regulate its function. A list of these regulators is given in Table 1 (interacting proteins for which the binding sites in MDM2 have been mapped are also shown in Fig. 1). Key examples of MDM2-regulating proteins are: • MDM4: a critical activator of MDM2 (discussed above) • The ARF (alternative reading frame) tumor suppressor: This protein is encoded, mainly within an alternative reading frame, within the CDKN2A gene which also encodes the p16INK4A tumor suppressor. ARF expression is induced by a variety of hyperproliferative stimuli including activated-oncogene products. ARF binds to MDM2 within the central acidic domain leading to inhibition of MDM2 ligase activity and, consequently, p53 ubiquitylation. ARF is also responsible for localizing/anchoring MDM2 into the nucleolus (i.e., into a

DNA Damage Signaling In response to DNA damage, MDM2 is phosphorylated by the DNA damage-activated protein kinase, ATM, at several C-terminal sites (mainly S395) and by the related kinase, ATR, at S407 (Fig. 3), leading to a transient attenuation of p53 turnover. Mechanistically, these phosphorylation events inhibit RING oligomerization and E3 ligase activity leading to decreased p53 nuclear export and attenuated degradation. Consistent with such a role, knock-in mice expressing an S394A substitution (S394 is the orthologous human S395 residue in mouse Mdm2), which cannot be phosphorylated, are radioresistant and fail to induce p53. In contrast, mice bearing an S394D substitution (where the presence of a negative charge can mimic constitutive phosphorylation), show a more robust induction of p53 in response to DNA damage, consistent with the idea that S394 (mouse) phosphorylation contributes significantly to p53 induction. Expression of the WIP1 protein phosphatase (wild-type p53-induced phosphatase, also known

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MDM2, Table 1 Brief summary of protein regulators of MDM2 Interacting protein ARF CtBP2 FKBP25 Gankyrin Lats2 Merlin MTBP Nucleophosmin Nucleostemin (GNL3) p19Ras P300 p53 mRNA PCAF PML PSME3 RASSF1A Ribosomal protein L5 Ribosomal protein L11 Ribosomal protein L23 Ribosomal protein S7 Ribosomal protein S14 RYBP TAF1 (TAFII250) Yin Yang 1 (YY1)

Function/outcome Inhibits E3 ligase function, sequesters MDM2 into nucleolus Cooperates with MDM2 to repress p53-mediated transcription Stimulates auto-ubiquitylation and proteasomal degradation of MDM2 Required for inhibition of MDM2-mediated p53 ubiquitylation by Rb Inhibits E3 ligase activity leading to activation of p53 Promotes MDM2 degradation Promotes stabilization of MDM2 and ubiquitylation and degradation of p53 Blocks p53/MDM2 interaction Inhibition of MDM2 Blocks association of MDM2 with p73-beta Required for p53 degradation Binds to RING finger and impairs E3 ligase activity. It also leads to increased p53-mRNA translation Ubiquitylates MDM2 leading to increased turnover Sequesters MDM2 into nucleolus Promotes ubiquitylation- and MDM2-dependent proteasomal degradation of p53 Disrupts interactions between MDM2, DAXX, and the deubiquitinase, HAUSP, thereby enhancing the self-ubiquitin ligase activity of MDM2 Inhibits MDM2-mediated ubiquitylation and degradation of p53 Binds to zinc finger and inhibits MDM2. Cooperates with Myc to induce p53 and suppress lymphomagenesis Inhibits MDM2-induced p53 polyubiquitination and degradation Inhibits MDM2 E3 ligase activity, leading to stabilization of MDM2 and p53 Inhibits E3 ubiquitin ligase activity Decreases MDM2-mediated p53 ubiquitylation, leading to stabilization of p53 Downregulates MDM2 auto-ubiquitylation leading to increased ubiquitylation and degradation of p53 Facilitates MDM2/p53 interaction and p53 ubiquitylation/degradation

as PPM1D) is also induced by p53 in response to DNA damage. WIP1 acts on both p53 itself and on MDM2 where it dephosphorylates Ser395, thereby reversing the effects of ATM/ATR. WIP1 induction is thought to be a major part of the mechanism that attenuates the p53 response and restores homeostasis of the p53/MDM2 feedback loop. Activation of the DNA damage pathway/pathways also leads to hypophosphorylation of several residues within the acidic domain that are essential for MDM2-mediated degradation of p53. Evidence suggests that under normal homeostatic

conditions, these modifications are promoted through the cooperative involvement of protein kinases GSK3-beta, CK1, and CK2. Following DNA damage, stimulation of the DNA-activated protein kinase leads to phosphorylation and activation of protein kinase AKT which, in turn, phosphorylates and inactivates GSK3-beta. These events are thought to underpin the hypophosphorylation of the acidic domain leading to a reduced capacity to degrade p53. It is also possible that DNA damage-induced changes in these modifications may cooperate with those promoted by ATM/ATR.

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MDM2, Fig. 3 Regulation of MDM2 by multisite phosphorylation. MDM2 is shown schematically, as in Fig. 1, together with the approximate positions of the groups of known sites of phosphorylation. The table shows the specific residues phosphorylated within each cluster (black

type), the relevant protein kinase/kinases where known (blue type), the biochemical function regulated by the modification/modifications (red type), and the relevant protein phosphatase/phosphatases where known (green type)

Survival Signaling MDM2 is a target of survival signaling stimuli mediated through the PI3-kinase/AKT pathway. AKT phosphorylates MDM2 at residues flanking its nuclear localization and export sequences (serines 166, 186, 188: see Fig. 2) leading to increased nuclear localization of MDM2 and, consequently, increased association with and degradation of p53. In this

manner, survival signaling, or indeed cancerassociated constitutive activation of AKT signaling, is able to increase the threshold for p53 induction and activation. Reciprocally, it is significantly easier to induce p53-mediated apoptosis following reduced survival signaling. The PIM family of oncogenic protein kinases, which are activated physiologically through increased gene

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expression in response to diverse signals, can also phosphorylate MDM2 at serines 166 and 186 (as for AKT, oncogenic activation of PIM may also lead to hyperactivation of MDM2). Additionally, these residues have been shown to be targeted by mTOR-activated S6K1 in response to DNA damage signals. Serines 166 and 186 can therefore act as a convergent signaling node in MDM2 that mediates, and possibly integrates, diverse signals that set a threshold for p53 induction and activity. From the perspective of potential disease susceptibility, there is also new evidence from mouse studies showing that glucocorticoids elevated during chronic restraint lead to increased phosphorylation of MDM2 at these residues via induction of serum- and glucocorticoid-induced protein kinase (SGK1). The increased MDM2 activity suppresses p53 and contributes to increased tumorigenesis in the mice. This may offer a potential explanation for the epidemiological observation that chronic psychological stress is associated with increased cancer susceptibility. Interaction with Other Proteins: Substrates and Regulators In addition to p53, MDM2 has been shown to interact with a host of other proteins. Many of these proteins are substrates for MDM2-mediated ubiquitylation and/or proteasomal targeting and degradation (see Table 2 and Fig. 1). (Others, as mentioned above and listed in Table 1, are key regulators of MDM2 or can act as cooperating partners.) As a consequence of some of the interactions summarized in Table 2, MDM2 is thought to encompass a number of p53-independent functions in a variety of processes including differentiation, transcriptional regulation, DNA repair, the maintenance of genome stability, and cell cycle control. MDM2 in Cancer MDM2 is abnormally upregulated in a wide range of human cancers through mechanisms including mainly gene amplification but also increased transcription, mRNA stability, and enhanced translation. Elevated MDM2 levels are thought to contribute to cancer development by suppressing

MDM2

p53 levels, thereby increasing the threshold for inducing p53. However, a proportion of malignancies have both overexpressed MDM2 and mutant p53, suggesting that one or more p53-independent function/functions of MDM2 may contribute to cancer development. Indeed, patients with both MDM2 overexpression and p53 mutation often have a worse prognosis than those with either abnormality alone. This is also reflected in animal models for cancer development: For example, a high proportion of mice expressing E-mu-Myc develop lymphomas in which Mdm2 is overexpressed concomitantly with loss or mutation of p53. Moreover, mice overexpressing Mdm2 in a p53-null background have a higher incidence of certain cancers as compared with p53-null mice. There is also evidence that overexpression of Mdm2 in mouse mammary epithelia of mammary gland (in both p53+/+ and / mice) leads to polyploidy. In addition to overexpression, mutations within the MDM2 gene have been observed in several types of human cancers. While the significance of these mutations is not fully understood, many occur within the region encoding the zinc finger. These mutant genes give rise to proteins that are impaired in their ability to interact with ribosomal proteins L5 and L11 and cannot, therefore, mediate the induction of p53 in response to ribosomal stress. These data fit with the idea that signaling through the ribosomal protein/MDM2 pathway may play a role in p53 activation during the development of at least some types of cancer. Genetic factors that affect the level of MDM2 expression in different individuals can also affect the susceptibility to cancer development. For example, the small nucleotide polymorphism, SNP309, represents a T to G substitution in the intronic promoter/enhancer region of MDM2, which generates a potent binding site for the transcription factor, Sp1, leading to increased MDM2 transcription. Appropriate cell lines harboring the G allele of SNP309 show increased levels of MDM2 resulting from increased transcription and an elevated threshold for p53 induction and apoptosis in response to chemotherapeutics. Multiple cancer studies have confirmed that

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MDM2, Table 2 Brief summary of targets/substrates of MDM2 Interacting protein Androgen receptor DHFR DNA polymerase e E2F1 FOXO3a HDAC1 HIF-1-alpha Histone H2B hnRNP K IGF1 receptor JMY MDM4 (MDMX) NBS1 Numb p21 p53 p63 p73 Rb Ribonucleotide reductase small subunit p53R2 Ribosomal protein L26 Ribosomal protein S7 Slug SP1 TBP TFIIE Tip60

Function/outcome Ubiquitylation and turnover Monoubiquitylation and inhibition Activation Stimulation via Rb turnover Ubiquitylation and turnover Recruits HDAC1 to deacetylate p53 Ubiquitylation and proteasomal degradation Monoubiquitylation leading to transcriptional repression Ubiquitylation and turnover Ubiquitylation and turnover Ubiquitylation and turnover Ubiquitylation and turnover, also partner protein required for efficient p53 degradation Inhibition of DNA repair leading to genomic instability Ubiquitylation and turnover Ubiquitylation-independent turnover Inhibition of transcription function, deacetylation, nucleocytoplasmic transport, increased turnover Inhibition of transcription function and increased turnover Inhibition of transcription function Ubiquitylation-independent turnover Ubiquitylation and turnover Drives its polyubiquitylation and proteasomal degradation. L26 therefore is no longer able to bind p53 mRNA and augments its translation Substrate for MDM2 E3 ubiquitin ligase: the ubiquitylated S7 selectively inhibits MDM2 degradation of p53 Ubiquitylation and turnover Inhibits DNA binding and SP1-mediated transcription Repression of transcription Repression of transcription Ubiquitylation and turnover

individuals harboring the G allele of SNP309 show increased risk, early onset, a relative lack of response to therapy, reduced survival, and poorer outcome as compared with individuals harboring the T allele. Additionally, there is a gender influence on susceptibility to various cancers arising from the G allele based on the observation that active estrogen signaling promotes MDM2 expression through the increased Sp1 binding at SNP309. Stratification analysis of cohorts of patients with various tumor types supports the idea that the G allele of SNP309 can accelerate tumorigenesis

specifically in individuals with active genderspecific hormone pathways such as estrogen signaling. Drug Target The principle for developing drugs aimed at inactivating MDM2 is that, by doing so, it should be possible to induce or reactivate p53 in cancers that retain wild-type p53 but have lost the ability (e.g., through MDM2 overexpression or loss of activators such as ARF) to induce a robust p53 response. It is possible that such drugs may, in themselves, be sufficient to induce p53 or,

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alternatively, may work in concert with other anticancer approaches. Similarly, induction of p53 using MDM2-targeted inhibitors may be of significant value in the development of cyclotherapybased approaches, i.e., where the activation of p53 in normal tissues causes a reversible cell cycle arrest that can protect normal cells (and thereby minimize side effects) from the action of other anticancer drugs such as antimitotics used to combat p53-mutant cancers. MDM2-targeted drugs may also have additional value independently of p53 status given the increasing range of p53-independent functions associated with MDM2 (discussed above). The best characterized small molecule inhibitor of MDM2 is Nutlin-3a, a compound that was developed based on the structure of the MDM2interacting region in the N-terminus of p53. Specifically, Nutlin-3a mimics the presence and spatial location of the three p53 residues (F19, W23, and L26) that fit within the p53-binding pocket of MDM2. It thus acts as a competitive inhibitor of p53 binding. Stapled peptides have also been developed as inhibitors of the p53/MDM2 interaction. Peptide stapling uses hydrocarbon cross-linkers to stabilize peptide structures with the effect of increasing targeting potency, inhibiting protease susceptibility, and, importantly, providing cell permeability. A key advantage of these reagents over small molecules is that they can be fine-tuned for greater specificity. There are several other MDM2-targeted molecules that block MDM2/p53 association including benzodiazepine, MI-219, WK298, AM-8553, and RG7112. The compound RITA can also block interaction with MDM2 but does so by binding to p53. There are also novel inhibitors available that act upon dimerization of MDM2 and/or MDM4. A number of MDM2-targeted compounds are currently undergoing clinical trials. Further Information There is a wealth of primary publications covering a period of over 20 years since the discovery of MDM2. There are also a number of excellent reviews. Some of these are given below for further reading.

Mdr1 Protein

References Bond GL, Levine AJ (2007) A single nucleotide polymorphism in the p53 pathway interacts with gender, environmental stresses and tumor genetics to influence cancer in humans. Oncogene 26:1317–1323 Bouska A, Eischen CM (2009) Murine double minute 2: p53-independent roads lead to genome instability or death. Trends Biochem Sci 34:279–286 Brown CJ, Cheok CF, Verma CS, Lane DP (2011) Reactivation of p53: from peptides to small molecules. Trends Pharmacol Sci 32:53–62 Lipkowitz S, Weissman AM (2011) RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis. Nat Rev Cancer 11:629–643 Miliani de Marval PL, Zhang Y (2011) The RP-Mdm2-p53 pathway and tumorigenesis. Oncotarget 2:234–238 Onel K, Cordon-Cardo C (2004) MDM2 and prognosis. Mol Cancer Res 2:1–8 Wade M, Wang YV, Wahl GM (2010) The p53 orchestra: Mdm2 and Mdmx set the tone. Trends Cell Biol 20:299–309 Wade M, Li YC, Wahl GM (2013) MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 13:83–96

Mdr1 Protein ▶ P-Glycoprotein

2ME ▶ Methoxyestradiol

2ME2 ▶ Methoxyestradiol

MEC ▶ Mucoepidermoid Cancer

Medullary Thyroid Cancer Targeted Therapy

Med28 ▶ Endothelial-Derived Gene-1

Medical Foods ▶ Nutraceuticals

Medullary Thyroid Cancer Targeted Therapy Cinzia Lanzi and Giuliana Cassinelli Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Definition Targeted therapies in oncology are therapeutic treatments intended to target molecular aberrations driving tumor pathogenesis or progression. In contrast to conventional cytotoxic therapies, which are essentially guided by the site of patient’s tumor, targeted therapies are based on molecular features of the tumor. According to this approach, selective antibodies or rationally designed drugs are currently used to inhibit the function of proteins involved in biochemical pathways thought to be essential for survival or distant dissemination of tumor cells. Therapeutic targets can be expressed by either ▶ cancer cells (e.g., proteins encoded by ▶ oncogenes) or by stromal cells present in the tumor microenvironment including endothelial cells forming the tumorassociated vasculature.

Characteristics Medullary thyroid cancer (MTC) is a neuroendocrine malignant tumor arising from parafollicular

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C-cells of the thyroid gland. It occurs sporadically in most cases, whereas in nearly 25% of cases, it is inherited in the context of the multiple endocrine 2 (MEN2) syndromes types 2A, 2B, and familial MTC. MTC represents the main cause of death in MEN2 patients. Surgery is the treatment of choice for both sporadic and hereditary MTC and the only curative modality available. Management of unresectable and advanced disease remains a challenge. Indeed, external radiation therapy has a limited role, and curative systemic treatments are not available. Only low rates (less than 20%) of partial, short-lasting, responses have been reported in patients receiving cytotoxic chemotherapeutics. The 10-year overall survival rate in patients presenting with locally advanced or metastatic disease is estimated in nearly 40%. Such a dismal prognosis prompts the preclinical and clinical research aimed at the identification of alternative, more effective, treatment options. Advances in the knowledge of pathogenetic mechanisms of MTC, and emerging evidence ascribing to ▶ angiogenesis a critical role in maintenance and dissemination of solid tumors, have provided rational bases for testing new targeted agents. The first targeted therapeutic agents, vandetanib and cabozantinib, have been approved by the US Food and Drug Administration (FDA) for the treatment of MTC in patients with advanced disease. Additional targeted agents are currently being evaluated in ▶ clinical trials for their efficacy and safety in MTC therapy. Molecular Therapeutic Targets in MTC Among molecules identified as possible therapeutic targets, ▶ protein kinases have a primary role. This class of enzymes, which catalyzes protein phosphorylation reactions, functions in physiological ▶ signal transduction pathways, regulating cell proliferation, survival, differentiation, and ▶ migration. Several protein kinases can otherwise function aberrantly driving key processes, defined as “hallmarks of cancer” by Hanahan and Weinberg (2011) and Lemmon and Schlessinger (2010), which are critical in tumor development and progression. In MTC as in other malignancies, the dysregulated function of tyrosine kinases is thought to sustain tumor growth,

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angiogenesis, ▶ invasion, and ▶ metastasis. In particular, available data from preclinical research and clinical trials support a role for ▶ receptor tyrosine kinases (RTKs) encoded by the ▶ RET proto-oncogene and by genes implicated in angiogenesis as valuable therapeutic targets in MTC. Ret and Angiogenesis-Related RTKs

The RET gene encodes a typical membrane RTK with extracellular, transmembrane, and cytoplasmic domains. Activation of the Ret ▶ receptor normally arises in the context of a complex including a co-receptor of the glial cell linederived neurotrophic factor (GDNF) family receptor-a and requires the interaction with a ligand of the GDNF family. The ligand binding induces dimerization of Ret molecules and a mutual transphosphorylation (referred to as autophosphorylation) of tyrosine residues essential for the receptor ▶ signal transduction. Tyrosine phosphorylated residues form, in fact, the docking sites for adaptor and transducing proteins mediating the activation of downstream signaling pathways involving RAS/RAF/ERKs, PI3K/ AKT, JNKs, ERK5, and PLCg, which in turn lead to gene expression regulation and biological responses (Arighi et al. 2005). Gain-of-function mutations of RET, leading to aberrant, ligandindependent activation of the Ret receptor, are frequent oncogenic events in tumors of the thyroid gland, i.e., ▶ papillary thyroid cancer and MTC. In hereditary MTCs, germline RET mutations occur in the vast majority of patients and are recognized as the disease initiating events. In sporadic MTC cases, somatic RET mutations are present in 40–60% of patients. The most common mutation in sporadic MTC, M918T, whose prevalence is nearly 90%, has been associated with a more aggressive behavior and lower survival. Mutations in the intracellular TK domain, typical of sporadic MTC and MEN2B, induce constitutive enzyme activation. Mutations at cysteine residues in the extracellular domain, typical of MEN2A and FMTC, cause receptor activation by inducing constitutive dimerization. Several lines of evidence from preclinical studies point to Ret mutant receptors as critical drivers

Medullary Thyroid Cancer Targeted Therapy

in MTC pathogenesis. For instance, the expression of RET oncogenes alone is sufficient to induce transformation in rodent fibroblasts, suggesting that these mutant receptors account for multiple mechanisms leading to the transformed cellular phenotype. Moreover, genetically engineered mice expressing thyroidtargeted MTC-associated RET oncogenes were shown to develop thyroid tumors that mimic the phenotype of human MTC, thereby providing support to the notion that RET activation is an early step in the pathogenesis process. These features, and the dependence of the transforming ability on constitutive tyrosine kinase activity, make the Ret mutant receptors attractive targets for therapeutic intervention in MTC (Lanzi et al. 2009). Several TK inhibitors undergoing clinical evaluation in MTC are indeed Ret-targeting agents. Nonetheless, a Ret-specific inhibitor is not presently available. Actually, all TK inhibitors clinically tested in MTC are multitarget agents sharing the ability to inhibit angiogenesis-related TK receptors (Table 1). Angiogenesis, the formation of new blood vessels from preexisting vessels, essential for both tumor growth and development of metastasis, provides additional relevant molecular targets for MTC therapy. Thyroid tumors have long been described as hypervascularized. In fact, MTC tends to metastasize early via hematogenous, in addition to lymphatic, route. However, little is known about the specific role of angiogenic factors in the pathogenesis or clinical course of MTC. Although the prognostic value of the angiogenic phenotype is debated, a few reports consistently showed overexpression of the ▶ vascular endothelial growth factor-A (VEGF-A) and its RTKs (VEGFR-1 and VEGFR-2) in sporadic and hereditary case series, supporting the idea that they are implicated in development and maintenance of MTC. VEGF-A-mediated pathway, which functions stimulating proliferation and survival of endothelial cells and increasing vascular permeability, is the major mediator of tumor angiogenesis. VEGF receptors are the most common targets shared by RTK inhibitors tested in the clinic in MTC patients. Other RTKs that play a

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Medullary Thyroid Cancer Targeted Therapy, Table 1 Multi-kinase inhibitors currently undergoing clinical trials Drug Vandetanibb, a (ZD6474) Cabozantinibc, a (XL184) motesanibd (AMG706) Sorafenibe (BAY439006) Sunitinibf (SU11428) Axitinibg (AG013736) Pazopanibh (GW786034) Lenvatinibi (E7080)

Company Astra Zeneca

Currently known targets VEGFR2-3, EGFR, RET

Phase of development II/III

Exelis

VEGFR2, RET, MET, KIT, TIE-2

III

Amgen Bayer, Onyx

II II

Pfizer Pfizer GlaxoSmithKline

VEGFR1-3, PDGFR, KIT, RET CRAF, BRAF,VEGFR2-3, PDGFR,FLT3, KIT, RET VEGFR1-3, PDGFR, KIT, RET, FLT3, CSF1R VEGFR1-3, PDGFR, KIT VEGFR1-3, RET, MET,KIT

II II II

Eisai

VEGFR1-3, RET, KIT, PDGFR, FGFR1-4

II

a

FDA approved ZD6474: [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy] quinazolin-4-amine] c XL184: N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide d AMG706: N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide e BAY43-9006: N-(3-trifluoromethyl-4-clorophenyl)-N’-(4-(2-methylcarbamoyl pyridine-4-yl)oxyphenyl) urea f SU11248: N-[2-(diethylamino)ethyl]-5-[(Z)-5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1Hpyrrole-3-carboxamide g AG013736: N-methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide h GW786034: 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzolsulfonamide i E7080: 4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxy-quinoline-6-carboxamide b

relevant role in tumor angiogenesis, such as the receptors for the ▶ fibroblast growth factor (FGFR) and for the ▶ platelet-derived growth factor (PDGFR), are additional common targets for RTK inhibitors under clinical evaluation (Table 1). Other Targets

The ▶ receptors of epidermal growth factor (EGFR) and hepatocyte growth factor (▶ Met) have been found overexpressed in the tumors of some MTC patients or in metastases compared to the matched primary tumors. Although the exact roles of EGFR and Met in MTC tumorigenesis have not been fully established, it is conceivable that co-targeting the cooperating or compensatory pathways mediated by these RTKs could be beneficial in subsets of MTC patients. RTK Inhibitors In the last years, multi-kinase inhibitors have entered clinical trials in patients with MTC (Table 1). Available data have confirmed the antitumor efficacy of the new therapeutic

approach. Several agents have demonstrated promising activity with clinical responses (according to Response Evaluation Criteria of Solid Tumor) and stabilization of disease (>6 months), accounting for benefit in the majority of patients (Schlumberger et al. 2012). To date, two drugs, vandetanib and cabozantinib, have received FDA approval based on the results of randomized placebo-controlled phase III trials. Vandetanib is an oral inhibitor of VEGFR2-3, Ret, and EGFR. In the phase III trial in patients with locally advanced or metastatic MTC, the median progression-free survival (PFS) was significantly prolonged from 19.3 months in the placebo arm to an estimated >30.5 months in the vandetanib arm. Partial responses were observed in 45% of patients. In this study, patients were not required to have progressive disease prior to entry. Cabozantinib is an oral inhibitor of VEGFR2, Ret, and Met. In the phase III trial in patients with radiographically confirmed progressive metastatic MTC, a significant prolongation in PFS was demonstrated with median PFS times of 4 and 11.2 months, in the

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Megapain/UCDEN (Ubiquitin Conjugate Degrading Endopeptidase)

placebo and cabozantinib arms, respectively. Partial responses were observed in 27% of patients. Additional new promising multi-target therapeutic agents under clinical investigation for treatment of patients affected by MTC are reported in Table 1. Although side effects from targeted therapy are generally less severe than with conventional cytotoxic ▶ chemotherapy, it is important to note that clinical trials of all the above agents reported significant toxicities which could be serious and dose limiting in some cases, requiring expert managing. Perspectives ▶ Receptor tyrosine kinase inhibitors have shown promise in the treatment of advanced MTC and are revolutionizing management paradigms. Further research is now needed to understand the basis of tumor response, with particular reference to the relative contribution of the inhibition of each target. A deeper knowledge of the relevant targets and the identification of new, more effective, inhibitors are important objectives in the attempt to optimize treatments and to improve outcomes. To date, in fact, none of the new therapies have shown an impact on overall survival of MTC patients. Future efforts are also expected to address the development of new treatment modalities including rational drug combinations of different targeted therapeutics, or targeted and conventional drugs, designed to interact synergistically to improve efficacy without enhancing toxicity.

References Arighi E, Borrello MG, Sariola H (2005) Ret tyrosine kinase signaling in development and cancer. Cytokine Growth Factor Rev 16:441–467 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 Lanzi C, Cassinelli G, Nicolini V, Zunino F (2009) Targeting RET for thyroid cancer therapy. Biochem Pharmacol 77:297–309 Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1133 Schlumberger M, Massicotte MH, Nascimento CL, Chougnet C, Baudin E, Leboulleux S (2012) Kinase inhibitors for advanced medullary thyroid carcinoma. Clinics 67:125–129

Megapain/UCDEN (Ubiquitin Conjugate Degrading Endopeptidase) ▶ Proteasome

MEK Kinases ▶ Mitogen-Activated Protein Kinase Kinase Kinases

Melanocytic Nevus ▶ Melanocytic Tumors

Melanocytic Tumors Wolter J. Mooi1 and Thomas Krausz2 1 Department of Pathology, VU Medical Center, Amsterdam, The Netherlands 2 Department of Pathology, University of Chicago, Chicago, IL, USA

Synonyms Melanocytic nevus; Melanocytoma; Melanoma; Mole; Nevocellular nevus; Nevus

Definition Melanocytic tumors are neoplastic proliferations of cells featuring phenotypic characteristics of melanocytes. The large majority originates in the skin. Benign melanocytic tumors are melanocytic nevi (or nevocellular nevi or, briefly, nevi or “moles”); malignant ones are melanomas.

Melanocytic Tumors

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Characteristics

available evidence suggests that the chance of melanoma is a few percent or less and is highest in large examples. Surgical removal of the nevus, or of its superficial part, improves cosmesis and may reduce melanoma risk, although the latter has not been established. Most surgeons prefer to operate large congenital nevi in the early infantile period. Acquired nevi tend to arise in childhood and adolescence, but some emerge later in life. Most start out as a small pigmented flat macule, which slowly increases in size and becomes raised, producing a papule. This initial growth phase is soon followed by stabilization of size and appearance. Most nevi are less than 6 mm across. Nevi are generally symmetrical, sharply defined, covered by an intact epidermis, and lack variations in color or degree of pigmentation. In case of doubt, an excisional biopsy (total surgical removal, with narrow margins) of the lesion allows histopathologic investigation, which usually, but not always, clinches the diagnosis. Nevus numbers are correlated with skin type, Caucasians with a fair skin generally having larger numbers than dark-haired Caucasians and non-Caucasians. Heavy exposure to ▶ UV radiation of sunlight, especially in early childhood, is correlated with increased nevus counts. In some families with an increased melanoma risk, nevi are very numerous and more irregular, clinically as well as histologically (dysplastic nevus syndrome; familial atypical multiple mole melanoma syndrome). Most melanocytic nevi harbor an activating B-RAF mutation, which results in increased signaling through the RAS-ERK pathway, which presumably drives, or contributes to, increased proliferation. Some variants, such as Spitz nevi (which are characterized by a large cell type), instead may have an activating HRAS mutation, while large congenital nevi tend to harbor and activate NRAS mutation. These mutations are mutually exclusive. In aggregate, these findings suggest that dysregulated RAS-ERK signaling is an important factor in the pathogenesis of a nevus. The characteristic cessation of nevus growth, which is caused by loss of proliferative activity, exhibits the central characteristics of cellular

Melanocytic tumors are neoplasms of cells showing the features of melanocytic differentiation, the most distinctive of which is the production of melanin. With few exceptions, this phenotype is thought to reflect their origin from a melanocyte. In mammals, most melanocytes reside in the basal epidermal cell layer and in the hair follicle. Smaller numbers are located in the dermal connective tissue, the uvea, the meninges, and the inner ear. In poikilotherms such as reptiles and fish, most melanocytes reside in the dermis. Cutaneous and extracutaneous melanocytic tumors are subdivided into melanocytic nevi (or nevocellular nevi, nevi, or “moles”), which are benign, and melanomas, which are malignant. Melanocytic nevi can be classified into congenital and acquired ones, the latter outnumbering the former by at least three orders of magnitude. Atypical mole syndrome is a disorder of the skin characterized by the presence of many molelike tumors (nevi). Most people have 10–20 moles over their bodies. People with atypical mole syndrome often have more than 100 moles, at least some of which are unusual (atypical) in size and structure. These moles vary in size, location, and color. They are usually larger than normal moles (5 mm or more in diameter) and have irregular borders. Changes in the appearance of these moles must be taken seriously by patients since such changes may foreshadow the onset of cancerous disease. Individuals with atypical mole syndrome are at greater risk than others for developing cancer of the skin in the form of malignant ▶ melanoma. Atypical mole syndrome is thought by some clinicians to be a precursor or forerunner of malignant melanoma. This type of cancer may spread to adjacent parts of the skin or, through the blood and lymph circulation, to other organs. Congenital nevi manifest at birth as a macule or larger patch of increased pigmentation. The largest examples, giant congenital nevi, may cover a substantial part of the body. The congenital nevus grows more or less in accordance with body growth, and it becomes somewhat raised and sometimes covered with hair. Melanoma arises in some congenital nevi, but this is uncommon: the

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senescence, with upregulation of p16INK4A, activation of senescence-associated b-galactosidase, and loss of proliferative activity. Germline inactivating mutations of INK4A or of the sequence coding for the p16INK4A binding site of CDK4 result in the melanoma-proneness familial dysplastic nevus syndrome mentioned above, whereas somatic mutations of INK4A or amplifications of CYCLIN-D1 are common in melanoma. In aggregate, these data suggest that unscheduled pRB-inactivation and inability to mount an oncogene-induced senescence response play an important role in melanomagenesis. ▶ Melanoma is a malignant tumor of melanocytes. Epidemiologic studies indicate that exposure to sunlight is an important factor in their pathogenesis. Fair-skinned and blond or red-haired individuals are especially at risk; melanoma is uncommon in non-Caucasians. Most arise in the skin and are detected when still relatively small, with a thickness of a few millimeters or less. Macroscopically, melanoma is often irregular in shape and color and may ulcerate or produce an itching or burning sensation. Melanoma may arise in a nevus; a change in a previously stable mole is suspicious of such malignant transformation. At the earliest phase, melanoma is confined to the epithelial compartment of the skin, but it subsequently invades into the dermis and underlying tissues; at this invasive phase, it may disseminate through the lymphatics and blood vessels. Lymphogenous metastases manifest in the skin and subcutis of the same body region (in-transit metastases) and in regional lymph nodes. Hematogenous metastases arise in a wide variety of sites, including some that are uncommonly affected by other tumors, such as the heart, intestines, and spleen. Primary melanoma is treated by wide local excision, usually with a margin of 1 cm of apparently uninvolved skin, in order to minimize the risk of local recurrence. Metastases to regional lymph nodes, manifest clinically or detected by sentinel node biopsy, are generally treated by regional lymph node dissection. This procedure still offers a significant chance of cure. Distant, hematogenous metastasis is not amenable to curative surgery, but a selected group of patients benefits from surgical removal

Melanocytic Tumors

of solitary metastases. The response to radiation therapy and standard chemotherapy regimens is usually modest. However, novel therapies that specifically target key signal tranduction pathways (Robert et al. 2015), or enhance antitumour immune responses (Hamid et al. 2013; Larkin et al. 2015), have shown substantial tumor responses. These successes have triggered much activity in this area clinical oncologic research.

Cross-References ▶ BRaf-Signaling ▶ Immunotherapy ▶ MAP Kinase

References Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, Dronca R, Gangadhar TC, Patnaik A, Zarour H, Joshua AM, Gergich K, Elassaiss-Schaap J, Algazi A, Mateus C, Boasberg P, Tumeh PC, Chmielowski B, Ebbinghaus SW, Li XN, Kang SP, Ribas A (2013) Safety and tumor responses with lambrolizumab (antiPD-1) in melanoma. N Engl J Med 369(2):134–44 Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P, Ferrucci PF, Hill A, Wagstaff J, Carlino MS, Haanen JB, Maio M, Marquez-Rodas I, McArthur GA, Ascierto PA, Long GV, Callahan MK, Postow MA, Grossmann K, Sznol M, Dreno B, Bastholt L, Yang A, Rollin LM, Horak C, Hodi FS, Wolchok JD (2015) Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med 373(1):23–34 Michaloglou C, Vredeveld LC, Soengas MS et al (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436:720–724 Mooi WJ, Krausz T (2007) Pathology of melanocytic disorders, 2nd edn. Hodder Arnold, London Pollock PM, Harper UL, Hansen KS et al (2003) High frequency of BRAF mutations in nevi. Nat Genet 33:19–20 Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, Lichinitser M, Dummer R, Grange F, Mortier L, Chiarion-Sileni V, Drucis K, Krajsova I, Hauschild A, Lorigan P, Wolter P, Long GV, Flaherty K, Nathan P, Ribas A, Martin AM, Sun P, Crist W, Legos J, Rubin SD, Little SM, Schadendorf D (2015) Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med 372(1):30-9

Melanoma Antigens

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Definition

Melanocytoma ▶ Melanocytic Tumors

Melanoma

▶ Melanoma antigens are a group of tumor antigens. Proteasomal cleavage of these intracellular proteins generates small antigenic peptides. The presentation of these peptides in conjunction with major histocompatibility complex (MHC) molecules facilitates an immune response promoting the destruction of the melanoma antigenexpressing cancer cells.

▶ Melanocytic Tumors

Characteristics

Melanoma Antigen Family A-11 ▶ MAGE-A11

Melanoma Antigen Gene Protein-11 ▶ MAGE-A11

Melanoma Antigen-A11 ▶ MAGE-A11

Melanoma Antigens Tobias Peikert and Ulrich Specks Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN, USA

Synonyms Melanoma-associated antigens

Background Over the years, several reports have documented cancer patients mounting immune responses against their malignancies. Most of these cases are characterized by massive lymphocytic infiltration of the tumor tissue, which leads to the destruction of the malignant cells and eventually to either partial or complete disease remission. Compared to other human malignancies, these immunologic phenomena are most frequently observed in malignant ▶ melanomas. This observation led researchers to focus their investigations on this particular tumor. Following experiments documenting the presence of tumor-specific cytotoxic T lymphocytes among the mononuclear cells infiltrating the tumor, investigators identified the first group of melanoma antigens as possible target antigens of these immune responses. Shortly thereafter, it became clear that most of these proteins are not specific for melanomas but are also present in a broad range of other malignancies. ▶ Proteasomes of these proteins produce several small unique 8–10-amino-acid-long peptides. Before being displayed on the cell surface in association with class I MHC molecules, these peptides are processed in the endoplasmic reticulum. Their subsequent presentation on the cell membrane of cancer cells results in the activation and proliferation of tumor-specific cytotoxic T lymphocytes (CD8+), mediating the destruction of these melanoma antigen-expressing cells. Classification Based on how these antigens are generated, the presence of tumor cell-specific gene expression,

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Melanoma Antigens

a

CD8+

T-cell

T-cell

Normal cell

CD8+

b

Cancer cell

c

Normal cell d

CD8+

CD8+

T-cells

T-cells

Normal cell

Cancer cell

Cancer cell

Normal cell

Cancer cell

Melanoma Antigens, Fig. 1 Melanoma antigens: (a) tumor-specific antigens – carcinogenesis generates genetically altered proteins which are recognized by cytotoxic T cells; (b) tumor-specific shared antigens (cancer/testis antigens) represent self-antigens with restricted gene expression in cancer cells and immunologically silent

male germ cells; (c) antigens encoded by genes overexpressed in tumor cells; and (d) antigens encoded by differentiation genes. Immune responses targeting antigens encoded by genes overexpressed in tumor cell and differentiation genes may lead to serious autoimmune reactions

the gene expression level, and whether the protein encoding the antigen represents a cell typespecific differentiation gene, melanoma antigens are classified into (Fig. 1):

novel antigenic epitopes and/or new MHC binding sites. Presentation of these new tumor-specific antigens on the cell surface together with the appropriate MHC molecules can trigger an antitumor immune response. This immune response typically begins with the binding of T-cell receptors to the antigen and is followed by the clonal expansion of tumor-specific cytotoxic (CD8+) T lymphocytes, which ultimately eliminate the melanoma antigen-expressing tumor cells. Rather than being universally expressed in all patients with certain malignancies, most of these novel tumor antigens are the result of specific mutations restricted to the tumor cells of individual patients. Therefore, most of these tumor-specific antigens do not represent unanimously applicable targets for cancer immunotherapy. However, some are present in a wide variety

• Tumor-specific antigens • Tumor-specific shared antigens (▶ cancer/testis antigens, CTA) • Antigens encoded by genes overexpressed in tumor cells • Antigens encoded by differentiation genes Tumor-Specific Antigens Tumor-specific melanoma antigens are the result of genetic alterations during carcinogenesis. Modifications of the structure and/or function of various physiologically expressed intracellular proteins due to these mutations can generate

Melanoma Antigens

of patients. A good example for this phenomenon is the oncogenic tyrosine kinase, ▶ BCR-ABL1, which is generated as the result of a translocation between chromosomes 9 and 22, t(9;22)(q34; q11), also known as the “Philadelphia chromosome.” This genetic abnormality is found in 95% of patients with chronic myelogenous leukemia (CML). Besides its role as an oncogenic tyrosine kinase, BCR-ABL1 also represents a tumorspecific antigen which is recognized by tumorspecific cytotoxic (CD8+) T lymphocytes. Tumor-Specific Shared Antigens (CTA) The second category of melanoma antigens are genes commonly found in a wide variety of malignancies, including melanomas, but absent in most adult tissues other than male germ cells and placental tissue. The genes are also known as CTA. Gene expression of CTA is almost exclusively restricted to tumor cells and male germ cells. The absence of gene expression and the lack of human leukocyte antigens in CTA-expressing germ cells preclude recognition of these antigens by cytotoxic T lymphocytes outside of CTA-expressing malignancies. Therefore, even though they represent selfantigens, CTA are considered to be tumor specific. The prototype of the CTA is the melanoma antigen (MAGE) gene family. The first members of this gene family were characterized in 1991 during the efforts to identify the immunological targets of cytotoxic T lymphocytes attacking the cells of the melanoma cell line MZ-2. Subsequently, many more members of this gene family have been identified. Up to date, three subfamilies with numerous genes (MAGE-A (1–15), MAGEB (1–17), and MAGE-C (1–7)) are known. Interestingly, all these genes are localized on the X chromosome. Their messenger RNA is assembled of three exons, and the entire open reading frame is localized on the third exon. A large area (65–85%) of the gene sequence is preserved between the different members of the MAGE gene family. This area, the MAGE-homology domain, encodes  170 amino acids and is positioned at the carboxyl-terminal end of the MAGE proteins. Based on the remarkable structural resemblance and the frequent co-expression of various

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members of the MAGE family within the same cell, it has been speculated that these proteins are involved in similar cellular pathways. However, the exact physiologic function of MAGE remains not fully understood. Several MAGE proteins have been shown to be involved in the regulation of the cell cycle and control of apoptotic pathways. In addition, the unique gene expression pattern of MAGE suggests that the physiologic role of the MAGE proteins is limited to pathways active during germ cell development and carcinogenesis. The silencing of MAGE gene expression in the majority of tissues is not attributable to a lack of the required transcription factors (Sp1 and Ets). These transcription factors are universally present in most cells; however, methylation of their binding sites at the gene promoter inhibits gene transcription. In contrast, DNA hypomethylation as frequently seen during carcinogenesis induces gene transcription by allowing transcription factor binding to the gene promoter. Alternatively, gene transcription can also be induced by inhibition of the DNA methyltransferase caused, for example, by 5-azacytidine. Other mechanisms potentially involved in the regulation of MAGE gene expression are genetic ▶ imprinting, suggested by the X chromosomal gene localization, and the presence of alternatively spliced promoters with variable transcription efficiency for some MAGE genes. Besides MAGE-A, MAGE-B, and MAGE-C (also called Type I MAGE genes), several additional MAGE gene subfamilies (MAGE-D to MAGE-L) have been described (Type II MAGE genes). In contrast to Type I MAGE genes, Type II genes are almost universally expressed in normal tissues. However, due to a lack of similarity between Type I and Type II MAGE genes, none of the antigenic peptides encoded by the Type I genes is present in Type II genes. This preserves the tumor specificity of the Type I MAGE genes. Other members of the cancer/testis antigens include the B melanoma antigens (BAGE), G antigens (GAGE), cancer/testis antigen 2 (LAGE-1), the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) gene, and the synovial sarcoma X (SSX) breakpoint genes.

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Antigens Encoded by Genes Overexpressed in Tumor Cells During carcinogenesis, there are numerous modifications to the cellular gene expression profile. Besides the activation of oncogenes and the silencing or mutation of tumor suppressor genes, cancer cells frequently overexpress genes that provide them with a selection advantage. These genes commonly enhance cell proliferation or inhibit apoptosis. Furthermore, these genes can also encode antigens, which are capable of triggering a cytotoxic T-lymphocyte-mediated immune response against the tumor. Examples for these antigens include ▶ survivin (inhibitor of apoptosis), ▶ telomerase (stabilizes chromosomes), and ▶ HER-2/neu (epidermal growth factor receptor, cell membrane-bound tyrosine kinase involved in cell growth and differentiation). HER-2/neu is overexpressed in 20–40% of all breast cancers. Given that these antigens represent self-proteins, the resulting immune response represents a breach of self-tolerance and is not tumor cell specific. Antigens Encoded by Differentiation Genes The last group of melanoma antigens includes proteins encoded by cell type-specific differentiation genes. For melanoma cells, this includes tyrosinase, Melan-A, gp100, and tyrosinaserelated proteins 1 and 2. An effective immune response targeting these antigens also results in the elimination of normal melanocytes and may present as vitiligo. Other tumor antigens encoded by differentiation genes include the ▶ prostatespecific membrane antigen (PSMA) and the ▶ carcinoembryonic antigen (CEA). Clinical Implications The elimination of cancer cells by the immune system represents an important defense mechanism against the development of malignancies. It has been well documented that immunocompromised individuals and patients receiving immunosuppressive medications are at an increased risk to develop certain types of cancer. In patients with established malignancies, the presence of tumorinfiltrating lymphocytes has been associated with better outcomes for several malignancies.

Melanoma Antigens

Peptides derived from melanoma antigens represent some of the immunological targets of these antitumor T lymphocytes. Occasionally, this vigorous immune response results in spontaneous tumor regression and remission. Unfortunately, these dramatic responses are only seen in very few individuals; more commonly, disease progression occurs despite the presence of antitumor T lymphocytes. The mechanisms by which the majority of malignancies escape the immune system are incompletely understood. It is possible that the loss of antigen or MHC class I expression by tumor cells and/or alterations in the local milieu of inflammatory cytokines leads to local anergy and allows tumor cells to proliferate despite the presence of antitumor T lymphocytes. Not surprisingly, melanoma antigens have been of great interest as targets for the development of anticancer vaccines. Prior to the characterization of the melanoma antigens, it has been shown that the adoptive transfer of antitumor lymphocytes results in a response of the tumor in ~30% of recipients. In addition, there was some success using either allogenic or autologous tumor cell vaccines. Following the characterization of the antigenic peptides contained in various melanoma antigens investigators developed various strategies to improve the immunogenic effects of these vaccines. These approaches include the use of peptide vaccines with or without immunogenic adjuvants, genetically altered antigenic peptides to increase MHC class I binding, transfer of antigen-transfected dendritic cells, as well as virus and DNA vaccines. Despite these efforts, clinical response rates remain suboptimal (20–30%), and further innovations are needed. Some of the problems to be resolved include the heterogeneous expression of melanoma antigens, local anergy, and the development of autoimmune phenomena.

References Boon T, Coulie PG, Van den Eynde BJ et al (2006) Human T cell responses against melanoma. Annu Rev Immunol 24:175–208 Chomez P, De Backer O, Bertrand M et al (2001) An overview of the MAGE gene family with the

Melanoma Drug Treatment identification of all human members of the family. Cancer Res 61:5544–5551 Forslund KO, Nordqvist K (2001) The melanoma antigen genes – any clues to their functions in normal tissues? Exp Cell Res 265:185–194 Van der Bruggen P, Traversari C, Chomez P et al (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643–1647 Van der Bruggen P, Stroobant V, Van Pel A et al (2006) Peptide database of T-cell defined tumor antigens. http:// www.cancerimmunity.org/peptidedatabase/Tcellepitopes. htm

Melanoma Drug Treatment Emilia Caputo Institute of Genetics and Biophysics – ABT, Napoli, Italy

Synonyms Melanoma therapy

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considered incurable, can be treated with potentially curative rather than palliative intent. Chemotherapeutics for Melanoma Treatment Dacarbazine, fotemustine, temozolomide, and nab-paclitaxel were used as chemotherapeutic agents in monotherapy for melanoma treatment. No difference in the overall survival (OS) value of melanoma patients treated with various chemotherapeutic combinations has been observed compared to the OS value resulting from dacarbazine in monotherapy. Treatment with dacarbazine in monotherapy represented the standard care for metastatic melanoma patients. Actually, it is used as the control arm in all studies using novel drugs and/or chemotherapeutic combinations. Fotemustine and temozolomide were mainly used in the treatment of patients with brain metastasis since these drugs are able to cross the blood–brain barrier (BBB). Actually, chemotherapy is indicated in the treatment of melanoma patients previously treated with B-RAF inhibitors (in melanoma patients carrying B-RAF V600E mutation) or with ipilimumab drug. Dacarbazine

Definition Melanoma drug treatment refers to the melanoma patient care by the application of pharmaceutical drugs.

Characteristics Melanoma is the most aggressive form of skin cancer. Approximately 70–80% of melanoma patients can be cured by surgical excision, when diagnosed at early stage of disease. Metastatic melanoma was treated with chemotherapeutic agents as standard care (Luke and Schwartz 2013). However, metastatic melanoma is refractory to chemotherapy with a median survival rate of about 6 months. Innovative drugs, such as immune (PD-1 inhibitors, ipilimumab)- and target-based therapeutics (B-RAF and MEK inhibitors), have been developed and have demonstrated how metastatic melanoma, once

This drug, also known as DIC or imidazole carboxamide, has been used as antineoplastic chemotherapy drug in the treatment of malignant melanoma, since 1976 (Fig. 1). This molecule was developed by Y. Fulmer Shealy, PhD at Southern Research Institute in Birmingham, Alabama. Dacarbazine gained FDA approval in May 1975 as DTIC-Dome. This chemotherapeutic agent belongs to the class of alkylating agents. It works by adding an alkyl group (CnH2n+1) to DNA, stopping cells from making new DNA molecules. DNA or deoxyribonucleic acid is the genetic material of a cell. If cancer cells cannot make DNA, they cannot split into two new cells, and subsequently they cannot grow. Dacarbazine is normally administered by intravenous infusion (IV) under the supervision of a doctor or nurse. This molecule is bioactivated in liver by demethylation to 5-(3-methyltriazen-1-yl)imidazole-4carboxamide (MTIC) and then to diazomethane, which is the alkylating agent. Like many chemotherapeutic drugs, dacarbazine may have

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Melanoma Drug Treatment

Melanoma Drug Treatment, Fig. 1 Timeline of approved drugs for metastatic melanoma treatment. Dacarbazine monotherapy has been the standard care for patients with advanced melanoma since 1976 (labeled in yellow), and only successively in 1999, temozolomide was approved. In 1998, the first immunotherapeutic agent (IL-2) gained FDA approval for melanoma therapy. In the last 4 years, various drug agents have been approved:

ipilimumab (antibody to CTLA-4) and vemurafenib (B-RAF inhibitor) in 2011, dabrafenib (B-RAF inhibitor) and trametinib (MEK inhibitor) in 2013, and pembrolizumab and nivolumab (antibody to PD-1) in 2014. Chemotherapeutics were labeled in yellow, immunotherapeutics drugs in red, and target-based therapeutic drugs in violet

numerous serious side effects, because it interferes with normal cell growth as well as cancer cell growth. Dacarbazine is highly emetogenic, and most patients need to be premedicated with antiemetic drugs. Other significant side effects include headache, fatigue, and occasionally diarrhea. Among the most serious possible side effects are immune suppression (reduced ability to fight infection or disease) and birth defects to children conceived or carried during treatment.

Temozolomide

Fotemustine

It is a nitrosourea alkylating agent approved for use in the treatment of metastatic melanoma. Fotemustine produces improved response rates. However, it does not increase survival. In patients with disseminated cutaneous melanoma treatment, the median survival rate is of about 7.3 months with fotemustine versus 5.6 months with dacarbazine. Fotemustine is more toxic compared to dacarbazine. It induces neutropenia (51% with fotemustine versus 5% with DTIC) and thrombocytopenia (43% vs. 6%, respectively) in melanoma patients (Avril et al. 2004).

It is bioactivated like dacarbazine in order to generate an alkylating agent that works by stopping the cell growth. This drug is administered orally, and it is adsorbed through the stomach and the small intestine. The drug passes through the intestinal wall and travels to the liver before being transported via the bloodstream to its target site. The absorption is fast and complete. Temozolomide was studied from Malcolm Stevens at Cancer Research Foundation of Birmingham University in England in 1978. Clinical trials testing this drug on patients with brain tumors ended in 1997. This drug was approved for its use in Europe and in the USA since 1999 (Fig. 1). Temozolomide may have numerous serious side effects, including fatigue, headache, constipation, occasional nausea, and thrombocytopenia. Nab-Paclitaxel

It is a new version of paclitaxel drug, belonging to taxane drugs. This molecule works by interfering with microtubules, which are part of the internal scaffold needed by cells during their division, and it leads then to cell death. The taxanes are water

Melanoma Drug Treatment

insoluble resulting in limited uptake and adverse reactions to the solvents used in their formulation. Nab-paclitaxel consists of paclitaxel bound to a protein called albumin, resulting in a stable complex, highly and readily bioavailable in contrast to water-insoluble paclitaxel. Nab-paclitaxel significantly improved progression-free survival (PFS) of melanoma patients, when compared with dacarbazine. The most common and severe toxicities noted with this drug are neuropathy and neutropenia. Immunotherapeutics for Melanoma Treatment Immunotherapeutic drugs are medicines able to stimulate a patient’s own immune system to recognize and destroy cancer cells more effectively. Several immunotherapeutics are used to treat patients with advanced melanoma (McDermott et al. 2014). IL-2

Interleukin-2 (IL-2) has been the first nonchemotherapeutic agent FDA-approved drug for advanced melanoma treatment in 1998 (Fig. 1). IL-2 is a protein, belonging to the family of type I cytokines. This protein is able to boost the immune system, by activating cytotoxic T cells. Man-made version of IL-2 is used in melanoma therapy. It is given at high dose as intravenous (IV) infusions. It may also be given along with chemotherapeutic agents (known as biochemotherapy) for the treatment of melanoma at stage IV. High-dose IL-2 administration can induce a reduction of advanced (stage III and IV) melanomas in about 10–20% of patients, with a median survival rate of about 59 months. Follow-up data derived from patients treated with high-dose IL-2 indicate long-term remissions in a small subset of melanoma patients. Serious side effects from high-dose IL-2 administration, such as systemic capillary leak syndrome (SCLS) due to increased capillary permeability, characterized by episodes of hypotension, edema, and hypovolemia, have limited its use. Because of this and other possible serious side effects, highdose IL-2 is given only in centers, having experience with this type of treatment. In Europe this

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therapeutic approach for metastatic melanoma treatment is not authorized. Interferon Alfa

It is used as an added (adjuvant) therapy after surgery to try to prevent potential cancer cells left in the body from spreading and growing. IF-a treatment may delay the recurrence of melanoma, but it has not been clearly established if it improves survival. However, in order for interferon treatment to be effective, it should be used at high doses. Unfortunately, many patients cannot tolerate these therapy side effects, including fever, chills, aches, depression, severe tiredness, as well as heart and liver problems. For this reason, it is critical, before to decide to use adjuvant interferon therapy, to take into account the potential benefits and side effects of this treatment. Ipilimumab

The most successful immunotherapeutic approach to date has been immune checkpoint inhibition. One of the best-studied checkpoints is cytotoxic T-lymphocyte antigen-4 (CTLA-4). It is a protein in the body which normally helps keep immune system cells (T cells) in check, mediating the immune tolerance. Consequently, it is possible to boost the immune response in the body, by blocking the CTLA-4 action. Drugs blocking CTLA-4 have been developed. Ipilimumab is one of the monoclonal antibodies raised against CTLA-4, able to block its action. This drug is given as an intravenous (IV) infusion, usually once every 3 weeks for four treatments. Ipilimumab treatment helped people live an average of several months longer compared to chemotherapy, in patients with melanomas that cannot be removed by surgery or that have spread to other parts of the body. The most common side effects from ipilimumab include skin rash, diarrhea, liver, and endocrinopathies. Other side effects are less common but can be more serious. This drug, by removing the brakes on the body’s immune system, may be responsible of serious side effects in other parts of the body, where the active immune system starts to attack and destroy parts of the body. In some people, these immune-related side effects have been fatal. Ipilimumab side effects

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most often occur during treatment, but some can occur up to a few months after treatment ended. PD-1 Inhibitors

These inhibitors (pembrolizumab and nivolumab) are monoclonal antibodies targeting programmed cell death-1 (PD-1), another immune checkpoint protein. By blocking PD-1, these drugs boost the immune response against melanoma cells in the body. These drugs are given as an IV infusion every 2 or 3 weeks. PD-1, when given to patients with advanced melanoma that has already been treated with ipilimumab, can cause a reduction in tumor size. It is not yet known if PD-1 helps patients live longer. Side effects can include fatigue, nausea, skin rash, joint pain, and diarrhea. Other, more serious side effects can occur less often. Like ipilimumab, PD-1 can cause the immune system to attack healthy parts of the body, causing serious or even fatal problems in other organs. However, these problems seem to happen less often than with ipilimumab. Target-Based Therapeutics for Melanoma Treatment Several molecular alterations associated with melanoma have been identified, and subsequently, various drugs targeting these molecular changes have been developed (Tronnier and Mitteldorf 2014). These drugs act differently from standard chemotherapeutic agents, which basically attack any dividing cells. Sometimes, targeted drugs work when chemotherapy does not. They can also have less severe side effects. Drugs Targeting Melanoma Cells Carrying Mutation in the B-RAF Gene

About half of all melanomas have changes (mutations) in the B-RAF gene. These changes cause the gene to make an altered B-RAF protein that forces the melanoma cells to grow and divide quickly. Various drugs (vemurafenib, dabrafenib, etc.) targeting directly this altered protein have been developed, and they are used in clinical trials. Vemurafenib This drug is used in patients with metastatic melanoma, carrying a B-RAF gene

Melanoma Drug Treatment

(mutation) change. It causes a reduction in tumor size in about half of the treated patients. It also prolongs the time before the tumors start growing again and helps some patients live longer, although the melanoma typically starts growing again eventually. This drug has been approved from FDA in August 2011 and from EMA in February 2012. It is administered orally, as a pill, twice a day. The most common side effects are photosensitivity, rash, and arthralgia. Some patients treated with vemurafenib develop new skin tumors called squamous cell carcinomas, which are usually less serious and invasive than the melanoma they already have and can be treated by removing them. A checking of the skin during treatment and for several months afterward is recommended. Less common but serious side effects can also occur, such as liver problems, heart rhythm problems, and grave allergic reactions. Dabrafenib This drug seems to work about as well as vemurafenib in terms of ability to shrink melanoma tumors when it is used by itself. It gained FDA and EMA approval in May and August 2013, respectively. It is given orally as a capsule, twice a day. Frequently, melanoma patients treated with dabrafenib develop thickening of the skin (hyperkeratosis), headache, fever, joint pain, nonmelanoma skin tumors, hair loss, and hand–foot syndrome (redness, pain, and irritation of the hands and feet), as common side effects. A peculiar side effect of dabrafenib treatment is severe fever. Although it also can cause squamous cell carcinomas of the skin, these may occur less often than with vemurafenib. More serious side effects can also occur with dabrafenib, and they include increased blood sugar levels, kidney failure, dehydration, and eye problems. Drug Targeting MEK Protein

MEK and B-RAF modulate the same signaling pathway inside cells, so drugs that block MEK proteins (MEK inhibitors) can also help treat melanomas with B-RAF gene changes. Trametinib This drug causes the reduction of melanomas that carried a modified version of

Melanoma Vaccines

B-RAF protein. It is given orally as a pill, once a day. Common side effects include rash, diarrhea, swelling, and increase of creatine phosphokinase (CPK) value. When used by itself, trametinib does not help to shrink melanoma tumors as the B-RAF inhibitors do. Dabrafenib/trametinib combination is promising, showing that some side effects (such as the development of other skin cancers) are actually less common with the combination. Drugs Targeting Melanoma Cells Carrying Mutations in the C-KIT Gene

A small portion of melanomas have changes in a gene called C-KIT that help them to proliferate. These gene changes are more common in acral melanomas (these melanomas start on the palms of the hands, soles of the feet, or under fingernails), in mucosal melanomas (they develop mainly inside the mouth or other mucosal (wet) areas), and in lentiginous melanomas (they start in areas that get chronic sun exposure). Adverse effects include itchy rash, headaches, neutropenia, and thrombocytopenia. Drugs targeting changes in C-KIT such as imatinib and nilotinib are available through clinical trials. Newer Treatments Newer types of immunotherapeutic drugs as well as drugs targeting different gene mutations associated to melanoma are available through clinical trials.

2713 malignant melanoma: a phase III study. J Clin Oncol 22:1118–1125 Hu-Lieskovan S, Robert L, Homet Moreno B, Ribas A (2014) Combining targeted therapy with immunotherapy in BRAF-mutant melanoma: promise and challenges. J Clin Oncol 32:2248–2254 Luke JJ, Schwartz GK (2013) Chemotherapy in the management of advanced cutaneous malignant melanoma. Clin Dermatol 31:290–297 McDermott D, Lebbé C, Hodi FS, Maio M, Weber JS, Wolchok JD, Thompson JA, Balch CM (2014) Durable benefit and the potential for long-term survival with immunotherapy in advanced melanoma. Cancer Treat Rev 40:1056–1064 Tronnier M, Mitteldorf C (2014) Treating advanced melanoma: current insights and opportunities. Cancer Manag Res 6:349–356

Melanoma Therapy ▶ Melanoma Drug Treatment

Melanoma Vaccines Thomas Tüting Laboratory for Experimental Dermatology, Department of Dermatology, University of Bonn, Bonn, Germany

Synonyms Specific immunotherapy for melanoma

Cross-References ▶ Adjuvant Therapy ▶ Alkylating Agents ▶ BRaf-Signaling ▶ Chemotherapy ▶ Combinatorial Cancer Therapy ▶ Melanoma Vaccines

References Avril MF, Aamdal S, Grob JJ, Hauschild A, Mohr P, Bonerandi JJ, Weichenthal M (2004) Fotemustine compared with dacarbazine in patients with disseminated

Definition Melanoma vaccines aim at inducing or enhancing cellular and humoral immune responses specifically against melanoma cells.

Characteristics Historical Background The earliest concepts for immunotherapy of malignant tumors were developed at the

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beginning of the twentieth century, when Paul Ehrlich suggested that the newly discovered antibodies be directed as “magic bullets” specifically against cancer cells. Various approaches to stimulate tumor immune defense have been experimentally developed in the last few decades. Patients with malignant melanoma have been of particular interest in the field of tumor immunology because clinical observations such as spontaneous regressions of primary tumors in the skin supported the idea that the immune system is capable of recognizing and eliminating melanoma cells. Immunological approaches were believed to be particularly suited to prevent the early metastatic spread of melanoma cells, which is associated with high mortality. Initial clinical studies performed with bacterial extracts derived from Corynebacterium parvum or bacillus CalmetteGuérin as an adjuvant immunotherapy were initiated in the 1970s but were largely unsuccessful. Recombinant cytokines such as interferon-alpha (IFNa) and interleukin-2 (IL-2) were introduced for immunotherapy of melanoma in the 1980s. Encouraging results were noted in some patients with inoperable metastatic melanoma, which showed complete and long-lasting remissions following treatment with high-dose IL-2. The occurrence of vitiligo-like depigmentation and other autoimmune phenomena in patients treated with IFNa or IL-2 appears to correlate with a favorable clinical course, suggesting that an activation of the immune system against pigment cells is involved. Tumor Immune Defense Against Transplantable Tumors in Mouse Models MacFarlane Burnet proposed in the 1950s that the immune system is principally able to recognize and eliminate cancer cells. This “immunosurveillance hypothesis” was controversially discussed for many years. The biologic understanding of immune defense against malignant tumor cells was widely studied in experimental mouse models involving the transplantation of chemically or virally transformed tumor cells. The analysis of humoral immunity against transplanted carcinogen-induced sarcomas revealed that the immune system is in principle capable of detecting molecular events associated

Melanoma Vaccines

with transformation such as the aberrant expression of p53. Adoptive transfer experiments showed that cellular components of the immune system were responsible for the rejection of transplanted tumor cells. In the 1980s it was discovered that cytotoxic T cells (CTL) recognize peptide fragments derived from intracellularly synthesized proteins bound to major histocompatibility (MHC) class I molecules on the cell surface and are able to specifically detect and destroy not only virusinfected but also tumor cells. Various experimental studies using well-characterized model antigens showed that recombinant vaccines were able to induce immunity against viral infection and transplanted tumor cells, provided that the tumor cells express the model antigen themselves. Culture of Melanoma-Specific Human T Cells and Identification of Melanoma Antigens In the 1980s melanoma-specific CTL could be isolated from peripheral blood or tumor tissue of melanoma patients and propagated in vitro with the help of recombinant IL-2, a T cell growth factor. In the 1990s a number of melanoma antigens recognized by these melanoma-specific CTL could be identified on the molecular level. These belonged to three principal categories: (i) tumor-specific antigens such as the MAGE family of proteins which were termed “cancer-testis antigens” because they are expressed by tumor cells as well as by germ cells in the testes; (ii) lineage differentiation ▶ tumor antigens such as the tyrosinase family of enzymes, gp100, or MART/Melan-A which are expressed by melanoma cells as well as melanocytes; and (iii) individually mutated tumor antigens which in some cases even participated in malignant transformation. The identification of melanoma antigens recognized by T cells provided the scientific basis for the development of T cell-directed melanoma vaccines. Rational Development of Melanoma Vaccine Strategies in Animal Models Strategies for melanoma vaccination were experimentally developed over many years using the transplantable mouse B16 melanoma cell line in the syngeneic, inbred C57BL/6 mouse strain. Unlike experiments with carcinogen-induced

Melanoma Vaccines

sarcomas, vaccinations with irradiated B16 melanoma cells were not able to protect mice against a subsequent challenge with viable B16 melanoma cells showing that these melanoma cells were poorly immunogenic. Cytokine gene transfer such as the transduction of B16 melanoma cells with GM-CSF significantly improved their immunogenicity. This was later shown to be due to the ability of GM-CSF to recruit and activate antigenpresenting dendritic cells (DC) which are critical for the induction of an effective cellular antitumor immune response. The establishment of protocols for the ex vivo culture of DC from precursors in the bone marrow or peripheral blood with the help of recombinant GM-CSF enabled their use as a biologic vaccine adjuvant. Injections with GM-CSF-derived DC pulsed with B16 tumor cell lysates were also able to promote protective immunity against a subsequent challenge with viable B16 melanoma cells. With the discovery of MHC class I-restricted antigen recognition by T cells and the molecular identification of different melanoma antigens, active specific approaches for melanoma vaccination were developed using well-characterized model antigens. Vaccines employing defined synthetic MHC class I-binding peptides with appropriate adjuvants, recombinant plasmid DNA, or recombinant viral vectors were able to successfully protect against a subsequent challenge with antigentransduced B16 melanoma cells. Cultured DC and stimulation of Toll-like receptors (TLR) with synthetic oligonucleotides, which activate the innate immune system, were required for the induction of an effective tumor immune response against lineage-specific differentiation antigens in a therapeutic experimental setting. Efficacy of Melanoma Vaccines in Clinical Trials Different strategies for therapeutic melanoma vaccination have been tested in clinical trials in the last 20 years. They can be grouped into vaccines on the basis of the following: 1. Autologous tumor cells 2. Allogeneic tumor cells 3. Synthetic or recombinant molecules

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The use of autologous tumor cells has the principal advantage that immune responses can potentially be stimulated against a broad spectrum of (unknown) tumor antigens. This includes the mutated proteins of the individual tumor. Both the generation of adequate amounts of autologous tumor cell lysates and the in vitro establishment of autologous melanoma cell lines require the acquisition of sufficient tumor material. This is only possible for patients with disseminated disease where tumor metastases can be easily excised. Clinical vaccine trials using autologous GM-CSF-transduced melanoma cells, cultured DC pulsed with autologous melanoma cell lysate, or purified heat-shock protein preparations have all shown that this strategy can successfully induce objective tumor regressions and long-lasting remissions in a small subset of patients. Unfortunately, most patients did not profit from the vaccine, but it needs to be considered that these trials were largely performed in patients with advanced disease. The idea of inducing a tumor-specific immune response using a vaccine approach appears to be particularly attractive in patients with clinical stage III melanoma after complete operative elimination of all detectable locoregional metastases. Autologous melanoma is usually not available in this situation. Therefore, well-characterized allogeneic melanoma cell lines derived from other patients have been used for vaccine purposes. This approach is potentially able to induce immune responses against cancer-testis antigens and lineage-specific differentiation antigens. Clinical trials involving large numbers of patients have been performed over many years. The results of these trials, reported in the last couple of years, claimed that this type of melanoma vaccine can be as effective as adjuvant treatment with recombinant IFNa. The molecular identification of melanoma antigens and the fact that many melanoma patients spontaneously show ▶ tumor antigen-specific cellular immunity have also promoted the development of vaccine strategies with synthetic peptides and recombinant viral vectors. Peptide-based vaccines could be made increasingly efficient with the help of recombinant cytokines (such as GM-CSF, IFNa, or IL-12), cultured DC, or

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synthetic CpG oligonucleotides as a TLR9 agonist. Various recombinant viral vectors including adenovirus, vaccinia virus, or canarypox virus carrying cDNA for defined melanoma antigens were also successfully used in clinical trials. Optimized melanoma vaccine strategies could effectively induce or enhance specific immune responses against the MAGE family of cancertestis antigens or against melanocyte differentiation antigens like tyrosinase, gp100, or MART/ Melan-A in vivo. However, despite objectively measurable vaccine effects, only very few patients responded clinically with tumor regression and a longer-lasting remission. Barriers for the Successful Implementation of Therapeutic Melanoma Vaccines Current research is directed towards understanding the reasons why melanoma vaccines are not as effective as expected in the clinical situation. Sensitive and reproducible procedures for the measurement of spontaneous and vaccine-induced immune responses against defined as well as unknown melanoma antigens were developed in the last decade. These include flow cytometric enumeration of specific CTL with recombinant MHC class I-peptide tetramers and evaluation of their ability to produce IFNg using intracellular cytokine staining or the Enzyme-Linked ImmunoSpot (ELISPOT) technique. The detection of melanoma-specific CTL present in very low numbers in blood or tumor tissue in vivo could be achieved by in vitro expansion in limiting dilution assays. Insights into the molecular and cellular mechanisms underlying the control of immune reactions and the maintenance of peripheral tolerance have also advanced the development of vaccines. It has become clear that the induction of CD8 + CTL is tightly controlled by regulatory lymphocyte populations via specific cell surface molecules and transcription factors such as CTLA-4 and FoxP3, respectively. In mice, selective elimination of regulatory T cells or blockade of CTLA-4 is able to circumvent peripheral tolerance mechanisms and enhance the induction of melanoma-specific cellular immunity in vivo. Stimulation of TLR was also shown to transiently

Melanoma Vaccines

suppress regulatory control and enhance vaccine responses. Further important insights could be gained with a detailed analysis of cellular immune responses in blood and tumor tissue of individual melanoma patients, which showed remarkable clinical regressions following melanoma vaccination. Injections of autologous tumor cells together with cultured DC as an adjuvant could enhance existing spontaneous CTL responses against various melanoma antigens. Vaccinations against the MAGE family of antigens were able to induce MAGE-specific CTL, which appeared to support the functional reactivation of preexisting CTL in the tumor tissue with other specificities. This observation demonstrated the importance of mechanisms regulating immune cell function within the tumor microenvironment, which is still not completely understood. Tumor cells need to communicate with endothelial cells, fibroblasts, and immune cells to support progressive tumor growth, invasive spread, and angiogenesis. The interaction between tumor growth control, inflammatory responses, and cytotoxic immunity in the pathogenesis of cancer is actively studied by many groups in the field. For example, the production of cytokines such as IL-6 and TGFb in tumor tissue is able to stimulate cell proliferation and simultaneously suppress the function of CTL. Various mechanisms contribute to the escape of melanoma cells from recognition and destruction by the cellular immune system. For example, melanoma cells are able to impair the function of DC and T cells and can downregulate MHC class I-restricted processing and presentation of antigens. Novel experimental mouse models have been generated in the last few years where melanomas can be induced in the skin by UV and spontaneously metastasize in lymph nodes and visceral organs on the basis of defined genetic alterations in the germline. These genetically engineered mouse melanoma models, which portray the clinical situation in patients much more closely than the transplantation of melanoma cell lines, may help to understand the role of molecular and cellular mechanisms regulating immune cell function in the pathogenesis of

Melanoma-Associated Retinopathy

melanoma and find ways to improve the therapeutic efficacy of melanoma vaccines. Summary and Future Perspective Various strategies for melanoma vaccination have been evaluated in clinical trials in the last 10–20 years. Several approaches, which had been rationally developed in experimental animal models, were clearly able to induce melanoma antigenspecific cellular and humoral immunity in patients. A favorable influence on the clinical course of the disease could only be achieved in a small minority of patients. Melanoma vaccines have disappointed the expectations. Most vaccine approaches have been evaluated in patients with advanced metastatic disease that had failed standard treatment options. Conceptually, melanoma vaccines should be much more effective in the adjuvant setting where the immune system can specifically detect and eradicate melanoma micrometastases in patients at high risk of recurrent disease. Melanoma vaccines may also become part of future treatment regimens for inoperable metastases which combine novel targeted antiproliferative, antiangiogenic, and immunostimulatory measures in order to attack tumors simultaneously from several sides. These approaches will have to aim at supporting the efficacy of melanoma-specific CTL in tumor tissue, for example, by creating the cytokine milieu associated with viral infections using appropriate synthetic oligonucleotides and by blocking regulatory mechanisms at the molecular or cellular level. Futuristic concepts include the adoptive transfer of ex vivo genetically engineered DC and CTL which are optimized for tumor cell destruction and may represent the “magic bullets” of the twentieth century.

References Bhardwaj N (ed) (2007) Review series tumor immunology. J Clin Invest 117:1130–1212 Blattmann NJ, Greenberg P (2004) Cancer immunotherapy: a treatment for the masses. Science 305:200–205 Boon T, Coulie PG, Van den Eynde BJ, van der Bruggen P (2006) Human T cell responses against melanoma. Annu Rev Immunol 124:175–208

2717 Parmiani G, Castelli C, Santinami M, Rivoltini L (2006) Melanoma immunology: past, present and future. Curr Opin Oncol 19:121–127 Rosenberg SA, Yang YC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10:909–915

Melanoma-Associated Antigen-A11 ▶ MAGE-A11

Melanoma-Associated Antigens ▶ Melanoma Antigens

Melanoma-Associated Retinopathy Claudia Pföhler Department of Dermatology, Saarland University Medical School, Homburg/Saar, Germany

Synonyms MAR; MAR syndrome

Definition Paraneoplastic syndrome in ▶ melanoma patients characterized by different visual signs and symptoms, a reduced b-wave in the electroretinogram, and serum antibodies against retinal proteins.

Characteristics Pathogenesis Formerly, melanoma-associated retinopathy (MAR) was supposed to result exclusively from antibody production against ▶ melanoma antigens that are also expressed in retinal tissue,

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Melanoma-Associated Retinopathy

Melanoma-Associated Retinopathy, Fig. 1 Immunofluorescence (a) and immunohistochemistry (b) using serum from different MAR patients on retinal tissue. Antibodies bind to photoreceptor cells (PR), cells of the inner

and outer plexiform layer (IPL and OPL), as well as on cells of the inner nuclear layer (INL) and the nerve fiber layer (NFL)

leading to the destruction of retinal cells and resulting in defective signal transduction. Indirect immunohistochemical investigation of serum samples from MAR patients on retinal tissue lead to the detection of these antibodies (Fig. 1). In some cases, a diffuse antibody involvement with a variety of different cell populations such as optic nerve, nerve fiber layers, photoreceptors, and bipolar cells could be observed. In single cases, a 35 kDa protein in Müller glial cells, a 22 kDa neuronal antigen, and retinal transducin could be identified as antigens possibly involved in the development of MAR. Experimental investigations of serum samples from patients with MAR using ▶ SEREX were able to identify mitofilin and titin as further target antigens. The

fact that none of these MAR-associated antigens detected to date by their capacity to elicit a humoral immune response is located on the cell surface questions a major pathogenetic role of the respective antibodies. Rather, it is probable that cellular, T cell-mediated processes are operative in the primary immune attack against the retina. Clinical Aspects Patients with MAR develop, often within a very short time period or even overnight, a multiplicity of visual disturbances. Patients describe frequently sudden onset shimmering, flickering, or pulsating photopsias in combination with difficulty seeing in the dark or night blindness. These first signs are often followed by progressive visual

Melanoma-Associated Retinopathy

loss over months. Further, some patients develop color vision deficiencies. The electroretinogram in MAR patients reveals a characteristic pattern of a markedly reduced b-wave in the presence of a normal dark-adapted a-wave, resembling that seen in patients with stationary night blindness. In most cases, patients have an established diagnosis of previous cutaneous melanoma and develop vision problems years later. Only in single cases, MAR symptoms preceded the diagnosis of melanoma in terms of a monitoric paraneoplastic syndrome. More often than not, the onset of MAR symptoms is associated with progression of disease and occurrence of metastases. The MAR syndrome with all its clinically apparent symptoms is very rare and to date only about 100 cases have been reported in the literature. However, clinical investigations including melanoma patients without visual disturbances could prove that subclinical MAR signs and symptoms appear more common than previously suspected. These subclinical symptoms are stage dependent and are predominantly found in patients with advanced disease. These clinical observations are confirmed by further experimental investigations. Antibody activity similar to that ascribed to the MAR syndrome appears frequently in patients with melanoma who have no MAR-like retinopathy but are potentially patients with a subclinical form of MAR. Diagnostic Procedure If clinical symptoms in a melanoma patient arise suspicion of MAR, the following ophthalmological examinations should be performed: • • • • •

Slit lamp and fundus examination Scotopic electroretinography Static and kinetic perimetry Nyctometry Color vision test (optional)

Serum samples from the individual patient should be investigated using indirect ▶ immunohistochemistry or immunofluorescence on retinal tissue. However, one should be aware that the

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presence of antiretinal antibodies alone is not sufficient to diagnose MAR, as antiretinal antibodies are also found in sera derived from healthy human subjects or patients with different ocular or nonocular inflammatory diseases. Therapy Reports about successful MAR therapy are very rare. The use of immunosuppressive agents was ineffective in most cases. However, in single cases the administration of corticosteroids, corticosteroids in combination with azathioprine, and plasmapheresis or the intravenous application of immunoglobulins could achieve improvement of vision disturbances. In addition, operative debulking of tumor masses or their irradiation could positively influence on MAR symptoms. Prognostic Relevance The prognostic relevance (▶ autoimmunity and cancer) of MAR symptoms remains unclear. Although the appearance of MAR symptoms is mostly associated with progression of disease, retrospective analyses did not find significant differences between the overall survival rates of melanoma patients with or without MAR. Further, it is still unclear if the antiretinal antibodies found in the serum from MAR patients have a protective effect for the individual patient. Background of this speculation is the observation of patients with non-small cell lung cancer or breast cancer who show extended overall survival if antitumoral antibodies are detected. However, it must be speculated that immunosuppressive therapy of MAR symptoms is associated with increased mortality. The comparison of melanoma patients with or without MAR concerning 5-year survival rates is not possible, as the number of MAR patients worldwide is too small for such analyses.

Cross-References ▶ Autoimmunity and Cancer ▶ Immunohistochemistry ▶ Melanoma Antigens ▶ SEREX

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Melatonin

References

Characteristics

Chan JW (2003) Paraneoplastic retinopathies and optic neuropathies. Surv Ophthalmol 48:12–38 Keltner JL, Thirkill CE, Yip PT (2001) Clinical and immunologic characteristics of melanoma-associated retinopathy patients. J Neuroophthalmol 21:173–187 Pföhler C, Preuss KD, Tilgen W et al (2007) Mitofilin and titin as target antigens in melanoma-associated retinopathy. Int J Cancer 120:788–795

Melatonin is among the most phylogenetically old (e.g., 3–4 billion years) and fundamental biological signaling molecules (hormones and cancer) found in a wide range of organisms as simple as bacteria and as complex as man. This compound is often present in high concentrations in many flowering and edible plants as well as in medicinal herbs; in this context, it is referred to as phytomelatonin. In humans, melatonin is produced during the night primarily by the pineal gland, a pea-sized neuroendocrine gland located deep within the center of the brain. Nighttime melatonin production follows a circadian (e.g., daily) rhythm that is synchronized by the light/ dark cycle. Light reaching specialized photoreceptors in the retina of the eye stimulates nerve impulses that are transmitted to a central biological clock mechanism residing in a small cluster of cells called the suprachiasmatic nucleus (SCN). The SCN is located in the brainstem, below the cerebral cortex, in a structure called the hypothalamus. The SCN is a central pacemaker or biological clock driving the circadian rhythm of nocturnal pineal melatonin production. Daylight resets the central biological clock in the SCN, whereas light, of sufficient intensity, wavelength, and duration, present during the night suppresses melatonin production. Although darkness is absolutely necessary for melatonin production to occur during the night, it is not sufficient to stimulate melatonin production during the day. Melatonin is soluble in both lipid and water compartments of living cells and thus has the ability to easily pass through all morphophysiological barriers including cell membranes. This pleiotropic molecule is involved in myriad functions including phase shifting of circadian rhythms, sleep/activity cycles, body temperature, seasonal reproductive rhythms, retinal physiology, immune activity, vascular tone, intermediary metabolism, free radical/antioxidant mechanisms, mitochondrial activity, and cancer development and growth. Melatonin’s mechanism of action in the regulation of many of these processes is

Melatonin David E. Blask1 and Richard G. Stevens2 1 Laboratory of Chrono-Neuroendocrine Oncology, Department of Structural and Cellular Biology, Tulane University School of Medicine, New Orleans, LA, USA 2 University of Connecticut Health Center, Farmington, CT, USA

Definition Melatonin is an indoleamine synthesized from tryptophan and secreted from the pineal gland during the night. Peak concentrations in blood reach 60–80 pg/mL at around 2–3 a.m. in humans, whereas daytime levels are barely detectable. The half-life of melatonin in the blood is  50–60 min. As a result of first-pass metabolism in the liver, 90% of melatonin is catabolized to 6-sulfatoxymelatonin, which is excreted in the urine. Most of the melatonin (e.g., 70%) circulating in the bloodstream is bound to albumin. The absolute oral bioavailability of melatonin (2–4 mg) is 15% with peak levels (2–4 ng/mL) being reached within  1 h of ingestion. The bioavailability of lower amounts (i.e., micrograms) of oral melatonin varies widely. Melatonin is regularly consumed from commercially available nutritional supplements by millions of people throughout the world primarily for sleep problems and/or jet lag. The long-term health implications of this are unknown.

Melatonin

thought to involve membrane-associated, inhibitory G protein-coupled melatonin receptors (MT1 and MT2), the activation of which leads to the suppression of production of the second messenger molecule, 30 ,50 -cyclic adenosine monophosphate (cAMP). As the neurohormonal expression of darkness, the timing and duration of circadian melatonin signal in essence “tell” all the cells, tissues, and organs of the body not only that it is dark but the length of the dark period as well. In this way, melatonin acts as one of the hands of the central circadian pacemaker to help coordinate physiological and metabolic activities in synchronization with the 24-h solar day. Melatonin Anticancer Action and Mechanisms Both the physiological melatonin signal as well as the administration of exogenous melatonin near the beginning of or during the dark phase suppress the initiation phase of tumorigenesis in experimental rat models of chemical ▶ carcinogenesis. This may be accomplished via melatonin’s ability to suppress the accumulation of DNA adducts (the resulting complex when chemicals bind to DNA) formed by carcinogens that cause damage to and permanent alterations in DNA (i.e., mutations and amplifications), which lead to neoplastic transformation. The inhibition of DNA adduct formation appears to relate to melatonin’s potent and direct free radical scavenging action (▶ Oxidative Stress). It may also indirectly detoxify carcinogens via activation of the glutathione and related antioxidative pathways. In addition to protecting cells from DNA damage, melatonin may also promote the repair of DNA once damage has occurred. Melatonin inhibits the proliferation of a number of human cancer cell lines in vitro such as breast, prostate, ovarian, liver, endometrial, choriocarcinoma, melanoma, colon, and bladder cancer cells. At pharmacological levels, melatonin exerts cytotoxic effects on cancer cells, whereas nocturnal, physiological levels induce oncostatic effects. One of melatonin’s important antiproliferative mechanisms is to delay the progression of cells from the G1/0 to the S phase of the cell

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cycle. Either alone or in combination with other agents, melatonin induces apoptotic cancer cell death (▶ Apoptosis). In some neoplastic cells, this indoleamine acts as a differentiating agent and diminishes their invasive/metastatic potential via alterations in adhesion molecule expression and the support of mechanisms responsible for gap junctional intercellular communication. A wide array of biochemical and molecular mechanisms of melatonin’s oncostatic action in vitro have been reported including the regulation of estrogen receptor expression and transactivation, calcium/calmodulin activity, protein kinase C activity, cytoskeletal architecture and function, intracellular redox status, melatonin receptormediated signal transduction cascades, aromatase and telomerase activity, and fatty acid transport and metabolism. A key to melatonin cancer inhibitory action on tumor growth in vivo involves the essential omega-6 ▶ polyunsaturated fatty acid (PUFA), ▶ linoleic acid (LA). As the most prevalent PUFA in the Western diet, LA levels greatly exceed those required to prevent essential FA deficiency (i.e., 1% of total calories). As a potent promoter of both murine and human tumorigenesis, LA exerts actions on cancer cells that are diametrically opposed to many of the oncostatic actions of melatonin listed above. Its oncogenic effects are related to its ability to upregulate the expression of genes involved in estrogen receptor (ERa) expression, cell cycle progression, G protein signaling, and the mitogen-activated protein kinase (MAPK) growth cascade. In both tissue-isolated ERa (+and) human ▶ breast cancer xenografts, melatonin acts via melatonin receptors to suppress tumor cAMP formation, leading to a suppression of LA uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Downregulation of LA uptake and metabolism reduces the activation of the epidermal growth factor receptor/MAPK pathway, culminating in tumor growth inhibition. The fact that LA upregulates whereas melatonin downregulates transcriptional regulation of ERa in human breast

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cancer cells via a melatonin receptor-mediated inhibition of cAMP in these cells would potentially provide ample opportunity for cross-talk among these pathways. Like rat hepatomas, human breast cancer xenografts exhibit a circadian rhythm of tumor proliferative activity, LA uptake, and metabolism and signal transduction activity that is driven by the nocturnal, circadian melatonin signal. Nocturnal Melatonin Suppression by Light at Night and Human Breast Cancer Risk The risk of developing breast cancer is up to five times higher in industrialized nations than in underdeveloped countries. Overall, nearly 50% of breast cancers cannot be accounted for by conventional risk factors. Westernized nations have increasingly become 24-h-per-day societies with greater numbers of people being exposed to more artificial light during the night both at home and particularly in the workplace. It has been postulated that light exposure at night may represent a unique risk factor for breast cancer in industrialized societies via its ability to suppress the nocturnal production of melatonin by the pineal gland. This hypothesis is based on studies showing that melatonin inhibits the development and growth of experimental models of breast cancer, whereas either surgical removal of the pineal gland or exposure to constant light stimulates mammary tumorigenesis in rodents. This postulate is further strengthened by epidemiological studies demonstrating that women working night shifts have a significantly elevated risk of breast cancer presumably due to their increased exposure to light at night (▶ Cancer Epidemiology). A similar observation has been made for prostate cancer in men (Prostate Cancer – Basic Characteristics and Experimental Models). Experimental studies have uncovered important new relationships between circadian biology, the endogenous nocturnal melatonin signal, and its suppression by light at night, relative to human breast cancer risk. The proliferative and metabolic activity of human breast cancer tissue, growing in a nude rat, perfused in situ with blood collected during completely

Melatonin

dark nights from premenopausal female subjects (i.e., high melatonin), is markedly reduced as compared to when the tissue is perfused with daytime blood (i.e., low melatonin). Subsequent exposure of the subjects to bright, fluorescent light during the night completely extinguishes the tumor inhibitory activity of their blood by lowering melatonin levels. Therefore, melatonin is the first soluble, nocturnal anticancer signal to be identified in humans that directly links the central circadian clock with some of the important mechanisms regulating breast carcinogenesis. These findings also provide the first definitive nexus between the exposure of healthy premenopausal female human subjects to bright, white light at night and the enhancement of human breast oncogenesis via circadian disruption (i.e., suppression) of the nocturnal, oncostatic melatonin signal. The suppression of circadian melatonin production by ocular exposure to bright white light at night, leading to augmented nocturnal tumor uptake of dietary LA and its conversion to mitogenically active 13-HODE, can now be afforded serious consideration as a new risk factor for human breast cancer. In non-shift working women, evidence suggests a decreased breast cancer risk associated with higher, first morning urine levels of 6-sulfatoxymelatonin or sleep duration 9 h per night (longer melatonin duration?). In some cancer patients, the nocturnal amplitude of circulating melatonin levels is reduced to various degrees. In breast cancer patients in particular, nocturnal circulating levels of melatonin are negatively correlated with breast cancer ERa content, while tissue levels of melatonin correlate positively with tumor ERa status and negatively with the nuclear grade and proliferative index. These findings suggest that cancer cells elaborate soluble factors that negatively feedback on the mechanisms regulating nocturnal melatonin production. Clinical Cancer Trials with Melatonin Over the past three decades, nearly 50 small clinical trials have been performed by a single group in Italy to test melatonin clinical efficacy in cancer

Melatonin

patients. In a minority of these studies, melatonin was given as a single agent, while in most cases it was administered in combination with other standard therapies, including chemotherapy, immunotherapy, or radiation therapy, exclusively to patients with advanced stage solid tumors that had become refractory to standard therapy alone. More than half of these trials consisted primarily of non-randomized broad phase II trials in which melatonin therapy was administered to patients with a wide range of malignancies; the remainder were randomized, controlled phase II trials that were disease-specific for lung cancer, colorectal cancer, breast cancer, glioblastoma, and brain metastases from solid tumors. In no case were the trials double-blind and/or placebo-controlled. An oral dose of anywhere from 10 to 50 mg of melatonin (usually 20 mg) was administered to cancer patients in the early evening either alone or concurrently with chemo- or radiation therapy. Although an objective partial tumor response was observed in a small percentage of patients receiving melatonin, the majority of tumor responses consisted of disease stabilization. Probably the most dramatic effect of melatonin treatment was a markedly improved 1-year survival in these patients compared with those receiving supportive care, chemotherapy, or radiotherapy alone. Other benefits that accrue from melatonin therapy as reported in these clinical trials are a reduction in the toxicities associated with chemotherapy including myelotoxicity, nephrotoxicity, thrombocytopenia, lymphocytopenia, stomatitis, neuropathy, and cancer cachexia. Probably the most clinically important aspect of these trials is that melatonin-treated cancer patients apparently achieve and maintain better performance status and experience less anxiety than those individuals not receiving the indoleamine. Performance status represents the general wellbeing and quality of life of the patient. In fact, patients who have a better performance status at the outset usually respond more favorably to melatonin alone or in combination with other more conventional therapies. The most common reported side effects of therapeutic (milligram) doses of melatonin

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include sedation, drowsiness, and mild hypothermia. In a randomized, double-blind, placebocontrolled trial, oral melatonin (10 mg) administered prior to sleep for 1 month was found to exert no toxic effects on a wide range of physiological and neurological parameters in healthy individuals. In the clinical cancer trials, in which up to 50 mg of melatonin was administered daily for 1–3 years, no adverse side effects warranting its discontinuation were reported. Summary and Conclusions The nocturnal, circadian melatonin signal is a newly identified inhibitory link between the central circadian pacemaker in the brain and the processes governing cancer development and growth in both experimental animals and humans. This anticancer signal in essence organizes the processes controlling oncogenesis within biological time structure and in doing so, provides the body with a degree of protection from the development and growth of cancer cells during each night. Melatonin blocks the ability of cancer cells to take up linoleic acid and convert it to the mitogenic signaling molecule 13-HODE. Bright light at night, which suppresses this oncostatic signal, represents a newly identified risk factor for breast cancer in night shift workers. Clinical trials indicate that nighttime melatonin supplementation may offer a promising new approach in the treatment of advanced stage malignancies and reduction in the toxicity of chemotherapy, immunotherapy, and/or radiation. Pharmacological doses of melatonin appear to be safe and generally well tolerated in cancer patients. The role of melatonin in human cancer prevention remains to be explored.

References Blask DE, Dauchy RT, Sauer LA (2005a) Putting cancer to sleep at night – the neuroendocrine/circadian melatonin signal. Endocrine 27:179–188 Blask DE, Brainard GC, Dauchy RT et al (2005b) Melatonin-depleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats. Cancer Res 65:11174–11184

M

2724 Lissoni P (2002) Is there a role for melatonin in supportive care? Support Care Cancer 10:110–116 Reiter RJ (2003) Melatonin: clinical relevance. Best Pract Res Clin Endocrinol Metab 17:273–285 Stevens RG (2005) Circadian disruption and breast cancer: from melatonin to clock genes. Epidemiology 16:254–258

Membrane Microdomain ▶ Lipid Raft

Membrane Raft ▶ Lipid Raft

Membrane Transport Proteins ▶ Membrane Transporters

Membrane Transporters Anne T. Nies Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Germany

Synonyms Membrane transport proteins

Definition Membrane transport proteins are embedded in the lipid bilayer of biological membranes and transfer ions and small molecules across these biological membranes. Several membrane transporters contribute to the resistance of tumor cells against anticancer drugs.

Membrane Microdomain

Characteristics General Features Membrane transporters mediate the movement of ions and small molecules across the plasma membrane and the membranes of intracellular compartments such as mitochondria, lysosomes, and vesicles. The human genome comprises at least 530 genes for plasma membrane transporters (1.7% of total genes) and 350 genes for intracellular membrane transporters (1.1% of total genes). Membrane transporters typically have several transmembrane segments, each consisting of a stretch of 20–25 hydrophobic amino acids that spans the lipid bilayer of a biological membrane. According to the direction of the transported molecules, membrane transport can be classified as uniport (one type of molecules is transported in one direction), symport (two different types of molecules are transported in the same direction), and antiport (two different types of molecules are transported in exchange with each other) (Fig. 1). Membrane transporters can also be classified as active (energy dependent) or passive (energy independent) (Fig. 1). Primary-active transporters are able to build up ion or solute gradients across membranes by directly utilizing the energy released during adenosine triphosphate (ATP) hydrolysis. They either belong to the ▶ ATP-binding cassette (ABC) transporter family or they are ion pumps (ATPases). ABC family members transport a large variety of small molecules including peptides, lipids, and endogenous and ▶ xenobiotic organic anions and organic cations, including drugs. ATPases pump ions such as Na+, K+, H+ (protons), Ca2+, and Cu2+ and contribute to maintain ion gradients across biological membranes. The Na+ gradient is preferentially used by the so-called secondary-active transporters that can couple the flow of Na+ down its concentration gradient with the transport of small molecules against their concentration gradients. One example is the Na+/glucose symporter in the brush border membrane of enterocytes in the small intestine that mediates the uptake of glucose into the body. The brush border is formed by the densely packed microvilli of the surface of columnar epithelial cells, e.g., in the intestine and in the

Membrane Transporters Membrane Transporters, Fig. 1 Classification of membrane transporters. (a) According to the direction of the transported molecules, one can differentiate between uniport, symport (coupled transport), and antiport (exchanger). (b) Transporters can also be classified as primary active, secondary active, and facilitative. See text for details

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a According to the direction of the transported molecules Symport

Uniport

Antiport

b According to the energy requirement Primary-active

Secondary-active

Facilitative

M ATP

ADP + Pi

proximal tubules of the kidney. Microvilli are small projections of the plasma membrane which greatly enlarge the surface area of the cell. Individual microvilli can only be distinguished using an electron microscope; in a light microscope, the microvilli are observed collectively as a fuzzy fringe at the surface of the epithelium which has, therefore, been termed brush border. Passive (facilitative) transporters mediate the movement of molecules down their concentration gradients. Most of the currently known secondary-active and passive transporters are subsumed into the solute carrier (SLC) group that comprises almost 400 members in 52 families. Similar to passive transporters, channels also mediate the movement of ions and small

molecules down their concentration gradients. However, several features clearly discriminate channels from membrane transporters. Channels are pores in biological membranes that have a rather limited specificity. Substance flow through channels is controlled by the channels’ open probability so that high transport rates of 107–109 molecules per second can be achieved. In contrast, transporters are able to specifically bind a substrate, but this high precision is traded in for slow transport rates of about 103–105 molecules per second. Evidently, no sugar channels have evolved, but different sugar transporters exist that can discriminate between sugars of the same size and selectively transport, e.g., glucose, but not fructose.

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Pharmacological Relevance of Membrane Transporters Especially the plasma membrane transporters decisively determine which substances enter or leave a cell at sufficient rates, because it is virtually impossible for ions and most small molecules to cross the lipid bilayer of a biological membrane at a sufficient rate by simple diffusion. A number of solute carrier (SLC) transporters serve as uptake transporters for nutrients and vitamins. ABC efflux pumps transport a wide variety of endogenous compounds out of cells and are localized in gastrointestinal and renal epithelia, hepatocytes, and blood–tissue barriers. The localization patterns underline that membrane transporters are essential for many physiological processes such as intestinal absorption, renal and hepatobiliary elimination and detoxification, and protection against toxins. Many SLC transporters and ABC efflux pumps transport ▶ xenobiotics so that they also decisively determine absorption, distribution, and elimination of drugs. They are, therefore, of important pharmacological relevance and can either be used as drug delivery systems or serve as drug targets. Role of Membrane Transporters in Drug Resistance Cancer cells are often resistant against a large variety of structurally diverse anticancer drugs. Two major mechanisms have been recognized that may cause this phenomenon of multidrug resistance, i.e., (i) an insufficient accumulation of anticancer drugs in the cancer cells and (ii) intracellular changes that impair the capability of drugs to kill the cancer cells, e.g., alterations in cell-cycle targets for cancer therapy, an increased repair of ▶ DNA damage, an inhibition of ▶ apoptosis, and an altered drug metabolism. The intracellular accumulation of anticancer drugs is decisively controlled by plasma membrane transporters. Cellular drug resistance can occur either by decreased drug uptake, typically via uptake transporters, or by enhanced drug efflux, typically via primary-active transporters. Several uptake transporters that transport nutrients as physiological substrates have been implicated in the sensitivity of cells to certain drugs. For example, the presence of nucleoside transporters (SLC28

Membrane Transporters

and SLC29 families) and folate transporters (SLC19 family) sensitizes cells to nucleoside analogs (e.g., ▶ cladribine, ▶ fludarabine, ▶ gemcitabine, and methotrexate, respectively). Sensitivity of cells to ▶ platinum drugs may be due to the presence of copper transporters (SLC31 family) and organic cation transporters of the SLC22 family in the plasma membrane. The relevance of uptake transporters for the sensitivity of cells to other anticancer drugs is incompletely understood and currently intensively investigated. In addition to plasma membrane transporters, some drugs may enter cells via ▶ endocytosis. This pathway is, e.g., utilized by some novel anticancer agents, the ▶ immunotoxins. Cellular resistance resulting from enhanced drug efflux is most often mediated by members of the ABC transporter family, namely, MDR1 ▶ P-glycoprotein (ABCB1), the multidrug resistance proteins (MRPs) of the ABCC subfamily, and breast cancer resistance protein (BCRP, ABCG2). Drug substrates include methotrexate, vinca alkaloids, ▶ anthracyclines (e.g., ▶ Adriamycin), podophyllotoxins, taxanes (e.g., ▶ paclitaxel), inhibitors of ▶ receptor tyrosine kinases (e.g., ▶ imatinib), and camptothecins (e.g., ▶ irinotecan). These drug efflux pumps have been identified in many human cancers so that they may contribute to drug resistance, although their precise role in establishing clinical drug resistance is still under investigation. Strategies to overcome multidrug resistance include its reversal by small molecules that function as inhibitors of ABC efflux pumps. Although already in their third generation, these reversal agents have so far not been beneficial in reversing drug resistance. Reasons for failure include the overlapping substrate specificities of several ABC drug efflux pumps and toxicity which is often caused because important physiological functions of the ABC efflux pumps are blocked by the reversal agents.

Cross-References ▶ Adriamycin ▶ Anthracyclines ▶ Apoptosis

Membrane-Linked Docking Protein

▶ Cladribine ▶ DNA Damage ▶ Endocytosis ▶ Fludarabine ▶ Gemcitabine ▶ Imatinib ▶ Immunotoxins ▶ Irinotecan ▶ Paclitaxel ▶ P-Glycoprotein ▶ Platinum Complexes ▶ Receptor Tyrosine Kinases ▶ Xenobiotics

References Deeley RG, Westlake C, Cole SP (2006) Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 86:849–899 Emami Riedmaier A, Nies AT, Schaeffeler E, Schwab M (2012) Organic anion transporters and their implications in pharmacotherapy. Pharmacol Rev 64:421–449 Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, Dahlin A, Evers R, Fischer V, Hillgren KM, Hoffmaster KA, Ishikawa T, Keppler D, Kim RB, Lee CA, Niemi M, Polli JW, Sugiyama Y, Swaan PW, Ware JA, Wright SH, Yee SW, Zamek-Gliszczynski MJ, Zhang L (2010) Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236 Hediger MA (2013) Special issue. The ABCs of membrane transporters in health and disease (SLC series). Mol Aspects Med 34 Kell DB, Dobson PD, Oliver SG (2011) Pharmaceutical drug transport: the issues and the implications that it is essentially carrier-mediated only. Drug Discov Today 16:704–714 Moitra K, Dean M (2011) Evolution of ABC transporters by gene duplication and their role in human disease. Biol Chem 392:29–37 Nies AT, Lang T (2014) Multidrug resistance proteins of the ABCC subfamily. In: You G, Morris ME (eds) Drug transporters: molecular characterization and role in drug disposition. Wiley, Hoboken, pp 161–185 Nies AT, Koepsell H, Damme K, Schwab M (2011) Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy. Handb Exp Pharmacol 201:105–167 Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM (2006) Targeting multidrug resistance in cancer. Nat Rev Drug Discov 5:219–234

See Also (2012) ATP-binding cassette transporters. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/

2727 Heidelberg, p 302. doi:10.1007/978-3-642-164835_441 (2012) Breast cancer resistance protein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 546–547. doi:10.1007/978-3-642-164835_719 (2012) Brush border. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 572. doi:10.1007/978-3-642-16483-5_744 (2012) Camptothecin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 603. doi:10.1007/978-3-642-16483-5_791 (2012) Methotrexate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2274. doi:10.1007/978-3-642-16483-5_3680 (2012) Multidrug resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2393. doi:10.1007/978-3-642-16483-5_3887 (2012) Multidrug resistance proteins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2393. doi:10.1007/978-3-642-164835_3888 (2012) Podophyllotoxins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2932. doi:10.1007/978-3-642-16483-5_4650 (2012) Solute carrier transporters. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3466. doi:10.1007/978-3-642-164835_5407 (2012) Taxanes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3614– 3615. doi:10.1007/978-3-642-16483-5_6648 (2012) Vinca alkaloids. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3908. doi:10.1007/978-3-642-16483-5_6187

Membrane-Linked Docking Protein Noriko Gotoh1 and Nobuo Tsuchida2 1 Division of Cancer Cell Biology, Cancer Research Institute, Kanazawa University, Kanazawa city, Ishikawa, Japan 2 Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

Definition Membrane-linked docking protein (MLDP) is a signal-transducing molecule and consists of the following family members: (i) FRS2/SNT (fibroblast growth factor receptor substrate

M

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Membrane-Linked Docking Protein

2/suc1-associated neurotrophic factor target), (ii) Dok (docking protein), (iii) TRAP (transmembrane adaptor protein), (iv) Gab (Grb2associated binder), and (v) IRS (insulin receptor substrate). The protein has the following structural motifs: (i) membrane-linking function and (ii) scaffolding or adaptor function, and (iii) multiple tyrosine residues capable of being phosphorylated by tyrosine kinases. However, the protein itself has no enzymatic activity as well as no or very short (less than 40 amino acid residues) extracellular domain. Although MLDP works as a mediator of signal transduction, the protein families represent groups of adaptor proteins N-termini of which are anchored in the membrane and the rest of which in the cytoplasm.

Characteristics Mechanism of Signal Transduction by MLDP MLDP is localized on the inner side of membrane since the protein contains (i) PH (Pleckstrin homology) domain, (ii) membrane-anchor domain of a stretch of hydrophobic amino acid residues, or (iii) a palmitylation/myristylation site at or close to N-terminus of MLDP. The upstream

Membrane-Linked Docking Protein, Fig. 1 Model of signaling through membrane-linked docking protein (MLDP)

signals are received by MLDP with two steps (Fig. 1). The first step is the docking at the inner side with transmembrane receptors. Growth factors, cytokines, or antigens bind to transmembrane receptors, resulting in dimerization or oligomerization. The receptors are phosphorylated by elevated tyrosine kinase activity themselves or by activated cytoplasmic tyrosine kinases. MLDP binds to the phosphorylated tyrosine site on the receptors using PTB (phosphotyrosine binding) domain. Once docked, several tyrosine residues of MLDP are phosphorylated by the activated ▶ receptor tyrosine kinase. In turn, MLDP provides with Tyr-phosphorylation sites as a platform to downstream effector proteins for their binding, such as to SHP2 (phosphotyrosine phosphatase = PTP) containing two ▶ SH2 domains. When inactive SHP2 is recruited to MLDP for its binding, conformational changes of SHP2 take place, thus resulting in the activation of phosphatase of SHP2. SHP2 subsequently activates ERK (one of the ▶ MAP kinases) pathway, resulting in cell proliferation. Another MLDP-binding protein, p85, regulatory subunit of phosphatidyl inositol (PI) 3-kinase, is also recruited after tyrosyl phosphorylation of MLDP, leading to activation of AKT survival pathway.

MLDP binds to upstream tyrosine kinase Ligand

Downstream SH2 containing proteins recruited and activated

Tyrosine kinase creates SH2 binding sites

Transmembrane domain Cell membrane Cytosol PTB domain

P

P

P

P

P

Receptor P

P P

P Membrane-linked docking protein (MLDP)

SH2 domain PTP domain Inactive

Active

Membrane-Linked Docking Protein

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FRS2/SNT FRS2/SNT family consists of two members: FRS2a/SNT-1 and FRS2b/SNT-2/FRS3. Each protein consists of a membrane-anchoring N-myristylation signal, a PTB domain, and a

Roles of Individual MLDP Family in Normal and Cancer Cells Five families of MLDPs, binding proteins to the PTB domain, and the binding molecules to the phosphorylated tyrosine residues exist (Fig. 2).

PTB

FRS2α

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Binding molecules to the PTB domain

Binding molecules to the phosphorylated tyrosine residues

FGFR, TrkA, B, RET

Grb2, Shp2

FGFR, TrkA, B, EGFR

Grb2, Shp2

SHIP, Dok1, 2

RasGAP, Csk, Nck

SHIP, Dok1, 2, EGFR, ErbB2

RasGAP, Csk, Nck

SHIP

Csk, Grb2

Myristylation

PTB

FRS2β

Y

ERK binding

Y

Proline-rich

PH

Dok2

PTB Y Y

Y

Y

Proline-rich

PH

Dok3

PTB

Y

Y

Y

Y LAT

Y

PTB Y Y

PH Dok1

Y

Y

Y

Y PLCγ1, Grb2 GADS

TM palmitylation

Y

Y SIT

Y

Y Grb2, Shp2, Csk

Disulphide bond

Y

Y

Y

Y

Glycosylation

Gab1 PH

Y

Y

Y

Grb2, Shp2, Gab2, PI3K,PICγ

Y

Proline-rich met binding

Gab2

Gab3

PH

PH

Y

Y

IRS1

PH PTB

IRS2

PH

Y

Y Y

PTB

Y

Y

Y

Grb2, Shp2, PI3K PLCγ

Y

Grb2, Shp2, PI3K

Y Y

Y

Y

Y

Y

Y

Y

Insulin receptor IGF-1R, IL-4R

PI3K, SHC, Grb2 Shp2

Y

Insulin receptor IGF1-R, IL-4R

PI3K, Shp2, Grb2

Insulin receptor IGF1-R

PI3K, Shp2, Grb2

Insulin receptor binding

IRS4

PH PTB

Y

Y

Y

Y

Membrane-Linked Docking Protein, Fig. 2 Schematic structures of several members of MLDPs. The phosphotyrosine-binding domain (PTB) binds to phosphorylated tyrosine residues on receptors or signaling proteins. The PH domain binds to phospholipids such as phosphatidyl inositol (PI)-3 phosphate. The proline-rich domains are important for binding to

Y

SH3-containing proteins such as Src family tyrosine kinases or Grb2. Y designates potential tyrosine (Y) phosphorylation sites. The ERK-binding domain in FRS2b, Met-binding domain in Gab1, or insulin receptor binding domain in IRS1 contain a unique sequence for binding to ERK, Met, or insulin receptor

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C-terminal side region containing tyrosine residues which serve as the binding sites, when phosphorylated, for the SH2 domains of Grb2 adaptor protein and SHP2 tyrosine phosphatase. Although both FRS2 family members are highly homologous, their different expression patterns point to the presence of specific roles for each member. Frs2a/Snt-1 is ubiquitously expressed at every developmental stage of the mouse, whereas high levels of expression of Frs2b/Snt-2 are confined to several tissues of neuronal or epithelial origin. Tyrosine-phosphorylated FRS2/SNT proteins serve as a platform for multiprotein complexes induced by activation of several receptor tyrosine kinases with their corresponding ligands, such as ▶ fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived growth factor (BDNF), and glial cell-derived growth factor (GDNF). The PTB domain of FRS2/SNT binds to phosphorylated tyrosine residues on TRKA, TrkB, and ▶ RET; although, in the case of FGF, it binds to both phosphorylated and unphosphorylated forms of FGF receptor. The SHP2-binding sites on FRS2a/SNT-1 play a primary role for Ras-ERK activation, and the Grb2binding sites on FRS2a/SNT-1 recruit Gab1 and ubiquitin ligase Cbl; tyrosine-phosphorylated Gab1 recruits and activates PI3-kinase (see “▶ PI3K Signaling”), whereas Cbl activates the ubiquitination, leading to degradation pathway. RET gene rearrangements lead to generation of chimeric oncoprotein, RET-PTC, in papillary thyroid carcinomas. Point mutations on RET gene result in the expression of constitutively active RET in inherited multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. FRS2a/ SNT-1 couples these oncogenic forms of RET with Ras-ERK activation and is implicated in oncogenesis. FRS2/SNT protein is also implicated in oncogenesis of another chimeric oncoprotein, nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), which was identified in anaplastic large-cell lymphoma with the t(2:5) ▶ chromosomal translocation. Contrary to the case of FRS2a/SNT-1, FRS2b/SNT-2 is not tyrosine phosphorylated

Membrane-Linked Docking Protein

significantly in response to epidermal growth factor (EGF) (one of the Epidermal Growth Factor Receptor Ligands) (see “▶ Epidermal Growth Factor-like Ligands”), but it inhibits EGF signaling and cell transformation via forming a complex with ERK2. FRS2b thus acts as an adaptor protein for negative regulation of EGF receptor tyrosine kinase signaling pathways. Dok Dok family consists of seven members: Dok1/ p62dok, Dok2/Dok-R/FRIP/p56dok, Dok3/Dok-L, Dok4/IRS5, Dok5/IRS6, Dok6, and Dok7. Among them, Dok1–3 or Dok4 and 5 comprise a subfamily based on their structural similarity. They are typical docking proteins with an N-terminal module composed of tandem PH-PTB domains followed by a region with binding motifs for SH2 domain. Dok1 and 2 also possess proline-rich sequences within their C-terminal tails that are for binding to SH3 domain (see “▶ SH2/SH3 Domains”). The PH domain of Dok1 binds to both PI-4,5-biphosphate (PIP2) and PI-3,4,5-triphosphate (PIP3) in the plasma membrane. Phospholipase C (PLC) g and PI3-kinase are also recruited to the plasma membrane where PIP3 is synthesized from PIP2. Dok1 is first identified as a tyrosinephosphorylated protein of 62 kDa (pp62) in leukemic cells isolated from patients of chronic myelogenous leukemia (CML) carrying a t(9;22) chromosomal translocation as a complex with a chimeric p210 (BCR-ABL) (see “▶ BCRABL1”) protein with elevated tyrosine kinase activity. Since Dok1 and Dok2 double knockout mice display CML-like disease and lymphoma, Dok proteins are considered essential for homeostatic control of activation and proliferation of hematopoietic cells. Dok 1, 2, and 3 proteins are key negative regulators to nonreceptor tyrosine kinases, forming multiprotein complexes in response to cytokines, growth factors, and immunogens, thus controlling the delicate balance between positive and negative signals. Dok1 and 2 constitutively bind to SH3 domains of Src family tyrosine kinases or c-Abl. Coaggregation of B cell receptor with FcgRIIB1 or T cell receptor stimulation

Membrane-Linked Docking Protein

induces tyrosine phosphorylation on Dok. The PTB domain of Dok binds to tyrosinephosphorylated SHIP1 (the SH2 domaincontaining inositol polyphosphate 50 -phosphatase), a negative regulator for B cell and T cell receptor signaling. The phosphorylated Dok1 and 2 bind to SH2 domains of Ras-GAP (▶ GTPase activating protein for Ras) and Csk, negative regulators for Ras and ▶ Src family kinases, respectively. Tyrosine-phosphorylated Dok1 and 2 also bind to SH2 domain of Nck adaptor protein, whereas Dok3 binds to Csk and Grb2 but not Ras-GAP. Dok also regulates signaling through receptor type tyrosine kinases (see “▶ Receptor Tyrosine Kinases”). Dok1 and 2 negatively regulate platelet-derived growth factor (PDGF), EGF, or insulin signaling. Upon EGF treatment, the PTB domain of Dok2 binds to tyrosine-phosphorylated EGF receptor, and subsequently Dok2 is phosphorylated, and then the phosphorylated Dok2 inhibits activation of downstream effectors. On the other hand, Dok4, 5, and 6 have positive effects on neurite outgrowth stimulated by activation of RET, TrkB, or TrkC receptor tyrosine kinases. In addition, Dok7 binds to musclespecific receptor kinase (MuSK) via the PTB domain and induces activation of MuSK for neuromuscular synaptogenesis. Mutations in DOK7 cause a congenital limb-girdle myasthenic syndrome with a characteristic pattern of weakness, particularly affecting the proximal muscle groups. Transmembrane Adaptor Proteins (TRAPs) TRAPs are membrane-associated molecules that are capable of binding with different SH2 domains after the tyrosyl phosphorylation and are key mediators of immunoreceptor-mediated signaling. They are divided into two groups: raft-associated TRAPs such as LAT (linker for activation of T cells), PAG (phosphoprotein associated with glycolipid-enriched membranes)/CBP (Csk-binding protein) and LAB (linker for activation of B cells)/LAT2 and nonraft associated TRAPs such as SIT (SHP2-interacting transmembrane adaptor protein). Lipid rafts are submicroscopic regions enriched in sphingolipids and cholesterol in the plasma membrane and contain

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several signaling proteins including Src family tyrosine kinases. The raft-associated TRAPs but not nonraft associated TRAPs have CXXC palmitylation motif in the juxtamembrane region for targeting the lipid raft. Each TRAP has a short extracellular domain, typical transmembrane domain, and a long cytoplasmic tail that has up to nine potential tyrosyl phosphorylation sites. LAT can be considered the master switch of T cell and mast cell activation. Stimulation of T cell receptor or high-affinity receptor for IgE (FceRI) on mast cells leads to tyrosine phosphorylation on LAT. Then LAT binds to SH2 domains of PLCg1, Grb2, and GADS, the Grb2-related adaptor protein. PLCg then hydrolyses PIP2, producing the second messengers IP3 and diacylglycerol. Grb2 binds to Ras activator SOS. GADS binds to SLP76, another adaptor protein, leading to activation of Itk cytoplasmic tyrosine kinase and Vav, an activator for Rac. NTAL is a LAT-like protein in non-T cells and tyrosine-phosphorylated PAG inhibits Src family tyrosine kinases via binding to SH2 domain of Csk. Tyrosine-phosphorylated SIT binds to Grb2, an SH2 domain-containing phosphatase Shp2 and Csk, and negatively regulates T cell signaling. Gab Gab family consists of Gab1, Gab2, and Gab3, has PH domain at N-terminus, and phosphotyrosine sites which, upon stimulation with growth factors, cytokines, or antigens, provide binding sites for SH2-containing proteins (SHP2, p85 subunit of PI3-kinase, Grb2, PLCg, or Crk). For tyrosine phosphorylation, Gab1 directly or indirectly binds to growth factor receptors for hepatocyte growth factor (HGF) (▶ MET), EGF, vascular endothelial cell growth factor (VEGF), and PDGF, in addition to Src family and JAK, though tyrosine kinases involved depend on cell types and ligands. Exact mechanisms are still to be clarified for signaling from activated tyrosine kinase to Gab proteins which contain no PTB domain, though Gab1 has direct HGF-receptor (MET)-binding domain. It has been suggested that Grb2 and/or Cbl is involved in coupling tyrosine kinase with Gab family. Gab proteins contain several proline-rich motifs that

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can mediate constitutive binding to the SH3 domain of Grb2. The Grb2–Gab complex targets the receptors containing Grb2 SH2 domain binding sites. In addition, tyrosine-phosphorylated FRS2a/SNT-1 docking protein binds to SH2 domain of Grb2, serving as another intermediate protein for coupling Gab to the receptors. Gab1 and Gab 2 are tyrosine phosphorylated by antigen receptors of B and T cells upon stimulation with various cytokines and antigens, whereas M-CSF stimulates Gab2 and Gab3. SHP2 activated by Gab family leads to cell proliferation by activating ERK, whereas PI3-kinase activation to cell survival by activating AKT. Overexpression of tyrosine kinase receptors that are often detected in cancer cells leads to enhanced tyrosine phosphorylation of Gab1, which stimulates proliferation and survival through SHP2-ERK, PI3-kinase-AKT, or Crk-Rac signal transduction pathway. Constitutive activation by in-frame fusion of RET tyrosine kinase, RET-TPC phosphorylate Gab1, leading to BRAF and ERK kinase stimulation. Trp-MET oncoprotein also stimulates tyrosyl phosphorylation of Gab1. Roles of Gab 2 in tumorigenicity have been reported: (i) frequent Gab2 amplification was reported in ▶ breast cancer and (ii) Gab2signaling pathway is used upon transformation by Bcr-Abl. In the former case, receptor activation of ERB2/▶ HER-2/neu enhanced further Gab2 signaling. IRS The protein family of IRS1, IRS2, IRS3, and IRS4 has been identified originally as proteins transducing insulin signals to intracellular effectors for the glucose homeostasis. This family commonly contains PH domain near N-terminus and PTB at the following position. IRS3 may not be expressed in human. Activated insulin receptor (IR) or IGF-1 receptor binds to IRSs by its PTB domain and subsequently induces phosphorylation on multiple tyrosine sites of each IRS, which are binding sites for PI3-kinase, SHC, an adaptor protein, and also for Grb2. IRS2 is unique in that it carries insulin receptor binding domain. IRS1 has a higher binding affinity to Grb2 when compared

Membrane-Linked Docking Protein

to IRS2, thus segregating IRS1 with mitogenic and IRS2 with metabolic outcomes. Oncogenic SV40 T antigen gene transforms human cells through IRS1 signaling, as the transforming ability was suppressed in several human cancer cell lines which do not express IRS1or at low levels. Constitutive phosphorylation of IRS1 and IRS2 appears to stimulate proliferation and motility in mammary cancer cells. IRS4 binds to nonreceptor tyrosine kinase, Brk (breast tumor kinase), which is overexpressed in a high percentage of breast cancers.

Cross-References ▶ BCR-ABL1 ▶ Breast Cancer ▶ Chromosomal Translocations ▶ Epidermal Growth Factor-like Ligands ▶ Fibroblast Growth Factors ▶ GTPase ▶ HER-2/neu ▶ MAP Kinase ▶ MET ▶ PI3K Signaling ▶ RAS Genes ▶ Receptor Tyrosine Kinases ▶ RET ▶ SH2/SH3 Domains ▶ Src

References Horejsi V, Zhang W, Schraven B (2004) Transmembrane adaptor proteins: organizers of immunoreceptor signaling. Nat Rev Immunol 4:603–616 Huang L, Watanabe M, Chikamori M et al (2006) Unique role of SNT-2/FRS2b/FRS3 docking/adaptor protein for negative regulation in EGF receptor tyrosine kinase signaling pathways. Oncogene 25:6457–6466 Sarmay G, Angyal A, Kertesz A et al (2006) The multiple function of Grb2 associated binder (Gab) adaptorscaffolding protein in immune cell signaling. Immunol Lett 104:76–82 Thirone ACP, Huang C, Klip A (2006) Tissue-specific roles of IRS proteins in insulin signaling and glucose transport. Trends Endocrinol Metab 12:70–76 Yamasaki S, Saito T (2004) Inhibitory adaptors in lymphocytes. Semin Immunol 16:421–427

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See Also (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163 (2012) ALK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 128. doi:10.1007/978-3-642-16483-5_178 (2012) Grb2 adaptor protein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1603. doi:10.1007/978-3-642-164835_2514 (2012) PH domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2835. doi:10.1007/978-3-642-16483-5_4486 (2012) Phosphatidyl inositol 3-kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2865. doi:10.1007/978-3-642-164835_4527 (2012) PTB domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3117. doi:10.1007/978-3-642-16483-5_4850 (2012) SH2 domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3397. doi:10.1007/978-3-642-16483-5_5280 (2012) SH3 domain. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3399–3400. doi:10.1007/978-3-642-16483-5_5281 (2012) Src family tyrosine kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3498. doi:10.1007/978-3-64216483-5_5467 (2012) T-cell receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3621. doi:10.1007/978-3-642-16483-5_5701

Membrane-Lipid Therapy Francisca Guardiola-Serrano1, David J. López1, Xavier Busquets2 and Pablo V. Escribá2 1 University of the Balearic Islands, Palma de Mallorca, Spain 2 Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain

Synonyms Bioactive lipid therapy; Lipid replacement therapy; Lipid therapy

Francisca Guardiola-Serrano and David J. López both authors contributed equally to this work.

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Definition Membrane-lipid therapy (MLT) involves the use of synthetic or natural compounds to modify the composition and structure of cell membranes as a means to treat diseases such as cancer. Modifying the lipid composition and structure of the membrane produces changes in the subcellular localization and activity of membrane proteins, affecting molecular entities involved in processes such as downstream signaling. Anticancer MLT drugs can specifically inhibit cell proliferation or induce cell differentiation and/or the programmed cell death of tumor cells.

Characteristics Diversity of Membrane-Lipid Composition and Structure Since the fluid mosaic model was first proposed by Singer and Nicolson in the early 1970s, additional details have emerged regarding the structure of the membrane highlighting its complexity. As such, membranes appear not to be as fluid as would be expected if they were merely a homogeneous mixture of lipids, the movement of lipids and proteins through the bilayer also seems somewhat restricted, and there is a higher density of proteins than previously thought. Indeed, we now know that ca. 40% of genes encode membrane proteins according to the Human Protein Atlas, and interestingly, ca. 50% of cancer-related proteins are membrane proteins (www.proteinatlas.org). Moreover, the lipid bilayer does not maintain a uniform thickness, since the flexibility of the acyl chains of phospholipids may vary the width of the membrane’s hydrophobic core in order to accommodate proteins of different sizes. In addition, not only does the lipid and protein composition vary between different cell types but also between different organelles or microdomains within a membrane. In fact, lipids and proteins segregate into regions that have specific compositions, such as large membrane domains (e.g., the basal, lateral, and apical membrane regions of polarized glandular, endothelial, and epithelial cells) and the smaller, yet highly abundant, membrane

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microdomains (lipid rafts, synaptosomes, caveolin domains, coated pits, receptor clusters, etc.). The number of lipid species exceeds the number of different amino acids and nucleic acid bases by orders of magnitude. Thus, the number of combinations of lipid entities, and the fact that their association is not restricted by covalent bonds, means that (i) membrane lipids can organize into many more primary and/or secondary structures than proteins and nucleic acids in vitro and (ii) the relative positions of lipids in membranes are dynamic. When dispersed in aqueous solution, lipids can organize in different ways as a function of their molecular structure, the water concentration, pH, ionic strength, or system pressure. In cell membranes, lipid molecules establish a dynamic lamellar structure, with many proteins using the transient or stable domains as platforms for their activity and for their interactions with other proteins. Moreover, the biophysical features of the membrane-lipid bilayer may affect its surrounding environment, and in fact, how lipids are organized (membrane structure) affects their interactions with membrane proteins, thereby modulating their activity (membrane functions). In general, modifications to the structure and/or lipid composition of the membrane are reflected by functional changes. For instance, oleic acid (18:1 cisD9) induces an important increase in the non-lamellar, inverted hexagonal phase propensity of membranes, thereby altering their interactions with G proteins that consequently affect their activity. By contrast, neither the trans analog of oleic acid (elaidic acid) nor stearic acid (18:0) markedly affect the lamellar phase of membranes, and accordingly, they do not influence G-protein activity, highlighting the specificity of this mode of action. In this sense, the lipid composition and structure of membranes most likely define the presence and activity of specific proteins, while the proteins themselves may in turn influence the lipid composition and structure of membranes, as occurs with phospholipases or other enzymes involved in lipid metabolism.

Membrane-Lipid Therapy

Mechanisms Involved in Membrane-Lipid Therapy MLT drugs can regulate features of cell membranes implicated in protein-protein and proteinlipid interactions (Figs. 1 and 2), thereby modulating cell signaling pathways such as those involved in the pathological state of cancer cells. These drugs can act through one or more of the following mechanisms, which may vary depending on the type of cancer cell and on the pathways altered in the abnormal state of the cell. MLT Drugs that Target the Cell Plasma Membrane

Changes in membrane-lipid composition and structure may modify the affinity of a specific protein for the membrane, or for its surrounding proteins, thereby modulating the signals transduced by the cell. For example, reductions in the phosphatidylethanolamine/sphingomyelin (PE/SM) ratio induced by the anticancer drug 2-hydroxyoleic acid hinder the receptor tyrosine kinase-Ras and Ras-Raf interactions at the membrane, provoking a reduction in MAPKassociated signaling. MLT drugs can modify the biophysical properties of the membrane in several ways, thereby controlling the activity and localization of proteins: Destabilization of Membrane Structure, as Occurs with Anticancer Drugs Like Anthracyclines and Hexamethylene Bisacetamide (HMBA) The membrane destabilization induced by these drugs regulates the interaction of G proteins and proteins like PKC with lipid membranes, thereby disrupting downstream signaling. Hyperfluidization of the Lipid Membrane Heat and chemical membrane fluidization augment the total ceramide levels in Jurkat cells, which drives the coalescence of raft microdomains in order to form large ceramideenriched membrane platforms that stimulate the apoptosis induced by the TNF-related apoptosisinducing ligand (TRAIL).

Membrane-Lipid Therapy

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M Membrane-Lipid Therapy, Fig. 1 Mechanisms of action of molecules acting via membrane-lipid therapy. MLT drugs may act through different mechanisms, either targeting plasma membrane lipids or proteins (1), nuclei and gene expression regulation (2), or protein lipidation enzymes that contribute to protein-membrane interactions (3). MLT drugs aim to change the composition and structure of the lipid bilayer in order to modify the localization

of membrane proteins in specific domains or to change protein-protein and protein-lipid interactions at the membrane. In addition, through lipid replacement, MLT drugs may also serve to repair the mitochondrial dysfunction caused by aging or other conditions, including cancer. The modifications produced by MLT drugs may affect gene regulation

Lipid-Raft Microdomain Remodeling Lipid rafts are liquid-ordered membrane microdomains enriched in cholesterol and sphingomyelin. These structures accommodate proteins that participate in signal transduction, and therefore, they serve as signaling platforms. MLT drugs can induce the coalescence of proteins inside rafts or they may exclude proteins from them. For example, the MLT drug 2-hydroxyoleic acid regulates the interaction of the Fasn death receptor with rafts in leukemia cells, inducing its ligand-free oligomerization and the ensuing activation of the extrinsic

caspase cascade, thereby provoking cell death. By contrast, natural omega-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic and eicosapentaenoic acid, exclude EGFR from raft microdomains, disrupting EGF/Ras/Erk signaling in breast cancer cells. Changes in Membrane-Lipid Composition Cancer cells have a lipid composition distinct to that of normal cells. Extensive changes in lipid content can modify the membrane structure, thereby affecting a number of cellular functions

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Membrane-Lipid Therapy

Membrane-Lipid Therapy, Fig. 2 Interaction of membrane lipids and proteins. Lipids with bulky polar head, like phosphatidylcholine (yellow) and phosphatidylserine (red), have a molecular shape similar to a cylinder. In turn, phosphatidylethanolamine (green) has a small polar head and increases the negative curvature strain of membranes (hexagonal phase propensity). 2-Hydroxyoleic acid (blue)

also favors non-lamellar phases. The scheme also shows a peripheral membrane protein interacting with fatty acid moieties of phospholipids (A) or deep hydrophobic regions of the membrane (B) in non-lamellar-prone regions. Electrostatic interactions with the polar head of charged phospholipids (C) also participate in the binding of this protein to the membrane

and potentially reversing the pathological state of the cell. These MLT drug modifications can take place in different ways, including:

the activity of SM synthase. Direct exposure to SM does not induce cell cycle arrest, whereas the enhanced sphingomyelinase activity provoked by 2-hydroxyoleic acid augments the SM available, and it inhibits cell growth and differentiation.

Regulation of Enzyme Activities that Modulate Lipids Lipids are not only biomembranebuilding blocks, but they also participate in cell signaling. Ceramide is one such example, the metabolism of which is tightly regulated in cells. An increase in the levels of this sphingolipid can drive a cell toward apoptosis, autophagy, or cell cycle arrest, while deficiencies in ceramide contribute to cell survival. In this sense, PUFAs have been shown to increase sphingomyelinase activity in cancer cells, leading to a decrease in the SM content and to the production of ceramide. Interestingly, cancer cells have less SM compared to non-tumor, resting cells. In this context, 2-hydroxyoleic acid increases the amount of SM in non-small lung cancer A549 cells by increasing

Lipid Replacement Therapy Is Used to Restore Damaged Lipids Through Supplementation with Membrane Phospholipids and Antioxidants The aim is to replace damaged membrane phospholipids that accumulate during aging and in other conditions in order to restore correct cell function. For instance, the adverse effects of some cancer therapies include the overproduction of reactive oxygen species (ROS) and thus excessive oxidative stress which damage healthy tissues. As a consequence several side effects appear in patients treated with antineoplastic drugs; the most common one is fatigue which is related to an inefficient production of energy at the

Membrane-Lipid Therapy

mitochondria. Mitochondria are one of the main targets of oxidative stress where it causes loss of mitochondrial transmembrane potential and function. Some studies are being held to treat these side effects by restoring mitochondrial function through lipid replacement therapy. MLT Drugs that Target the Nucleus

Many aspects of lipid metabolism and function are initiated in the nucleus, and indeed, nuclear lipids with regulatory effects have been well documented. Thus, MLT drugs could act in the nucleus in several ways: Lipid-Mediated Regulation of Nucleic Acid Function MLT drugs could bind to transcription factors, such as PPARs, and induce gene expression. For example, omega-3 PUFAs are described to activate the PPARg transcriptional pathway and to induce the synthesis of the transmembrane protein syndecan-1, a heparan sulfate proteoglycan that acts as a tumor suppressor by inducing apoptosis and inhibiting cell growth. The Regulation of Gene Expression to Alter Membrane-Lipid Composition Cancer cells have an intense requirement for lipids that is often satisfied through a robust program of lipid synthesis, principally to sustain the generation of membranes. Hydrophobic MLT drugs may modulate the lipid-binding transcription factors PPARs, RXR, RAR, and LXR, regulators of the expression of genes controlling the synthesis, and oxidation of fatty acids. MLT Drugs Target Protein-Lipid Interactions

Even if it were considered as conventional therapy, the binding of certain drugs to proteins that modify lipid-protein interactions may be well classed as MLT. By preventing correct protein lipidation, these drugs inhibit protein binding to the membrane, thereby hindering interactions with downstream signaling proteins. For example, drugs that act as farnesyl transferase and geranylgeranyl transferase inhibitors impair the

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binding of Ras to the membrane, preventing its isoprenylation and later interaction with upstream and downstream signaling proteins. Development of Membrane-Lipid Therapy Drugs and Ongoing Studies In the 1980s, it was shown that the semisynthetic anthracycline doxorubicin could kill cancer cells by directly interacting with the cell membrane. Since then, several drugs have been designed in order to regulate membrane-lipid structure and to abolish the off-target interactions and side effects of anthracyclines (Fig. 3). Cytotoxic drugs inhibiting protein-membrane interactions are being evaluated in clinical trials. For instance, inhibitors of farnesyl transferase like lonafarnib/Sch. 66336 and Zarnestra or tipifarnib have been evaluated, or are under evaluation, in several clinical trials, in combination with other drugs to treat glioblastoma, chronic myeloid leukemia, or non-small cell lung cancer (www. ClinicalTrials.gov identifiers: NCT00050336, NCT00038597, NCT00038493, NCT00079339, etc.). The type and abundance of the different lipid classes, and their fatty acid composition, define the membrane microdomains that generate platforms to which proteins can bind and where signal transduction can take place. Accordingly synthetic drugs have been designed to regulate the organization of membrane microdomains in a similar way to that of natural-occurring lipids. The omega-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are among the natural-occurring lipids currently being used in cancer therapy, and studies are ongoing to analyze the potential of DHA in preventing recurrence in breast cancer survivors (www.ClinicalTrials.gov identifier NCT01849250). While there are several benefits to use nutritional factors as therapeutic treatments for cancer, the main concern is their metabolism. Therefore, current research focuses on improving their efficacy by combining them with traditional therapies, such as cytotoxic drugs or radiotherapy. Indeed, a phase II clinical trial concluded that DHA

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Membrane-Lipid Therapy

Membrane-Lipid Therapy, Fig. 3 Chemical structures of 2hydroxyoleic acid (1), hexamethylene bisacetamide (2), edelfosine (3), miltefosine (4), daunomycin (5), propofolDHA (6), and NEO6002 (7). Carbon atoms are shown in gray, hydrogen in light gray, phosphorous in orange, oxygen in red, and nitrogen in light blue

enhanced the antimetastatic potential of anthracyclines, with no adverse side effects. In order to improve the efficacy of natural fatty acids, and in line with the studies of anthracyclines as antitumor drugs, a series of 2-hydroxylated fatty acids have been rationally designed. This research led to the development of 2-hydroxyoleic acid, an oleic acid derivative with a hydroxyl group on the alpha carbon that is currently in a phase I/IIa clinical trial for the treatment of glioma and other solid tumors (www.ClinicalTrials.gov identifier NCT01792310). 2-Hydroxyoleic acid regulates the lipid bilayer by reducing the lamellar-to-inverted hexagonal phase transition temperature (reducing the surface packing pressure). In addition, it enhances SM synthase activity to produce a concomitant increase in the SM content specifically in cancer cell membranes. Interestingly, the increase of this sphingolipid is not observed in non-tumor cells,

and it appears to be a functional switch that changes the cell’s status from proliferating to quiescent. 2-Hydroxylinoleic acid, ABTL0812, is another hydroxylated fatty acid derivative capable of regulating the structure of the cell membrane, and it has successfully completed a phase I/Ib clinical trial for advanced cancer (www. ClinicalTrials.gov identifier NCT02201823). One interesting chemical modification of a natural lipid is the hybrid compound constituted by the anesthetic propofol covalently bound to DHA. Interestingly, both molecules have antitumor activity in vitro, and this and other related derivatives have been studied as antitumor agents. Another example is the molecule NEO6002, which results from the combination of gemcitabine, a nucleoside analog, with cardiolipin, a phospholipid typically found in mitochondria. The lipid modification favors the internalization of the compound into the cell and

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seems to be less toxic and more effective than gemcitabine alone. Single alkyl phospholipids are synthetic resistant analogs of lysophosphatidylcholine that act at the cell membrane to induce apoptosis of tumor cells. The prototype was edelfosine, yet other analogs like miltefosine or perifosine have since appeared. Miltefosine predominantly exerts its cytotoxic activity by acting on enzymes linked to phospholipid turnover and membrane signal transduction, and it was clinically effective in patients with skin metastasis of breast cancer and cutaneous lymphomas. In addition, there is an ongoing phase II clinical trial to assess the combination of perifosine and torisel for malignant gliomas (www.clinicaltrial.gov, NCT02238496). Specific alterations to the lipid composition and lipid metabolism of cells have been associated with cancer. For instance, antigen-519, a molecular biomarker of certain breast cancers, is fatty acid synthase (FASN), and selective inhibitors of this enzyme inhibit cell growth and induce apoptosis in cancer but not in normal cells in vitro. Moreover, a commercially available FASN inhibitor, orlistat, blocks cell growth and induces apoptosis of breast cancer cells when administered in combination with the antibody trastuzumab. Some other genes involved in lipid metabolism, such as olr1 and glrx, are upregulated in breast and prostate tumors, and the enzymes involved in lipid metabolism ACACA, FASN, INSIG1, and SREBP1 are strongly expressed in breast cancer and are associated with poor patient survival. Moreover, the median survival of glioma patients markedly and significantly decreases as the number of deregulated membrane-lipid metabolism genes increases. These data highlight the relevance of membrane lipids in the cell physiology and etiopathology of tumors.

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▶ Caveolins ▶ G Proteins ▶ Gemcitabine

References Escribá PV, Sastre M, Garcia-Sevilla JA (1995) Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures. Proc Natl Acad Sci U S A 92:7595–7599 Escribá PV, González-Ros JM, Goñi FM, Kinnunen PK, Vigh L, Sánchez-Magraner L, Fernández AM, Busquets X, Horvath I, Barceló-Coblijn G (2008) Membranes: a meeting point for lipids, proteins and therapies. J Cell Mol Med 12:829–875 Escribá PV, Busquets X, Inokuchi J-i, Balogh G, Török Z, Horváth I, Harwood JL, Vígh L (2015) Membrane lipid therapy: modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment, progress in lipid research. In press. doi:10.1016/j.plipres.2015.04.003, http://dx.doi. org/ Ibarguren M, López DJ, Escribá PV (2014) The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health. Biochim Biophys Acta 1838:1518–1528 Lladó V, López DJ, Ibarguren M, Alonso M, Soriano JB, Escribá PV, Busquets X (2014) Regulation of the cancer cell membrane lipid composition by NaCHOleate: effects on cell signaling and therapeutical relevance in glioma. Biochim Biophys Acta 1838:1619–1627

MEN1 ▶ Multiple Endocrine Neoplasia Type 1

MEN1B ▶ p27

Cross-References

MEN4 ▶ 2-Hydroxyoleic Acid ▶ Apoptosis

▶ p27

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Menopausal Symptoms After Breast Cancer Therapy Vineeta Singh1, Christobel Saunders2 and Martha Hickey3 1 School of Surgery and Pathology, QEII Medical Centre, Sir Charles Gairdner Hospital, Nedlands, WA, Australia 2 School of Surgery and Pathology, QEII Medical Centre, University of Western Australia, Crawley, WA, Australia 3 Obstetrics and Gynaecology, The University of Melbourne, Parkville, VIC, Australia

Definition Menopause is the permanent cessation of menstruation resulting from loss of ovarian activity. Natural menopause is diagnosed following 12 months of amenorrhea, which is not due to a pathological cause. Menopause secondary amenorrhea (early menopause, surgical menopause) can also be induced by surgery, chemotherapy, and radiotherapy.

Characteristics Introduction ▶ Breast cancer is one of the most common causes of morbidity and mortality in women of middle years in the western world. There has been a reduction in annual breast cancer death rates in almost all western countries, which may be due to the increasing use of breast screening and adjuvant systemic therapy (admixture populations). As a result, more women are becoming longterm breast cancer survivors. A common side effect of breast cancer treatment is the earlier onset of menopause or menopausal symptoms. Many women who are premenopausal at diagnosis will develop premature menopause resulting from chemotherapy, endocrine therapy, bilateral oophorectomy, or ovarian radiation and may experience severe and long-lasting menopausal symptoms. All

Menopausal Symptoms After Breast Cancer Therapy

endocrine therapy will generally induce menopausal symptoms, often for the duration of its use. This can occur in women at any age, regardless of menopausal status. Premenopausal women who are older at the time of chemotherapy are more likely to experience chemotherapy-induced ovarian failure leading to permanent menopause. Those who do not have ovarian failure may subsequently go through menopause at a younger age. Younger patients require higher cumulative dosage of chemotherapy to induce ovarian failure and may have temporary menopausal symptoms. However, it is not possible to predict reliably whether an individual woman will go through menopause as a result of her chemotherapy treatment. A significant number of peri- and postmenopausal women are taking hormone replacement therapy (HRT) when they are diagnosed with breast cancer. Cessation of HRT commonly leads to a return of menopausal symptoms. Recurrence of menopausal symptoms and premature menopause in breast cancer patients can have significant negative impact on quality of life, body image, sexual function, and self-esteem. The clinical challenge lies in providing safe and effective therapy to these patients. The safety of conventional hormonal treatments for menopausal symptoms has not been established following breast cancer. Furthermore, many women will have hormone receptor-positive tumors, and endocrine therapy aims to reduce circulating estrogen and/or suppress ovarian activity. HRT may undermine the efficacy of these treatments. Hence, there is an urgent need to provide some alternative form of treatment to alleviate menopausal symptoms in breast cancer patients. Treatment Options Menopausal symptoms are variable in nature and severity, and each woman requires careful individual assessment. This should include an assessment of the likely cause, nature, frequency, severity of symptoms, and impact on quality of life of these symptoms. For some women, reassurance that her symptoms are normal and are likely to reduce over time, combined with practical suggestions for minimizing their impact, is

Menopausal Symptoms After Breast Cancer Therapy

sufficient management. Available treatment interventions fall into two main groups. Synthetic Hormonal Compound

Tibolone has weak estrogenic, progestogenic, and androgenic actions. It shows similar efficacy to HRT in reducing hot flushes and vaginal dryness and may improve sexual function more effectively than HRT. It improves bone density but its impact on fracture rates is not known. Unwanted side effects of tibolone include a reduction in circulating high-density lipoproteins, uterine bleeding, body pain, and headache. The safety of tibolone in women with a prior history of breast cancer is subject of a large randomized controlled trial (the LIBERATE study) due to be reported at the end of 2007. Nonhormonal Therapies

A variety of nonhormonal therapies aimed at reducing vasomotor symptoms or urogenital atrophy have been subjected to randomized controlled trials. The most common vasomotor symptom is hot flushes. However, the basic physiology underlying hot flushes is poorly understood. It is hypothesized that reduced estrogen levels cause an induction of noradrenergic activity leading to the feeling of warmth, heat loss, and sweating. Because the placebo effect is profound and prolonged in treatments for hot flushes, it is essential that new therapies should be tested for adequate durations (at least 12 weeks) in randomized placebo controlled trials. Phytoestrogens. Phytoestrogens are plant derivatives either in the form of ▶ isoflavones found in soy products or lignans found in flax seeds. They exhibit both estrogenic and antiestrogenic effects, depending on their concentration. Although some studies have shown small benefits in hot flushes from using phytoestrogens, a met-analysis failed to demonstrate any benefit over placebo. Their safety in women with a history of breast cancer is unknown. Black cohosh (Cimicifuga racemosa). It was traditionally used by native North Americans for treating range of menstrual problems. It appears to act by competing for the ▶ estrogen receptor and binds to gamma-aminobutyric acid (GABA),

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serotonin, and dopamine receptors. Again, results have been conflicting with some studies finding black cohosh mildly effective in decreasing severity and frequency of hot flushes, genitourinary symptoms, and mood disturbances in few studies. However, a meta-analysis of randomized controlled trials has shown no overall benefit of black cohosh over placebo. Black cohosh has been associated with several episodes of hepatotoxicity and liver failure. Neuroendocrine agents. (i) Clonidine – It is a central alpha-adrenergic agonist, found to be moderately more effective than placebo in treating hot flushes. Some studies have also shown improved quality of life in breast cancer patients treated with clonidine for hot flushes. Clonidine is one of the very few nonhormonal therapies licensed for the treatment of hot flushes. There are no safety data regarding clonidine and breast cancer. (ii) Selective serotonin and noradrenaline reuptake inhibitors (SSRI/SNRI) – Over the menopause transition, serum serotonin levels fall and monoamine oxidase activity increases. Venlafaxine is an SNRI, which has demonstrated short-term (up to 6 weeks) superiority over placebo in reducing hot flushes. Side effects of venlafaxine are largely dose related and include nausea, constipation, dry mouth, and decreased appetite. Other SSRI including paroxetine, fluoxetine, and citalopram have also demonstrated short-term efficacy in reducing hot flushes although their safety in some breast cancer patients has been questioned due to their interactions with the metabolism of tamoxifen. The GABA analogue gabapentin may be more effective than SSRI/SNRI and is well tolerated by most women, though side effects may include somnolence, dry mouth, heart palpitations, and dizziness. Gabapentin effectively reduced hot flushes for at least 12 weeks in randomized placebo controlled trials of women some of whom have had a personal history of breast cancer. Treatment of Urogenital Atrophy and Related Symptoms Lack of estrogen results in vaginal atrophy, urinary frequency, dysuria, and incontinence. In breast cancer patients, urogenital atrophy is relatively uncommon in tamoxifen users, due to the

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estrogenic effects of tamoxifen in the vagina. Urogenital atrophy is more common in those using ▶ aromatase inhibitors. Topical estrogens are more effective than nonhormonal preparations in the treatment of urogenital atrophy. They have been widely used following breast cancer but reports, which state that topical estradiol may increase circulating estrogen levels in women using aromatase inhibitors, have raised some concern. Topical estradiol is also available and may be a safer choice following breast cancer. Sexual dysfunction is often multifactorial, and the management of urogenital atrophy should include a discussion of relationship problems, loss of libido, and depression. Physiotherapy and lubricants such as olive or almond oil may also be helpful for some women. Treatment of Bone Loss ▶ Osteoporosis is a common condition seen in older postmenopausal women. Endocrine therapy for breast cancer may increase the risk of osteoporosis. Abrupt withdrawal of circulating estrogens occurs following chemotherapy-induced ovarian failure, oophorectomy, or gonadotropinreleasing hormone (GnRH) or following treatment with an aromatase inhibitor (AI) in the postmenopausal setting. Chemotherapy alone does not cause osteoporosis if not associated with ovarian failure. Bone loss and increased fracture risk (especially nonhip fractures) are major concerns for long-term breast cancer survivors. Tamoxifen shows agonistic estrogenic activity in bone and reduces risk of fracture in postmenopausal but not in premenopausal women. In postmenopausal women, AIs have a deleterious effect on bone density and may increase the risk of fracture. A baseline bone mineral density to be repeated up to annually is advised. A bisphosphonate in conjunction with AI may mitigate the deleterious effect of AI on bone. Lifestyle modifications to minimize bone loss including diet, supplementary calcium weightbearing exercise, stopping smoking, and minimizing alcohol consumption should be encouraged. Calcium and vitamin D supplements should also be advised to any women at risk.

Menopausal Symptoms After Breast Cancer Therapy

Cardiovascular Complications In women with early breast cancer, one of major reported causes of deaths is cardiovascular diseases. The American Heart Association guidelines stratify women in three groups based on their 10-year probability of having a coronary event. Universal recommendations for all groups are moderate exercise, cessation of smoking, body weight aimed at less than 25 BMI (body mass index) and waist circumference less than 35 in., limited intake of saturated fat and cholesterol, and dietary modification to include grains, fruits and vegetables, and fish. Some drugs used in the treatment of breast cancer can have adverse cardiovascular effects. Tamoxifen increases the incidence of thromboembolic events and may increase triglyceride levels and lower low-density lipoproteins (LDLs). AIs have less impact on lipid profile, but their longterm effects on cardiovascular disease are not yet known. Tibolone may also have an adverse effect on LDLs in some women. Conclusion Menopausal symptoms are common in women treated for breast cancer. Estrogen-containing HRT is the most effective treatment for menopausal symptoms but is not recommended following breast cancer, even for those with estrogen receptor-negative disease. There is an urgent need for safe and effective nonhormonal treatments. Several nonhormonal treatments have been studied with varied results and outcomes. Lifestyle modifications are an integral part of treatment especially for osteoporosis and cardiovascular diseases. Younger women who develop premature menopause often need psychological assessment and support. Topical estrogens are effective for vaginal atrophy but their safety in women using aromatase inhibitors is not established. Gabapentin and clonidine appear effective for hot flushes but have significant side effects. Several short studies support the efficacy of SNRI and SSRI but their medium to long-term efficacy is not established. Very few studies have addressed effective nonhormonal treatments for symptoms other than hot flushes. Because of the complexity of these cases, management of

Merlin

menopausal symptoms following breast cancer is best conducted within a multidisciplinary environment and with individualized care.

References Brufsky A et al (2007) Zoledronic acid inhibits adjuvant letrozole-induced bone loss in postmenopausal women with early breast cancer. J Clin Oncol 25(7):829–836 Couzi RJ, Helzlsouer KJ, Fetting JH (1995) Prevalence of menopausal symptoms among women with a history of cancer breast and attitude towards estrogen replacement therapy. J Clin Oncol 13(11):2737–2744 Friedrich M et al (1998) The influence of tamoxifen on the maturation index of vaginal epithelium. Clin Exp Obstet Gynecol 25(4):121–124 Hickey M, Saunders CM, Stuckey BG (2005) Management of menopausal symptoms in breast cancer patients: an evidence based approach. Lancet Oncol 16(9):687–695 Nelson HD, Vesco KK, Haney E et al (2006) Non-hormonal therapies for menopausal hot flashes. Systemic review and meta-analysis. JAMA 295(17):2057–2070

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nervous system. Biallelic inactivation of NF2 is also uniformly observed in sporadic vestibular schwannomas and in a large percentage of sporadic meningiomas. In addition, somatic genetic changes resulting in biallelic inactivation of NF2 occur in about 50% of malignant pleural mesotheliomas, which are mesodermally derived tumors unrelated to those found in the NF2 syndrome. Development of bilateral vestibular schwannomas is a hallmark of the NF2 syndrome. Mutations of the NF2 gene have also been reported, at lower frequencies, in other malignancies such as carcinomas of the kidney, breast, colon, thyroid, and liver. Because the NF2 gene product exhibits significant homology to the highly conserved ezrinradixin-moesin (ERM) protein family, it was named Merlin, for Moesin-ezrin-radixin-like protein. It has also been dubbed schwannomin for its suppressive effect against schwannoma formation.

Characteristics

Merlin Guang-Hui Xiao and Joseph R. Testa Fox Chase Cancer Center, Philadelphia, PA, USA

Synonyms NF2 gene product; Schwannomin

Definition Merlin is the protein product of the NF2 ▶ tumor suppressor gene. Germline mutations of the NF2 predispose affected individuals to the development of ▶ neurofibromatosis type 2, an autosomal dominant disorder that affects 1 in 33,000–40,000 people. Development of bilateral vestibular schwannomas is a hallmark of this NF2 syndrome; additional tumor types include schwannomas of other cranial, spinal, and cutaneous nerves, cranial and spinal meningiomas, as well as ependymomas and gliomas of the central

Merlin is a 595-amino-acid protein made up of three structural domains: an N-terminal domain that exhibits highest homology to ▶ ERM proteins, an a-helical region and a unique C-terminal domain. Like ERM proteins, but distinct from all known tumor suppressors, Merlin is cytoskeleton associated and localizes predominately at the cell membrane, especially at ruffling edges. The growth-suppressing function of Merlin has been connected to its role in contact inhibition of cell proliferation. Notably, the primary phenotypic consequence of Merlin deficiency in mouse primary cells is loss of contact-dependent growth inhibition, which is accompanied by a lack of cellular adherens junction formation. In agreement with this contention, phosphorylation and expression levels of Merlin are regulated by cell density, and Merlin is required for the establishment of stable adherens junctions. Biochemical experiments have connected Merlin mechanistically to signaling of the small GTPase known as Rac, which activates an array of intracellular signaling pathways involved in cell

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motility, invasiveness, and proliferation. Like ERM proteins, Merlin also interacts with CD44, a cell surface receptor for hyaluronan and many other extracellular matrix components. It has been proposed that binding of Merlin to CD44 mediates contact inhibition at high cell densities. At low cell density, Merlin is growth permissive and exists in a complex with ezrin, moesin, and CD44. It has been demonstrated that CD44 function is mediated by Rac1 activation. The binding of hyaluronic acid to CD44 activates Tiam1, a Rac1-specific guanine nucleotide exchange factor, thereby stimulating Rac1 activity and cytoskeleton-mediated cell migration. In addition to CD44 signaling, Merlin mediates contact inhibition of cell proliferation by blocking recruitment of Rac to the plasma membrane, while Merlin mutants allow for recruitment of Rac to matrix adhesions and support continued growth of confluent cells. Mechanistically, Merlin competes with Rich1, a Rac1 GTPase activating protein, for Angiomotin binding at tight junctions. Merlin releases Rich1 from Angiomotin, allowing Rich1 to suppress Rac1 activity and Ras-MAPK signaling. Merlin’s tumor suppressor activity is regulated by various post-translational modifications. In response to active Rac or Cdc42, but not active Rho, Merlin is phosphorylated on serine 518 (S518), and this phosphorylation is mediated by p21-activated kinase (Pak), a common downstream target of both Rac and Cdc42. Merlin S518 has also been found to be a substrate of PKA. Phosphorylation of Merlin by Rac/Pak or PKA signaling inactivates Merlin tumor suppressor activity by changing its conformation from an “active” open state into a “less active” closed state. Indeed, a phospho-mimic mutant Merlin S518D impairs Merlin’s ability to suppress cell cycle progression and motility, whereas a S518A phosphorylation-defective form of Merlin can act as a constitutively active form of the tumor suppressor in modulating cell proliferation. Intriguingly, wild-type Merlin and Pak appear to operate in a negative feedback loop, because unphosphorylated Merlin can directly bind Pak and inhibit its activity. Conversely, the myosin phosphatase MYPT1-PP1δ has been shown to

Merlin

dephosphorylate Merlin at S518 and activate its tumor suppressor function. In addition to Pak and PKA, AKT kinase phosphorylates Merlin on residues T230 and S315, which promotes Merlin degradation by polyubiquitination. It has been reported that Merlin can be sumoylated on lysine residue K76 and that this post-translational modification is essential for Merlin’s tumor suppression function. Interestingly, AKT or Pak2 kinase activity appears to be required for sumoylation of Merlin, although the possibility of a feedback loop between Merlin and AKT requires further investigation. Over the years, Merlin has been recognized as a plasma membrane and cortex localized tumor suppressor. A role for Merlin in the nucleus has been uncovered. With optimized immunostaining methodology, prominent nuclear staining of Merlin has been observed in multiple cell types. The active form of Merlin translocates to the nucleus, binds to and inhibits the E3 ubiquitin ligase CRL4DCAF1, which is implicated in cell proliferation, DNA replication, and ubiquitination. Silencing of CRL4DCAF1 caused profound growth inhibition in Merlin-null schwannoma cells from NF2 patients. Intriguingly, re-expression of Merlin and depletion of DCAF1 induce a similar gene expression program. Merlin is a multifunctional tumor suppressor that interacts with numerous proteins implicated in cell morphology, motility, proliferation, metastasis, and survival. In addition to its wellestablished role in inhibiting the Rac1-Pak axis, Merlin’s tumor suppressive activity has been linked to growth factor receptor signaling. Merlin inhibits focal adhesion kinase (FAK)-Src, RasERK and PI3K-AKT signaling, as well as the activation of mTORC1 independently of AKT. Regarding FAK-Src signaling, it is notable that Merlin deficiency has been found to be predictive of FAK inhibitor sensitivity in mesothelioma. Evidence suggested that weak cell-cell adhesions in Merlin-deficient tumor cells lead to a greater dependence on cell-extracellular matrix (ECM)induced FAK signaling and to a heightened vulnerability to FAK inhibitor treatment. Merlin loss-driven tumorigenesis has also been linked to CRL4DCAF1-mediated inhibition of the

Mesoblastic Nephroma

Hippo pathway kinases LATS1 and LATS2. CRL4DCAF1 has been shown to target LATS1 and LATS2 for ubiquitylation and inhibition in the nucleus, thereby activating YAP-driven transcription. Conversely, re-expression of Merlin induces phosphorylation and inactivation of YAP in NF2-mutant cells. It is also noteworthy that studies have uncovered recurrent somatic mutations of LATS1 and LATS2 in malignant mesotheliomas that lack point mutations in NF2, suggesting that genetic alterations affecting Hippo signaling are mutually exclusive. Merlin interacts with various elements of this pathway, which is thought to play a critical role in tumor suppression by restricting proliferation and promoting apoptosis by inactivating the Yap oncoprotein. When Merlin is mutated or not expressed in MM, oncogenic Pak and FAK signaling is activated, whereas tumor suppressive Hippo signaling is inactivated, resulting in transcription of oncogenic genes and miR-29, leading to activation of Akt and mTORC1. Thus, the emerging picture of Merlin’s tumor suppressive function is one of significant complexity.

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Definition A type of stem cell found in bone marrow (marrow stromal cells) and many other locations, e.g., adipose tissue. Defined by their ability to adhere to plastic when cultured, and of trilineage differentiation into fat, cartilage, and bone. Found to be highly malleable and can be switched to many other different cell types. Possess an extensive proliferative potential and ability to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, and neurons.

Cross-References ▶ Stem Cell Plasticity ▶ Stem Cell Telomeres

Mesenteric Fibromatosis ▶ Desmoid Tumor

References Li W, et al (2010) Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. Cell 140:477–490 McClatchey AI, Giovannini M (2005) Membrane organization and tumorigenesis – the NF2 tumor suppressor, Merlin. Genes Dev 19:2265–2277 Petrilli AM, Fernandez-Valle C (2016) Role of Merlin/NF2 inactivation in tumor biology. Oncogene 35: 537–548 Shapiro IM, et al (2014) Merlin deficiency predicts FAK inhibitor sensitivity: a synthetic lethal relationship. Sci Transl Med 6: 237ra68 Zhang N, et al (2010) The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev Cell 19:27–38

Mesoblastic Nephroma Rhoikos Furtwängler Universitätsklinikum des Saarlandes, Klinik für Pädiatrische Onkologie und Hämatologie, Homburg/Saar, Germany

Synonyms Atypical congenital mesoblastic nephroma; Congenital mesoblastic nephroma; Fetal hamartoma of the kidney; Leiomyomatous hamartoma of the kidney; Malignant mesenchymal nephroma of infancy

Mesenchymal Stem Cells Definition Synonyms MSCs

Mesoblastic nephroma is a rare mesenchymal tumor of the kidney, which typically occurs in

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neonates and young infants and constitutes the most frequent renal tumor in the first 3 months of life.

Characteristics Mesoblastic nephroma (MN) is a rare malignant tumor of the kidney, which originates from the mesenchymal anlage (metanephrogenic blastema). The typical patient is a neonate or young infant. In the first 3 months of life, MN is the most frequent renal tumor; hence, preoperative chemotherapy is not recommended for renal tumors in this age group. It accounts for 5% of all childhood renal tumors. According to the German Childhood Cancer Registry, the cumulative incidence of MN is estimated at 1 in 150,000 children (0–14 years) in Germany. Contrary to ▶ nephroblastoma, MN has a 2–1 male preponderance. Metastatic disease at diagnosis is an extreme rarity. Diagnosis The clinical course of the MN even in the connatal or neonatal period is mainly indolent. Incidental diagnosis because of a palpable abdominal mass or a routine ultrasound scan is frequent. Abdominal protrusion, pain, emesis, hypertension, and hematuria can be clinical symptoms of MN forcing parents to contact a pediatrician. A growing number of MNs are diagnosed even antenatally, as prenatal ultrasound scans become more and more standard care. Nevertheless MN can be the reason for preterm deliveries, and in reports throughout the 1970s and 1980s a remarkable number of MNs had been found in stillbirths. Ultrasound scan and MRI scan are the gold standard in the diagnosis of childhood renal tumors. Differential diagnosis includes ▶ neuroblastoma, ▶ nephroblastoma, nephroblastomatosis, ▶ cystic nephroma, ▶ rhabdoid tumor (RT), clear cell sarcoma of the kidney (CCSK), and other rarer renal tumors. In ultrasound scan, MN usually appears as a solid, intrarenal heterogeneous structure of mixed echotexture with circumferential echopoor areas. Furthermore, a

Mesoblastic Nephroma

so-called vascular ring sign can be seen in colorcoded duplex sonography. The latter corresponds to a heterogeneous circumferential contrast enhancement in MRI scan. A pseudocapsule, often seen in the case of nephroblastoma, is normally not visualized in MN. However, nephroblastoma cannot be distinguished from MN by imaging alone, contrary to the typical neuroblastoma (retroperitoneal and extrarenal mass with calcifications that often involves the vena cava and/or aorta and displaces the kidney). Unfortunately, there is no specific MN marker yet, but catecholamines in urine and blood, Neuron-Specific-Enolase (NSE), meta-Iodo-Benzyl-Guanidin (mIBG) scan, and fine needle aspiration can be of additional use in distinguishing neuroblastoma from MN. Final diagnosis, especially differing MN from other renal tumors, can only be made by histology; therefore, primary surgery is the next step in diagnosis. Histology and Genetics MN histology can be polymorphic and challenging. This has been the reason for a variety of synonyms for MN over the years. However, classical mesoblastic nephroma is actually distinguished from a cellular and a mixed-type mesoblastic nephroma. The classical type MN is characterized by leiomyomatous histology with spindle cells in bundles, the absence of necrosis, and rare mitoses. It grows in a rather displacing than infiltrative manner, but tends to develop finger-shaped projections, thereby trapping normal renal tissue. The cellular-type MN shows contrary to the classic-type MN a high cellularity and mitotic index and an atypical growth pattern (invasion of other adjacent structures other than connective tissue, hemorrhage, necrosis, and fleshy areas); thus, it can infiltrate the adipose capsule of the kidney and beyond. Earlier, this MN subtype had also been called atypical congenital MN. The mixed-type MN has a predominantly classic histological pattern but with some islets of cellular-type MN within. The local tumor extension is staged according to the Children’s Oncology Group (COG) or International Society of Paediatric Oncology (SIOP)

Mesoblastic Nephroma

working classification of renal tumors of childhood. Macroscopic residual tumor, rupture, and positive resection margins are reasons for a local stage III. Studies have shown that cellular- and occasionally mixed-type MN often differs genetically from classic MN. The translocation t(12;15)(p13; q25), detectable in most cellular- and occasionally in mixed-type MN, gives rise to an ETV6-NTRK3 gene fusion. Moreover, trisomy 11 is found in those MNs too. The translocation t(12;15)(p13; q25) can be of use to differ cellular MN from the sometimes histologically similar clear cell sarcoma of the kidney. This translocation has also been proven in (congenital) infantile fibrosarcoma, thus unraveling a genetic link between those two histologically similar lesions. A study has shown receptor tyrosine kinase activation of GAB2, STAT, and Hippo pathway; this could potentially be of future use in diagnosis and targeted therapy. Treatment Primary surgery is the gold standard in MN treatment, leading to high cure rates by complete resection. Complete nephrectomy including the adipose capsule is the gold standard, since MN’s finger-shaped projections can be inadvertently left behind in the case of partial nephrectomy. Furthermore, sometimes cystic growth pattern, especially in cellular-type MN, makes the tumor vulnerable and can cause rupture during surgery. MN is susceptible to chemotherapy which is indicated in the case of non-resectable tumor or relapse. Adjuvant chemotherapy is discussed controversially in patients with stage III cellular-type MN and older age. Standard nephroblastoma and sarcoma treatment regimens are effective in the treatment of MN (dactinomycin, vincristine, cyclophosphamide, doxorubicin, ifosfamide, etoposide, and carboplatin). As MN can be treated by surgery and chemotherapy, radiation therapy is not indicated in these very young patients. Prognosis Though MN, if completely resected, has an excellent prognosis (overall and event-free survival rate

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>95%), metastatic and local relapses occur, but almost exclusively in cellular-type MN. Contrary, classical and mixed-type MN may be followed even in the case of positive surgical margins or local spillage. Second-look surgery can be indicated to achieve downstaging. Relapsed patients can develop metastasis to the lung, more seldom to the liver or the brain. Incomplete resection, cellulartype MN, and older age correlate with relapse but are probably not independent risk factors.

Cross-References ▶ Cystic Nephroma ▶ Nephroblastoma ▶ Neuroblastoma ▶ Rhabdoid Tumor

References Furtwaengler R, Reinhard H, Schenk I et al (2006) Mesoblastic nephroma – a report from the Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH). Cancer 106:2275–2283 Gadd S, Beezhold P, Jennings L et al (2012) Mediators of receptor tyrosine kinase activation in infantile fibrosarcoma: a Children’s Oncology Group study. J Pathol 22:119–130 Knezevich SR, Garnett MJ, Pysher TJ et al (1998) ETV6NTRK3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma. Cancer Res 58:5046–5048 Schenk JP, Schrader C, Furtwaengler R (2005) MRI-morphology and staging of congenital mesoblastic nephroma: evaluation of a collection with 20 patients. RöFo 177:1373–1379 Vujanic GM, Sandstedt B, Harms D et al (2002) Revised International Society of Paediatric Oncology (SIOP) working classification of renal tumors of childhood. Med Pediatr Oncol 38:79–82

See Also Clear cell sarcoma of the kidney. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 880. doi:10.1007/978-3-642-16483-5_1210 Metanephrogenic blastema. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2259. doi:10.1007/978-3-642-16483-5_3668 SIOP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3413. doi:10.1007/9783-642-16483-5_5319

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Mesothelin

Characteristics

Mesothelin Mesothelin results from the cleavage of a 69 kDa preproprotein encoded by the human MSLN gene (NC_000016) that spans over 16 exons and occupies about 8 kb of human chromosome 16 (Fig. 1). The alternative splicing of MSLN gene results in at least two mesothelin transcript variants, variant 1 encoded by MSLN1 (NM_005823) and variant 2 encoded by MLSN2 (NM_013404) (Fig. 2). Variant 1 is predominant and variant 2 differs from variant 1 by 24 bp inserted in exon 11. The cleavage of MSLNencoded preproprotein at the cationic motif TILRPRFRREVE in exon 9 releases the megakaryocyte potentiating factor (MPF), a 31 kDa soluble protein, while mesothelin remains membrane bound. MSLN gene is composed of 16 exons spanned over 7733 bp. In human, MSLN gene is located in 16p13.3. MSLN1 and MSLN2 gene variants encode a 69 kDa preproprotein that is cleaved in position 937 after the cationic motif RXRR into two mature proteins, MPF and mesothelin. MSLN1 encodes mesothelin variant 1 and MSLN2 encodes variant 2, a less abundant, alternate splice with a 24-bp insertion in position 1282. Soluble mesothelins arise through a cleavage of GPI-anchored variants 1 or 2 or, less frequently, an 82-bp insertion in position 1828 of MSLN1

Nathalie Scholler Center for Cancer, SRI Biosciences, Menlo Park, CA, USA

Synonyms CAK1 antigen; MPF; MSLN; SMR; Soluble mesothelin-related proteins

Definition The name mesothelin was given by K. Chang and I. Pastan to a 40 kDa, GPI-anchored glycoprotein (GPI-anchored protein) that is physiologically expressed at the cell surface of mesothelial cells lining the pleura, pericardium, and peritoneum (Chang and Pastan 1996). Mesothelin is an epithelial marker highly expressed by ▶ cancer cells from diverse origins, including ovarian adenocarcinomas, pancreatic adenocarcinomas, and ▶ mesotheliomas. Soluble forms of mesothelin can be found in fluids from patients affected by these cancers.

Exon 7 Exon 8 Exon 9 Exon 6

Cleavage site (RXRR)

Exon 5

Exon 10 Exon 11

Exon 4

24-bp insertion site of variant 2

Exon 3

Exon 12

Exon 2

Exon 13 Exon 1

Exon 14

Exon 15 Exon 16 82-bp insertion site of variant 3

Signal peptide MSLN gene - 7733 bp

Mesothelin, Fig. 1 Exon map of MSLN gene

Mesothelin

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Mesothelin 1282

MSLN1 (variant 1)

2052 bp

MSLN2 (variant 2)

2076 bp

(variant 3)

1195 bp

1828

RXRR 24-bp insert

GPI-anchored 82-bp insert 212-pb frameshift

SMR

Hydrophilic C-terminal domain

Mesothelin, Fig. 2 MSLN variants

Mesothelin, Fig. 3 Glycosylphosphatidylinositol (GPI) anchor

C H2N

Protein

C

NH2 CH2 CH2 Phosphoryl-ethanolamine residue

O P

O

O 6

Manα1-2

R8 O

HO HO

R1, R2 R3 R4, R7 R5 R6 R8

R7 O Manα1-6

HO HO

O

= = = = = =

Fatty acid or Ceramide ± Fatty acid at C2 or C3 ± Phosphoothanolamine ± Galα1-2Galα1-6 [Galα1-2]Galα1-3 ± GalNAcβ1-4 ± Manα1-2

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2

O 6

Manα1-4

R6

R4 O 4 CH2OH

O GlcNα1-6

R3

OH HO

R5 O

O

HO NH2

6

Phosphatidyl-inositol group

Carbohydrate-containing linker

–O

3

OH

O

2

O –

O

P

O

O CH2CH R1

resulting in a 212-bp frameshift mutation that transforms the GPI anchor motif into a hydrophilic domain (SMR or variant 3). The insertion of an 82-bp fragment results probably from a lack of splicing of the last intron (Figs. 1 and 2). The core of a GPI anchor is composed of a hydrophobic phosphatidylinositol group and a

CH2 R2

carbohydrate-containing linker made of glucosamines and mannoses linked to a phosphorylethanolamine residue (Fig. 3). The GPI anchor is linked to the C-terminal amino acid of a mature protein via the phosphorylethanolamine residue. R1 and R2 fatty acids anchor the protein to cell membranes.

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Mesothelin homologues were described for chimpanzees (MSLN gene, Pan troglodytes, accession DQ052446), macaque (LOC698095 gene, Macaca mulatta, accession XM_ 001087333), bovine (LOC516237 gene, Bos taurus, accession XM_594389), dog (LOC611363 gene, Canis familiaris, accession XM_849019), rat (Msln gene, Rattus norvegicus, accession NM_031658), mouse (Msln gene, Mus musculus, accession NM_018857), and chicken (LOC 416534 gene, Gallus gallus, accession XM_414835). Mouse mesothelin is 55% homologous to its human counterpart and its protease target sequence TVIHPRFRRDAE is conserved. Tissue Expression Serial analysis of gene expression (SAGE), oligonucleotide, and cDNA arrays have been used by independent laboratories to identify large sets of genes expressed at higher levels in cancer tissues compared with normal tissues. MSLN transcripts were found consistently highly overexpressed in nonmucinous ovarian carcinomas and invasive ductal adenocarcinoma (pancreatic ductal adenocarcinoma (DA)) and significantly overexpressed in mesotheliomas and pulmonary, gastric/esophageal, and colorectal adenocarcinomas. By realtime PCR-based analysis, normal transcript levels of MSLN and CCL23, GAGED2, SPAG6, ST18, WT1, and PRAME genes were found to be associated with continuous complete remission of pediatric acute myeloid leukemia, while elevated levels of at least one of these genes were found prior to relapse in most patients. The upregulation of MSLN transcripts correlates well with mesothelin cell-surface expression. Studies by immunohistochemistry (IHC) confirm that mesothelin is significantly more expressed by pancreatic cancer cells as compared to very weak or no expression in chronic pancreatitis or normal pancreatic ducts; mesothelin is found with a higher frequency in invasive intraductal papillary mucinous neoplasms (IPMN) than in noninvasive. Comparative analyses with tissue microarrays showed that the mesothelin protein expression in ovarian cancer depends on the histological type (75% of expression in serous papillary tumors, 30% in endometrioid, and less than

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20% in mucinous). Diffuse mesothelin staining in primary high-grade ovarian serous carcinomas may be correlated with prolonged survival. Finally, mesothelin was also found to be expressed at a low level by a variety of adenocarcinomas, including endometrium, stomach/esophagus, pulmonary, ▶ Brms1, and colorectal. None or very rare mesothelin expressions (less than 5%) were reported for carcinomas of the prostate, bladder/ureter, liver, kidney, and thyroid. Regulation Mechanisms that regulate MSLN transcription levels and mesothelin cell-surface expression or release as a soluble form in patient fluids are not well understood. Several pathways have been explored. MSLN gene was found hypomethylated in pancreatic ductal adenocarcinoma, consistently with the inverse correlation between mRNA expression and DNA methylation described in numerous cancers. Also, mesothelin upregulation in carcinomas has been associated with a misregulation of Wnt signal transduction pathway. In mouse mammary epithelial cells, Wnt-5a downregulates mesothelin expression, perhaps through antagonism of the Wnt/beta-catenin pathway, while in human colon cancer cells, the forced expression of an N-terminal b-catenin binding site-deficient high mobility group (HMG)-box T-cell factor 1 is associated with the upregulation of several GPI-anchored adhesion molecules, including mesothelin. Furthermore, the overexpression of mesothelin in exon 9 GISTs suggests that mesothelin regulation could be linked to the intracellular signaling cascade triggered by ligand-independent activation of KIT receptor tyrosine kinase. Finally, the presence of soluble mesothelin derived from GPI-anchored forms in ovarian and mesothelioma patient fluids could also be related to the overexpression of GPI PLD observed in some cancer cells. Function Mesothelin knockout mice have no obvious phenotype and produce normal offspring without anatomical or histological abnormalities, which suggests that mesothelin is a nonessential protein. However, mesothelin binds to CA125 in a specific

Mesothelin

and N-linked glycan-dependent manner; thus, CA125-expressing tumor cells could bind specifically to mesothelin-expressing peritoneal lining. Consequently, CA125/mesothelin-dependent cell attachment may play an important role in the peritoneal implantation of ovarian tumor cells. Some indirect experimental evidences suggest that mesothelin could also have a role in neoplastic progression. First, mesothelin upregulation in pancreatic cancer corresponds to the transition from carcinoma in situ to DA. Second, mesothelin expression was found to be upregulated after carcinogenic treatment in rats and its expression correlated with the risk of cancer development. Virgin and parous rats were compared for breast cancer incidence and mesothelin expression, before and after carcinogen exposure. Mesothelin was downregulated before and after treatment in parous rats but only before treatment in virgin rats. It was increased after treatment in virgin rats, as were their cancer development risks. Finally, mesothelin is an immunogenic molecule. This observation is consistent with mesothelin GPI anchor but puzzling considering its overexpression by cancer cells. In fact, most tumor antigens are weak immunogens and this has been a burden for the development of ▶ cancer vaccines. Nevertheless, anti-mesothelin autoantibodies have been found in about 40% of mesothelioma and epithelial ovarian cancer patients, and in some patients with pharynx/larynx ▶ squamous cell carcinoma. In addition, mesothelin-specific CD8 T-cell responses are identifiable with a HLA-A2 mesothelin epitope in both normal and cancer patients and have been reported to be increased after vaccination with pancreatic cancer lines in the presence of GM-CSF. Diagnostic Marker The differential expression of mesothelin at the cell surface of some cancer cells and in patient fluids makes it suitable as a cancer marker. Mouse monoclonal antibodies (mAb) are commercially available for immunohistochemistry (IHC) staining, for example, K1 mAb from Abcam and 5B2 mAb from BioGenex, and for ▶ ELISA assay (Mesomark™ kit from Fujirebio Diagnostics, Inc., corresponding to the assay described by Scholler et al. (1999)).

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Anti-mesothelin mAbs stain mesotheliomas with a high sensitivity and a specificity of 75%. Several studies suggest than mesothelin can be useful for differential diagnostics, alone or combined with a biomarker panel. For example, mesothelin staining can help to identify the origin of mucinous tumors or metastatic adenocarcinomas. Mesothelin staining is much less frequent (less than 20%) in primary ovarian mucinous tumors than in metastatic pancreatic mucinous carcinomas (more than 70%). Used with PSCA, mesothelin appears highly specific for pancreatic adenocarcinoma in fine needle aspirate (FNA) specimens thus useful in categorizing suspicious lesions. In combination with p53, TAG-72, mCEA, and loss of Dpc4, mesothelin staining distinguishes welldifferentiated liver metastasis of DA from bile duct adenomas (BDA) or hamartoma of the liver. Mesothelin, calretinin, and cytokeratin 5/6 were reported to be the best positive mesothelioma markers for differentiating epithelioid mesotheliomas from renal cell carcinomas; mesothelin, WT1, p63, and MOC31 distinguish between epithelioid mesotheliomas and squamous carcinomas of the lung; mesothelin, calretinin, BG8, and MOC 31 distinguish between epithelioid mesothelioma and adenocarcinoma. Finally, a larger panel including mesothelin, CA125, CDX2, cytokeratins 7/20, ▶ estrogen receptor, gross cystic disease fluid protein 15, lysozyme, prostate-specific antigen, and thyroid transcription factor 1 was reported to correctly classify 88% of breast, colon, lung, ovary, pancreas, prostate, and stomach adenocarcinomas. Mesothelin measured in fluids is a promising marker for ovarian carcinomas and mesotheliomas. Mesothelin serum levels are elevated in most late-stage ovarian cancer patients and in most patients with malignant mesotheliomas (MM) at diagnosis, and serum levels correlate with tumor size and increase during tumor progression. This suggests that mesothelin serum levels could be helpful to monitor disease progression and to screen asbestos-exposed individuals for early MM. In addition, the presence of mesothelin in MM pleural fluid can help to better

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discriminate mesothelioma from pleural metastasis. Results, taken together with mesothelin cellsurface expression on pancreatic tumor, suggest that mesothelin could also be a serum marker for some pancreas cancers. A pancreatic tumor cell line was reported to release soluble mesothelin in culture supernatant using an acoustic wave device immunosensor, and mesothelin mRNA was isolated from pure pancreatic juice of pancreatic tumors and was found more abundantly in DA than in IPMN. Finally, various studies combined mesothelin with other biomarkers to form a composite marker (CM) and demonstrated that the use of CM can improve diagnostic test sensitivity. For ovarian carcinoma diagnostic, mesothelin titers have been evaluated in combination with CA125, HE4, M-CSF, ▶ kallikrein, and/or soluble EGF receptor; for mesothelioma diagnostic, mesothelin has been combined with ▶ osteopontin. Interestingly, mesothelin tumor cell expression and serum levels do not strictly correlate. Although mesothelin serum levels are more frequently increased in both MM and ovarian cancer patients whose tumor expressed mesothelin (30% expression by tumor cells), some patients without detectable mesothelin expression on tumor cells have elevated titers of serum mesothelin. The absence of detectable mesothelin by IHC could be due to technical artifact, or alternatively, in some cases soluble mesothelin might be mainly released from normal mesothelial cells that are in contact with the tumor microenvironment, such as in ▶ pleural effusion or peritoneal fluid. Therapeutic Applications Because of its high expression in mesothelioma, ovarian, and pancreatic carcinomas, its immunogenicity, and its nonessential function, mesothelin is an antigen of choice for targeted therapies and cancer vaccines. The specific binding of mesothelin to CA125 suggests that agents able to compete with or to block CA125/mesothelindependent cell attachment could prevent or delay the development of peritoneal metastasis. Antimesothelin natural and recombinant antibodies such as single chain Fragment variables (scFv)

Mesothelin

or single domain antibodies (nanobody) have been generated through mouse immunization and/or screening of recombinant antibody libraries expressed by phage or by yeast. Resulting recombinant antibodies have been used in various antibody-based therapies, including fused to toxin, conjugated with drugs, and expressed as a chimeric antigen by T cells. For example, single chain Fragment variables (scFv) isolated in the Pastan's group or in the Scholler's group were fused with Pseudomonas exotoxin A (SS1P and RG7787), 125I or 111In, or fused to Mycobacterium tuberculosis heat shock protein 70 (scFvMTBHsp70) and used alone or in combination with ▶ Taxol in vitro and in vivo in mouse model systems. SS1P synergized with Taxol in vivo but not in vitro which underlines the importance of the tumor microenvironment for therapeutic strategies. Phase I and II trials with anti-mesothelin recombinant immunotoxins (RIT) were completed in patients with mesothelin-expressing tumors without evidence of non-manageable side effects; however most patients developed neutralizing antibodies against the bacterial toxin prompting to the development of a less immunogenic molecule (RG7787). Phase II clinical trials for patients with advanced malignant pleural mesothelioma are ongoing with anetumab ravtansine (BAY 94-9343), an antibody-drug conjugated (ADC) consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Following the success of CAR T cells targeting refractory hemotologic malignancies, mesothelin-targeting CARs are now actively developed by several groups in the hope to treat multiple solid malignancies Finally, mesothelin also shows promises as a therapeutic or preventative vaccine target against cancer. Strategies are in development and include DNA vaccines, mesothelin virus-like particles, mesothelin presented by bacterium Listeria monocytogenes, and protein or peptide-based mesothelin vaccines. DNA vaccine with a singlechain trimer of HLA-A2 linked to human mesothelin peptides were successfully used to prevent the growth in vivo of HLA-A2-positive human mesothelin-expressing tumor cell lines in

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HLA-A2 transgenic mice. These results suggest possible clinical translation of mesothelintargeted therapy and DNA vaccines for immunotherapy of gynecologic cancers against mesothelin.

Cross-References ▶ BRMS1 ▶ Cancer ▶ Cancer Vaccines ▶ DNA Vaccination ▶ ELISA ▶ Estrogen Receptor ▶ Kallikreins ▶ Lung Cancer ▶ Mesothelioma ▶ Osteopontin ▶ Pleural Effusion ▶ Prostate Cancer Clinical Oncology ▶ Squamous Cell Carcinoma ▶ Taxol ▶ Thyroid Carcinogenesis ▶ Wnt Signaling

References Chang K, Pastan I (1996) Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 93:136–140 Ikezawa H (2002) Glycosylphosphatidylinositol (GPI)anchored proteins. Biol Pharm Bull 25:409–417 Rump A, Morikawa Y, Tanaka M et al (2004) Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J Biol Chem 279:9190–9198 Scholler N, Fu N, Yang Y et al (1999) Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci U S A 96:11531–11536

See Also (2012) CA125. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 577–578. doi:10.1007/978-3-642-16483-5_761 (2012) DNA methylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1140. doi:10.1007/978-3-642-16483-5_1682 (2012) Frameshift mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1454. doi:10.1007/978-3-642-16483-5_2266

2753 (2012) GIST. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1548. doi:10.1007/978-3-642-16483-5_2411 (2012) Glycoprotein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2451 (2012) GPI-anchored protein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1587. doi:10.1007/978-3-642-16483-5_2490 (2012) GPI PLD. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1587. doi:10.1007/978-3-642-16483-5_2489 (2012) HLA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1706. doi:10.1007/978-3-642-16483-5_2765 (2012) Hypomethylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1791. doi:10.1007/978-3-642-16483-5_2922 (2012) KIT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1945–1946. doi:10.1007/978-3-642-16483-5_3228 (2012) Knock-out. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1957. doi:10.1007/978-3-642-16483-5_3237 (2012) Ovarian adenocarcinomas. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2670. doi:10.1007/978-3-642-164835_4294 (2012) Pancreas. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2762–2763. doi:10.1007/978-3-642-16483-5_7055 (2012) Pancreas ductal adenocarcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2779. doi:10.1007/978-3-642-164835_4360 (2012) Wnt/beta-catenin pathway. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3957. doi:10.1007/978-3-642-164835_6256

Mesothelioma Joseph R. Testa1 and Michele Carbone2 1 Fox Chase Cancer Center, Philadelphia, PA, USA 2 University of Hawaii Cancer Center, Honolulu, HI, USA

Synonyms Malignant mesothelioma

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Definition Mesotheliomas are tumors derived from mesothelial cells that form the membranes surrounding the lungs, pericardium, and peritoneum. Mesotheliomas are highly aggressive malignancies, with a median survival of 9 months from diagnosis. The incidence of mesothelioma is higher in men than in women (8:1), and these tumors usually occur during the seventh and eighth decades of life. Mesothelioma is usually associated with occupational ▶ asbestos exposure or to exposure to erionite, a mineral fiber that shares some physical characteristics with crocidolite asbestos. The latter is an amphibole form of asbestos. Iron predominates over magnesium in its composition. Following inhalation, it is strongly carcinogenic. Genetic predisposition to mineral fiber carcinogenesis may contribute to multiple cases of mesothelioma within the same family. There are 3,000 cases of mesothelioma per year in the USA and more than 1,000 in England and Italy. The incidence of mesothelioma continues to increase in spite of measures adopted in the 1970s and 1980s to eliminate (Italy) or reduce (USA) the use of products containing asbestos.

Characteristics Pathogenesis Asbestos

▶ Cancer epidemiology studies and experiments performed in vitro and in animals have clearly linked exposure to crocidolite asbestos, a form of amphibole asbestos, and to erionite, a type of zeolite, to the development of mesothelioma. Other forms of amphibole asbestos, such as tremolite, have also been linked to mesothelioma, but some studies suggest that the risk may be lower compared to crocidolite. Whether other forms of asbestos, such as anthophyllite or chrysotile (the latter is a serpentine (winding or snake-like), cause mesothelioma is still debated. Chrysotile is a magnesium silicate, but in some deposits, varying amounts of iron have replaced magnesium. In the absence of iron, it is thought to be non-

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carcinogenic. The mechanisms responsible for asbestos carcinogenicity have been linked to the secretion of TNF-alpha by mesothelial cells and ▶ macrophages exposed to asbestos, which in turn leads to ▶ NFkB activation. The activation of the NFkB pathway in mesothelial cells appears to allow these cells to survive the cytotoxicity and genetic damage caused by asbestos, and these damaged cells may proliferate into a mesothelioma. Programmed necrosis of mesothelial cells induced by asbestos also causes high mobility group box 1 (HMGB1) release and resultant inflammation linked to the pathogenesis of mesothelioma. Asbestos has been shown to induce the expression of the genes FOS and JUN, which encode the ▶ AP-1 transcription factor that activates various genes critical in the initiation of DNA synthesis. The persistent induction of AP-1 following asbestos exposure may enhance cell division and favor malignant growth. Asbestos is a ubiquitous carcinogen. Therefore, virtually everyone may have some level of exposure. It is hypothesized that there is a threshold level of exposure above which the risk of developing mesothelioma increases. But the threshold is unknown, and individual genetic susceptibility may influence this threshold. Although asbestos fibers induce cytotoxicity in mesothelial cells, cell survival activated by key signaling pathways may promote transformation. Activation of the ▶ AKT signal transduction pathway is one of the major ways in which cell survival is promoted, and activation of AKT has been observed in about 65% of mesotheliomas, which has therapeutic implications, because AKT signaling contributes to tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to ▶ chemotherapy or radiotherapy. Thus, the AKT signaling pathway could be a novel therapeutic target to overcome mesothelioma resistance to conventional therapies. Chromosome Changes

In vitro studies have shown that human mesothelial cells acquire extensive numerical and structural chromosomal abnormalities shortly after exposure to low concentrations of asbestos fibers.

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Mesothelioma, Fig. 1 Mesothelioma pathogenesis. Arrowheads indicate a stimulatory effect. Crossed bars indicate an inhibitory effect. The presence of asbestos or erionite leads to the release of TNF-a by mesothelial cells and macrophages. TNF-alpha causes NFkB activity that in turn protects mesothelial cells from the toxic effects of asbestos. This mechanism prevents asbestos-induced mesothelial cell death. Programmed necrosis of mesothelial cells induced by asbestos also causes release of HMGB1 and resultant inflammation linked to the pathogenesis of mesothelioma. Mesothelial cells that have developed genetic damage because of exposure to mineral fibers can now divide and occasionally progress to become a mesothelioma. In cells that survived asbestos exposure,

asbestos induces autophosphorylation of the epidermal growth factor receptor (EGF-R), which activates the ERK kinases and leads to AP-1 induction and cell division. AKT, a critical regulator of cell survival, is also activated. Most cells exposed to asbestos die due to apoptosis or cell lysis because of the effects of these carcinogens and because of the DNA damage they accumulate (X symbols in the nuclei). Occasionally, a cell that has accumulated numerous chromosomal changes and other genetic alterations escapes cell death to form a malignancy. Individuals exposed to both asbestos and with inherited mutation of BAP1 are at higher risk of mesothelioma when compared to individuals exposed to only one of these carcinogens

While many of the DNA alterations caused by asbestos will either be of no significance or lead to cell death, a few cells may develop perturbations of key cell-cycle regulatory genes, leading to tumor formation and/or ▶ progression. Cytogenetic analysis has revealed multiple chromosome alterations in most human mesotheliomas. Although a specific chromosomal change is not shared by all mesotheliomas, several prominent sites of chromosomal loss have been identified. Deletion of specific regions in the short (p) arms of chromosomes 1, 3, and 9 and long (q) arm of 6, 13, and 15 is repeatedly observed. Loss of a copy of chromosome 22 is the single most consistent numerical change seen in mesotheliomas, and monosomy 4 and monosomy 14 are also common. These recurrent losses frequently occur

in combination in a given tumor. Loss and/or inactivation of ▶ tumor suppressor genes residing in recurrent sites of chromosomal deletion are thought to contribute to the development of mesothelioma (Fig. 1). Tumor Suppressor Genes

Most mesothelioma cell lines exhibit homozygous deletion of the 9p21 chromosome region. ▶ CDKN2A, which encodes the alternative ▶ tumor suppressor gene products p16INK4a and p14ARF, appears to be the critically affected locus in this region. Genes encoding p15INK4b (CDKN2B) and methylthioadenosine phosphorylase (MTAP) are also frequently co-deleted with CDKN2A/ARF in these tumors, although whether CDKN2B and MTAP play a significant

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role in mesothelioma pathogenesis is uncertain at this time. The p16 ▶ INK4 protein binds to the ▶ cyclin-dependent kinase CDK4 to inhibit the catalytic activity of the CDK4/cyclin D enzymes. Therefore, loss/inactivation of p16INK4a leads to cell-cycle deregulation through the loss of a key inhibitor of G1/S phase progression. More than 80% of mesothelioma cell lines have homozygous deletions of one or more p16INK4a exons, and most of the remainder have greatly downregulated expression of p16INK4a due to promoter hypermethylation. ▶ Immunohistochemistry studies suggest that loss of p16INK4a expression is a universal finding in mesothelioma cells, and ▶ chromosomal fluorescence in situ hybridization (FISH) has revealed absence or reduced copy numbers of the p16INK4a gene in most mesothelioma cells. Collectively, these data suggest that loss of at least one copy of p16INK4a occurs in most of these tumors, with the remaining allele silenced by promoter hypermethylation. In a given tumor, all or a subset of cells may contain homozygous loss of p16INK4a, with these cells presumably having a proliferative advantage when placed into long-term culture. Alteration of p16INK4a appears to play a critical role in mesothelial cell tumorigenesis, since experimental re-expression of p16INK4a in mesothelioma cells results in cell-cycle arrest and programmed cell death (▶ apoptosis), as well as inhibition of tumor formation or diminished tumor size. Homozygous deletion of p16INK4a also leads, in most cases, to inactivation of p14ARF, because these two genes share exons 2 and 3, although their reading frames differ. The product of the p14ARF tumor suppressor gene is required for activation of p53 in response to the action of oncogenic proteins such as ▶ RAS. Unlike p14ARF, mutation of the ▶ TP53 gene is uncommon in mesothelioma and has been observed in a subset of tumors that retain the p14ARF locus. Since the product of the p16INK4a gene induces a G1 cell-cycle arrest by inhibiting the phosphorylation of pRb, homozygous loss of p14ARF and p16INK4a would collectively affect both the p53- and pRb-dependent cell regulatory pathways, respectively. In vitro studies have shown that transfer of p14ARF into mesothelioma cell lines induces G1-phase arrest and

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apoptosis. Together, the available evidence suggests that alteration of either product of the CDKN2A locus, i.e., p14ARF or p16INK4a, contributes to the pathogenesis of mesothelioma. Homozygous deletion of p16INK4a/CDKN2A was shown to be a significant independent adverse prognostic factor in mesothelioma. Although the ▶ neurofibromatosis 2 tumor suppressor gene, NF2, predisposes affected individuals primarily to tumors of neuroectodermal origin, somatic mutations of NF2 have occasionally been identified in seemingly unrelated malignancies. NF2 somatic mutations predicting either interstitial in-frame deletions or truncation of the NF2 gene product (▶ merlin) have been reported in 40–55% of mesothelioma cell lines. In many cases, it was possible to confirm the mutation in matched primary tumor DNA. Western blot analyses revealed complete absence of merlin protein expression in cell lines that exhibited alterations of the NF2 gene, suggesting that truncated forms of the protein are unstable. LOH analyses documented allelic loss of the NF2 locus in more than 70% of mesothelioma cases. All cases exhibiting mutation and/or aberrant expression of NF2 showed allelic losses, implying that inactivation of NF2 in mesothelioma occurs via a “two-hit model.” One mechanism by which merlin acts as a tumor suppressor is by inhibiting the activity of ▶ p21-activated kinase (Pak), a downstream target of Rho, Rac, and Cdc42 ▶ GTPases. Merlin exerts an antiproliferative effect, in part, via repression of Pak-induced ▶ cyclin D1 expression. Moreover, Pak regulates ▶ motility in mammalian cells, which raised the intriguing possibility that merlin loss of function, due to biallelic inactivation of NF2, may contribute to ▶ invasion and/or ▶ metastasis of mesothelioma. Re-expression of merlin in an NF2-deficient mesothelioma cells inhibits cell motility, spreading, and invasiveness. Expression of merlin attenuates the phosphorylation of ▶ focal adhesion kinase, a key component of cellular pathways affecting migration and invasion, at a critical phosphorylation site and disrupted the interaction of FAK with oncogenic binding partners. Altogether, these findings suggest that merlin inactivation is a critical step in mesothelioma

Mesothelioma

pathogenesis and is related, at least in part, with upregulation of FAK activity. Merlin also suppresses cellular proliferation by inhibiting the activation of RAS. Merlin counteracts the ▶ ERM (ezrin, radixin, and moesin)-dependent activation of RAS, which correlates with the formation of a complex comprising RAS, ERM proteins, Grb2, SOS, and filamentous actin. Thus, part of the tumor suppressor function of merlin appears to be its interference with Ras- and Rac-dependent signal transfer. Asbestos-exposed Nf2 heterozygous (+/) knockout mice exhibited accelerated mesothelioma formation compared with asbestos-treated wild-type littermates, and loss of the wild-type Nf2 allele, leading to biallelic inactivation, was observed in all of the asbestos-induced mesotheliomas from Nf2 knockout mice. Like in the human disease counterpart, mesotheliomas from Nf2 knockout mice show frequent homologous deletions of the Cdkn2a (p16Ink4a/p19Arf) locus and adjacent Cdkn2b (p15Ink4b) tumor suppressor gene, as well as reciprocal inactivation of the ▶ TP53 gene in a subset of tumors that retain Arf. As in the human disease, mesotheliomas from Nf2 knockout mice also showed frequent activation of Akt kinase. Thus, this mouse model of environmental carcinogenesis faithfully recapitulated many of the molecular features of human mesothelioma and thus has significant implications for preclinical testing of novel therapeutic drugs. The involvement of similar somatic genetic and cell signaling perturbations in human mesothelioma and in mesothelioma of asbestos-exposed Nf2 knockout mice suggests that specific sets of molecular events cooperate in mesothelioma pathogenesis. In 2011, we reported germline (inherited) mutations of the BRCA1-associated protein 1 (BAP1) tumor suppressor gene in two families with multiple mesotheliomas and/or uveal (ocular) melanomas as well as other tumors, the first proof that genetic factors can influence the risk of mesothelioma. A concurrent study reported germline BAP1 mutations in two families with benign melanocytic tumors as well as cutaneous and uveal melanoma. Since 2011, a steadily increasing number of papers have confirmed

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these findings and/or extended the disease phenotype to other cancer types. Collectively, these findings have led to the identification of a BAP1 tumor predisposition syndrome characterized by mesothelioma, cutaneous and uveal melanoma UM, renal cell carcinoma, and other tumors due to heterozygous germline mutations of BAP1. Somatic mutations of BAP1 are observed in up to 65% of mesotheliomas. Notably, MM associated with germline BAP1 mutations have been shown to have a significantly better prognosis compared to sporadic mesotheliomas. BAP1 encodes a nuclear ubiquitin carboxy-terminal hydrolase, and current evidence indicates that inactivation of BAP1 has been shown to cause alterations in DNA repair, chromatin remodeling, and cell cycle regulation, which collectively contribute to tumor formation. Studies with genetically engineered heterozygous (+/mut) Bap1 mice have shown to have a significantly higher incidence and accelerated rate of asbestos-induced mesotheliomas than wild-type littermates, validating the notion that genetic factors can predispose to the carcinogenic effects of asbestos. Moreover, mesothelioma cells from Bap1+/mut mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure. Diagnosis Chest pain and accumulation of fluid in the pleura or abdomen are often the first symptoms. Radiological tests and/or cytology often reveals that the patient has a tumor and may raise the suspicion of mesothelioma, but the final diagnosis relies on thoracoscopy or laparoscopy and histological evaluation of the tumor biopsy. Because of their undifferentiated state, mesothelial cells can evolve along either an epithelial cell type or a fibroblast type of differentiation. Morphologically, mesotheliomas are distinguished as epithelial and sarcomatous, the latter also known as fibrous or spindle cell mesothelioma. Sufficient

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sampling of cells, however, will often reveal both components, thus the terminology of biphasic or “mixed-type” mesothelioma. Epithelial mesotheliomas, especially when well differentiated, have median survivals of 2 years or more; fibrous mesotheliomas have a median survival of 6 months from diagnosis and are resistant to therapy. Patients with biphasic mesotheliomas have a median survival slightly more favorable than patients with fibrous mesothelioma. Histologically, epithelial mesotheliomas must be differentiated from carcinomas of the lung and ▶ breast cancer, biphasic mesotheliomas from ▶ synovial sarcoma and ▶ carcinosarcoma, and sarcomatous mesotheliomas, from other types of sarcomas. Electron microscopy (EM) reveals long branching microvilli in epithelial mesotheliomas and is very useful to distinguish these tumors from carcinomas. ▶ Immunohistochemistry also helps to distinguish mesotheliomas from carcinomas. All epithelial mesotheliomas stain diffusively and strongly positive for calretinin, a ▶ vitamin Ddependent ▶ calcium-binding protein involved in calcium signaling; it is an immunohistochemical marker for ▶ mesothelioma and can be used to help differentiate different types of ▶ lung cancer. All epithelial mesotheliomas also are positive for the ▶ Wilms tumor (WT1) protein (nuclear staining is specific, membranous staining is not), for keratin 7, Cam5.2, CK5/6, and pankeratin AE1/AE3 (membranous staining). All of these markers can also be found in various other tumors, including carcinomas; therefore, the diagnosis of mesothelioma requires that the epithelial markers are negative. Mesotheliomas are negative for ▶ CEA, LeuM1 (CD15), BerEp4 (up to 5% of mesothelioma may show focal positivity with this marker), Moc31, TTF-1, and B72.3. Carcinomas are positive for some of these epithelial markers and are usually negative for calretinin and for ▶ Wilms tumor protein. Sarcomatous mesotheliomas stain positive for cytokeratin, which distinguish them from other sarcomatous tumors of the pleura, and may also stain positive for ▶ Wilms tumor protein (WT1) and calretinin. All mesotheliomas are malignant. However, histologically, malignant mesotheliomas have occasionally been associated with long

Mesothelioma

survivals of years or even decades. Whether the latter should be called mesotheliomas is debatable. There are also some unrelated tumors called “benign mesotheliomas,” which are histologically different from mesotheliomas and are clinically benign. It is unfortunate that the term mesothelioma is often used to identify these lesions, because it generates confusion and patient anxiety. Multicystic mesothelioma, also called multilocular peritoneal inclusion cyst, is a totally benign mesothelial lesion characteristically formed by multiple cysts arranged in grapelike clusters. Adenomatoid mesotheliomas are benign mesothelial lesions of the genital system. Mesothelioma of the atrioventricular node is neither a mesothelioma nor a tumor. This lesion represents congenital heterotopia of the endodermal sinus in the atrioventricular node. Well-differentiated papillary mesothelioma is found more often in the abdominal cavity of young women. Histologically, it is formed by multiple papillary structures covered by cytologically benign mesothelial cells. The lesion is benign, but there are cases in which several years after diagnosis the patient developed a true mesothelioma. Clinical Approaches To date, therapeutic options for mesothelioma remain very limited. Standard therapy with Alimta and ▶ cisplatin is associated with a prolonged survival of about three months. Patients with early-stage disease are good candidates for surgical resection. Surgery should be performed by surgeons experienced with this type of procedure to reduce the risk of operative mortality, which in experienced hands is about 2%. Either pleurocotomy or extrapleural pleurectomy has the potential to cure very early disease. However, mesotheliomas are usually diagnosed at an advanced stage when curative resection is not possible. Chemotherapy has been disappointing to date, although new drugs (e.g., Onconase) are presently being tested alone or in combination with conventional chemotherapeutic agents. The discovery of two serological markers, ▶ mesothelin and ▶ osteopontin, that are elevated in patients with mesothelioma has raised the

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possibility of screening individuals at high risk of mesothelioma (e.g., asbestos workers) for early detection, which might be associated with a prolonged survival and possibly a cure. Prospective clinical trials are ongoing in Cappadocia, Turkey, where there is a mesothelioma epidemic associated with erionite exposure and genetic predisposition, to validate these ▶ serum biomarkers. Moreover, clinical trials are planned to test among high-risk mesothelioma cohorts, the possible benefit of chemoprevention using Onconase, an RNAse inhibitor that has had beneficial effects in some mesothelioma patients in the absence of significant adverse side effects.

▶ Neurofibromatosis 2 ▶ Nuclear Factor-kB ▶ Osteopontin ▶ p21 ▶ Progression ▶ RAS Genes ▶ Retinoblastoma Protein, Clinical Functions ▶ Serum Biomarkers ▶ SV40 ▶ Synovial Sarcoma ▶ TP53 ▶ Tumor Necrosis Factor ▶ Tumor Suppressor Genes ▶ Vitamin D ▶ Wilms’ Tumor

Biological

and

Cross-References ▶ Akt Signal Transduction Pathway ▶ Aneuploidy ▶ AP-1 ▶ Apoptosis ▶ Asbestos ▶ Breast Cancer ▶ Calcium-Binding Proteins ▶ Cancer Epidemiology ▶ Carcinoembryonic Antigen ▶ Carcinosarcoma ▶ CDKN2A ▶ Chemotherapy ▶ Chromosomal Fluorescence in Hybridization ▶ Cisplatin ▶ Cyclin D ▶ Cyclin-Dependent Kinases ▶ ERM Proteins ▶ Fluorescence in Situ Hybridization ▶ Focal Adhesion Kinase ▶ GTPase ▶ Immunohistochemistry ▶ INK4A ▶ Invasion ▶ Lung Cancer ▶ Macrophages ▶ Merlin ▶ Mesothelin ▶ Metastasis ▶ Motility

References

Situ

Altomare DA, Vaslet CA, Skele KL et al (2005) A mouse model recapitulating molecular features of human mesothelioma. Cancer Res 65:8090–8095 Carbone M, Emri S, Dogan UA et al (2007) A mesothelioma epidemic in Cappadocia: scientific developments and unexpected social outcomes. Nat Rev Cancer 7:147–154 Carbone M, Yang H, Pass HI, et al. (2013) BAP1 and cancer. Nat Rev Cancer 13:153–159. Pass HI, Lott D, Leonardo F et al (2005) Asbestos exposure, pleural mesothelioma and osteopontin levels. N Engl J Med 353:1564–1573 Testa JR, Cheung M, Pei J, et al. (2011) Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet 43:1022–1025. Xu J, Kadariya Y, Cheung M, Pei J, et al. (2014) Germline mutation of Bap1 accelerates development of asbestosinduced malignant mesothelioma. Cancer Res 74:4388–4397. Yang H, Bocchetta M, Krocznyska B et al (2006) TNF-alpha inhibits asbestos-induced cytotoxicity via a NFkB dependent pathway, a possible mechanism for asbestos induced oncogenesis. Proc Natl Acad Sci U S A 103:10397–10402

See Also (2012) Allelic loss. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 137. doi:10.1007/978-3-642-16483-5_186 (2012) Biopsy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 415. doi:10.1007/978-3-642-16483-5_644 (2012) Calretinin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 602. doi:10.1007/978-3-642-16483-5_6743

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2760 (2012) Carcinogen. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 644. doi:10.1007/978-3-642-16483-5_839 (2012) Cell cycle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) CD15. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 693. doi:10.1007/978-3-642-16483-5_921 (2012) Cdc42. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 704. doi:10.1007/978-3-642-16483-5_955 (2012) Chromosomal aberrations. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 838. doi:10.1007/978-3-642-16483-5_1138 (2012) Crocidolite. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 998. doi:10.1007/978-3-642-16483-5_1378 (2012) Cytokeratins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1051. doi:10.1007/978-3-642-16483-5_1472 (2012) Deletion. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1080. doi:10.1007/978-3-642-16483-5_1553 (2012) Epithelial cell. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1291– 1292. doi:10.1007/978-3-642-16483-5_1958 (2012) Erionite. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1307. doi:10.1007/978-3-642-16483-5_1986 (2012) Exon. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1360. doi:10.1007/978-3-642-16483-5_2059 (2012) FISH. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1415–1416. doi:10.1007/978-3-642-16483-5_2197 (2012) FOS. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1446. doi:10.1007/978-3-642-16483-5_2252 (2012) Genetic susceptibility. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1528. doi:10.1007/978-3-642-16483-5_2384 (2012) Heterotopia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1689. doi:10.1007/978-3-642-16483-5_6744 (2012) Homozygous deletion. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1729. doi:10.1007/978-3-642-16483-5_2807 (2012) JUN. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1929. doi:10.1007/978-3-642-16483-5_3186 (2012) Knock-out mouse. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1957. doi:10.1007/978-3-642-16483-5_3239 (2012) Laparoscopy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1979. doi:10.1007/978-3-642-16483-5_3276 (2012) Loss of heterozygosity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/

Mesothelioma Heidelberg, pp 2075–2076. doi:10.1007/978-3-64216483-5_3415 (2012) Mesothelial cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2241. doi:10.1007/978-3-642-16483-5_3646 (2012) Monosomy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2372. doi:10.1007/978-3-642-16483-5_3831 (2012) Mutagenic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3909 (2012) Onconase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2632. doi:10.1007/978-3-642-16483-5_4232 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Point mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2934. doi:10.1007/978-3-642-16483-5_4653 (2012) PRb. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2967. doi:10.1007/978-3-642-16483-5_4708 (2012) Promoter hypermethylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4769 (2012) Radiotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3158. doi:10.1007/978-3-642-16483-5_4926 (2012) Resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3263. doi:10.1007/978-3-642-16483-5_5052 (2012) Rho. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3302. doi:10.1007/978-3-642-16483-5_5099 (2012) Sarcoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3335. doi:10.1007/978-3-642-16483-5_5161 (2012) Simian virus 40. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3411. doi:10.1007/978-3-642-16483-5_5307 (2012) Targeted therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3610. doi:10.1007/978-3-642-16483-5_5677 (2012) TNF-?. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3713. doi:10.1007/978-3-642-16483-5_5841 (2012) Transcription factor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3752. doi:10.1007/978-3-642-16483-5_5901 (2012) Tremolite. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3782. doi:10.1007/978-3-642-16483-5_5968 (2012) Tumor suppressor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3803. doi:10.1007/978-3-642-16483-5_6056 (2012) Two-hit model. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3821. doi:10.1007/978-3-642-16483-5_6070

MET (2012) Zeolite. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3975. doi:10.1007/978-3-642-16483-5_6297

MET Kyle Furge and George F. Vande Woude Van Andel Research Institute, Grand Rapids, MI, USA

Definition Met is a member of the ▶ receptor tyrosine kinase family. Like other members of this protein family, Met possesses a highly glycosylated extracellular ligand binding region, a hydrophobic membranespanning region, an intracellular region that contains a tyrosine kinase domain, and a C-terminal multisubstrate binding site that mediates interactions with several signal transduction pathways upon receptor activation. Activation of receptor tyrosine kinases results from binding a protein growth factor. The growth factor that activates Met is hepatocyte growth factor/scatter factor, referred to as HGF/SF, as it was identified independently as both a growth factor for hepatocytes (HGF) and a fibroblast-derived cell motility factor, scatter factor (SF). It was later discovered that HGF and SF were identical proteins and HGF/SFMet signaling can induce different biological affects depending on the cell context. Since then, HGF/SF-Met signaling has been implicated in a variety of cellular responses including proliferation, motility, ▶ invasion, chemotaxis, and morphogenic differentiation. Through these actions, Met regulates a diverse series of biological processes ranging from lumen formation to neuronal development to ▶ cancer cell invasion and ▶ metastasis.

Characteristics Met was identified in the early 1980s as an ▶ oncogene. A chromosome rearrangement

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resulted in the fusion of the N-terminal proteinprotein dimerization motif of tpr (translocated promoter region) to the C-terminal tyrosine kinase domain of met. The resulting chimeric protein, Tpr-Met, has high constitutive tyrosine kinase activity and can potently transform cells in vitro. Isolation of the tpr-met cDNA led to the identification of the full-length Met receptor. Human met spans over 120 kb of genomic sequence located on chromosome 7q31. The gene consists of 21 exons that when spliced together produce a primary mRNA transcript that encodes the complete receptor. Several met splice variants have been identified, but the physiological role of these variants is not well understood. Mature, full-length human Met is produced by proteolytic cleavage of a single 1408 amino acid, partially glycosylated precursor protein into an N-terminal a-chain, which is entirely extracellular, and a larger C-terminal b-chain (Fig. 1). The b-chain consists of an extracellular portion, a membrane-spanning segment, and an intracellular region that contains the tyrosine kinase domain and the C-terminal binding site. Similar to other receptor tyrosine kinases, such as the insulin receptor, the a- and b-chains of Met are joined via a disulfide linkage and further glycosylated during transport to the cell surface. While at the cell surface, Met clusters together and may interact with cell ▶ adhesion molecules, such as cadherin, to help regulate cell-cell interactions. Met is normally expressed in the epithelial cells of several embryonic and adult tissues (kidney, liver, lung, skin, stomach, and placenta). In contrast, HGF/SF expression is usually restricted to the surrounding mesenchyme (fibroblast stroma and other mesenchymal cells). Several aspects of organogenesis, such as tissue growth and morphogenic differentiation, are regulated by interactions between the organ epithelia and the surrounding mesenchyme. Endocrinemediated signaling between HGF/SF and Met is believed to play an important role in regulating normal epithelial-mesenchyme interactions. If HGF/SF-Met signaling is inhibited by gene disruption in mice, the mouse embryos die in early gestation with several defects in liver and placental organogenesis. Furthermore, while not directly

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MET Met receptor α

Semaphorin



β



Semaphorin PSI

Cystein-rich Cbl IPT/TIG

Transmembrane Juxtamembrane

Kinase

C-terminal binding site

∗ ∗ ∗ ∗ ∗ ∗ ∗

pY1003 PLC-γ pY1234 pY1235 pY1349 pY1356

Gab1 Gab2 Gab10

Pl(3)K-Akt

Shp2

SOS-Ras Src

Crk-C3G-Rap1 -Cdc42 / Rac1 -Raf

Ship1

MET, Fig. 1 The Met receptor tyrosine kinase stimulates multiple signal transduction pathways. The Met receptor is a membrane-spanning a/b heterodimeric protein that binds the HGF/SF growth factor. The extracellular region of Met contains a SEMA domain (gray boxes), a PSI domain, and four tandem IPT/TIG domains (white ovals). The intracellular region of Met contains a tyrosine kinase domain (black box). Phosphorylation of tyrosine residues (-pY) within the kinase domain, following growth factor binding and receptor dimerization, activates the tyrosine kinase

domain. Phosphorylation of the tyrosine near the membrane-spanning region serves o inhibit the kinase activity of this receptor, through Cbl binding and subsequent receptor degradation. Phosphorylation of tyrosine residues in the C-terminal end of Met activates a multisubstrate binding site that mediates interactions with several adapter and signal transduction proteins to stimulate growth, motility, and morphogenic differentiation. Asterisks (*) indicate where missense mutations have been identified in various human tumors

observed in the mouse mutants (partly due to the early embryonic lethality and partly due to compensation by other factors), HGF/SF-Met signaling has been implicated in regulating epithelialmesenchymal transitions and other epithelialmesenchyme interactions such as mammary gland duct formation, lung tubule formation, and kidney development in organ culture models. In addition, several epithelial cell types undergo significant morphogenic differentiation to form branched tubule structures complete with interior lumens when grown in a three-dimensional matrix and stimulated with HGF/SF (Fig. 2). While other growth factors such as epidermal growth factor receptor (EGFR) ligands may induce proliferation and/or movement, HGF/SF is somewhat unique in its ability to induce all of the cellular responses required for branching morphogenesis, which

refers to the formation of treelike networks of epithelial tubes through reiterated cycles of branch initiation, branch outgrowth, and branch arrest. This process relies on the precise spatiotemporal control of gene expression, cell proliferation, and migration and is essential for the physiological function of many organs including the lung, the vascular system, and the kidney. However, HGF/SF-Met signaling is not exclusively involved in regulating epithelial morphogenesis. Met is also expressed in skeletal muscle and in the developing nervous system, and HGF/ SF-Met signaling is required for the ▶ migration of myogenic precursors in limb bud formation and has been implicated in neuronal development. HGF-Met signaling also stimulates the proliferation of hepatocytes, renal epithelial cells, and vascular endothelial cells and increases the motility

MET

MET, Fig. 2 Met-induced branching morphogenesis of cells grown in a three-dimensional matrix. Several epithelial cell lines (cell line C127 is shown) that express Met undergo significant changes in cellular morphology and three-dimensional organization following stimulation with HGF/SF

of both epithelial and vascular endothelial cells and may play a significant role in wound repair and tissue regeneration. The ability of HGF/SF to induce increased growth, motility, and capillarylike tubule formation in vascular endothelial cells in vitro and to promote blood vessel formation in vivo suggests that Met may stimulate angiogenic processes as well. Cellular and Molecular Features Normal Met activation by HGF/SF is believed to occur through receptor dimerization and induction of transphosphorylation of tyrosine residues, which are critical for growth factor-mediated signal transduction (Fig. 1). Much work has been done to determine which of the phosphorylated tyrosine residues in the intracellular region of Met

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(there are 16 of them) are important for stimulating cellular responses. Phosphorylation of two tyrosines located within the activation loop of the tyrosine kinase domain (amino acid positions 1234 and 1235 in human Met) greatly enhances the intrinsic kinase activity of the receptor and is critical for potent receptor activation. Phosphorylation of two closely spaced tyrosines at the C-terminal of Met (amino acid position 1349 and 1356 in human Met) generates a multisubstrate docking site that several adaptor proteins, including Gab1, Grb2, Shp2, and Shc, can bind. In turn, these adaptors recruit numerous signal transduction proteins including phosphotidylinositol-3OH kinase (PI3K), phospholipase C-g (PLC-g), gain-of-function mutations, Rac1/Cdc42, Rap1, and other signaling molecules. Several assays that monitor HGF/SF-Met-meditated cell proliferation, cell scattering, and branching morphogenesis have been instrumental in identifying the signaling pathways that are responsive to Met activation. However, detailed analysis of which protein pathway mediates each of these cellular responses is complicated by prevalent pathway and ▶ receptor cross-talk (Peschard and Park 2007). Generally, activation of the Ras and ▶ B-Raf signaling pathway via the Grb2/SOS complex is important for both cell proliferation and motility. Activation of PI3K is important for cell survival and prevention of ▶ apoptosis, while activation of the Rac1/Cdc42 pathways and disassembly of ▶ E-cadherin/catenin complexes at the cell surface promotes loss of intercellular adhesion and gain of cell motility. Induction of branching morphogenesis likely requires all of these factors and additional factors downstream of Gab1, as overexpression of Gab1 can stimulate both cell scattering and branching morphogenesis in the absence of HGF/SF. As several of the proteins activated by Met have also been implicated in tumor generation and progression, downregulation of the activated Met signal is important to prevent hyperactivation of these protein pathways and subsequent cellular transformation. One important means of downregulating growth factor receptors is degradation of the active receptors. ▶ Endocytosis can clear activated growth factor receptors either

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transiently, if they are recycled back to the cell surface, or permanently, if they are sorted to the lysosome and degraded. Alternatively, activated growth factor receptors can undergo ▶ ubiquitination and subsequent degradation by the proteasome. For Met, ubiquitination is mediated in part by phosphorylation of a serine residue located in the receptor juxtamembrane region (serine 1003 in human Met). Phosphorylation of this serine allows the binding of the Cbl E3 ubiquitin ligase. Binding of Cbl results in ubiquitination and degradation of the receptor. Clinical Relevance While Met plays several important roles in normal growth and development, abnormal Met signaling likely contributes to generation and progression of human tumors. As previously mentioned, Met was originally isolated as the constitutively activated oncoprotein Tpr-Met. However, Met missense mutations have been implicated in the cause of hereditary and sporadic human papillary renal carcinoma (HPRC). The missense mutations are located in the tyrosine kinase domain of Met and produce a constitutively active receptor that promotes tumor formation and/or progression (Fig. 1). Other missense mutations, in the kinase and other domains, have also been identified in gastric, hepatocellular, and other cancers. In the absence of an activation mutation, abnormal expression of Met and HGF/SF has now been identified in most solid tumors. A comprehensive table describing the different Met expression and sequence abnormalities in a wide spectrum of human cancers has been collected and can be found at http://www.vai. org/met/. During tumorigenesis, Met can become constitutively activated by coexpression of HGF/SF in the same cell, forming an autocrine stimulatory loop. Numerous tumor types, including several different carcinomas (lung, breast, and others), sarcomas (osteosarcoma, ▶ Kaposi sarcoma, and others), and ▶ brain tumors (glioblastoma multiforme), express Met and/or HGF/SF. Moreover, in model systems, MetHGF/SF-expressing cells are not only invasive in vitro but also rapidly metastasize when injected

MET

into mice. Part of the mechanism behind the invasive phenotype of HGF/SF-Met expressing cells is upregulation of the urokinase proteolysis network. Induction of the proteolysis network following Met activation can degrade the surrounding extracellular basement membrane and facilitate invasion and metastasis. Overexpression of Met is also observed in several gastric carcinoma cell lines as well as carcinomas of the lung, pancreas, thyroid, colon, and stomach and is thought to participate in cell transformation and tumorigenicity. As HGF/SF has been found in tumor stroma and normal human serum, it is possible that activation of Met in some of these cells may result from paracrine or endocrine stimulation. The paracrine model may be responsible for some ▶ breast cancers. Identification of HGF/SF and Met in breast cancer tumors is a strong negative prognostic indicator of reoccurrence and survival. The effects of HGF/SF in tumor progression may in part result from an increase in tumor ▶ angiogenesis. Invasive breast cancers that contain high levels of HGF/SF or brain tumors that are engineered to overexpress HGF/SF have increased microvessel density and increased levels of other vascular markers. Other effects, such as sustained proliferation and/or misregulated differentiation, may also contribute significantly to HGF/SF-Met-mediated tumor growth/progression. As such, treatments that inhibit HGF/SF-Met signaling may be therapeutic in preventing the onset and progression of many cancer types and may also play significant roles in preventing cell metastasis and angiogenesis in these cancers.

Cross-References ▶ Adhesion ▶ Angiogenesis ▶ BRaf-Signaling ▶ Brain Tumors ▶ Breast Cancer ▶ Cancer ▶ E-Cadherin ▶ Endocytosis ▶ Epidermal Growth Factor-like Ligands

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▶ Epithelial-to-Mesenchymal Transition ▶ Gastric Cancer ▶ Invasion ▶ Kaposi Sarcoma ▶ Lung Cancer ▶ Metastasis ▶ Migration ▶ Oncogene ▶ Receptor Cross-Talk ▶ Receptor Tyrosine Kinases ▶ Ubiquitination

(2012) SEMA Domain. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3359–3360. doi:10.1007/978-3-642-16483-5_5230 (2012) Transphosphorylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3774. doi:10.1007/978-3-642-164835_5954

References

A statistical technique where all data from all available studies of something are combined, regardless of data quality. The results of several case-control studies, or cohort studies or both, are combined to generate a summary measure of the odds ratio. The individual odds ratios are combined but are weighted by some function of the variance of each odds ratio. The technique is used by researchers to get a maximum of statistical information without worrying about distortion in the results.

Birchmeier C, Birchmeier W, Gherardi E et al (2003) Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4:915–925 Gherardi E, Birchmeier W, Birchemeier C et al (2012) Targeting MET in cancer: rationale and progress. Nat Rev Cancer 12:89–103 Peschard P, Park M (2007) From Tpr-Met to Met, tumorigenesis and tubes. Oncogene 26:1276–1285 Zhang Y, Vande Woude GF (2002) HGF/SF-met signaling in the control of branching morphogenesis and invasion. J Cell Biochem 88:408–417

Meta-Analysis Definition

See Also (2012) Branching Morphogenesis. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 481. doi:10.1007/978-3-642-164835_711 (2012) Cadherins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 581–582. doi:10.1007/978-3-642-16483-5_770 (2012) Cell Scattering. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 739. doi:10.1007/978-3-642-16483-5_1010 (2012) Gain-of-Function Mutation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1489. doi:10.1007/978-3-642-164835_2303 (2012) Morphogenic Differentiation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2372–2373. doi:10.1007/978-3-64216483-5_3835 (2012) PSI Domain. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3116. doi:10.1007/978-3-642-16483-5_4845 (2012) Rap1. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3168. doi:10.1007/978-3-642-16483-5_4947 (2012) Renal Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3225–3226. doi:10.1007/978-3-642-16483-5_6575

Cross-References ▶ Adjuvant Chemoendocrine Therapy ▶ Cancer Epidemiology ▶ Surrogate Endpoint

Metabolic Polymorphisms and Cancer Susceptibility Roland C. Wolf Biomedical Research Centre, University of Dundee, Dundee, UK

Definition The genetic basis for interindividual differences in cancer susceptibility.

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Characteristics History The term pharmacogenetics (synonym pharmacogenomics) has been used for approximately the last 40 years to describe genetic or inherited factors that determine individuality in response to drugs and other therapeutic agents. In studies starting in the mid-1950s, it became apparent that some individuals were hypersensitive to certain drug treatments. This sensitivity was also observed within other family members and was subsequently shown to be an inherited trait. Early epidemiological studies were focused on the drugs used in the treatment of malaria and tuberculosis. Interindividual variation in the ability to metabolize these drugs was subsequently attributed to inherited differences in the enzymes glucose 6-phosphate dehydrogenase, which generated sensitivity to the antimalarial drug primaquine, and N-acetyltransferases, which generated sensitivity to antitubercular drugs such as isoniazid. The term pharmacogenetics was first coined by Vogel in the late 1950s. In the 1960s and 1970s, there was increased interest in this research area, and many genetic variants in a range of genes involved in determining drug response were identified. Important Genetic Factors In order to elicit a therapeutic response, a drug first needs to enter the circulation, i.e., to cross the gastrointestinal (GI) tract. We now know that the entry of many drugs into the body is regulated by a multigene family of drug transporters (sugar ABC transporters) that have the capacity to pump drugs in and out of cells. In addition, the GI tract also expresses enzymes (sugar ABC transporters) that can metabolize and inactivate drugs. Variability in the level of expression or activity of these drugmetabolizing enzymes is therefore an important determinant of circulating drug concentrations. Once a drug enters the body, it is transported to the liver, which is the main organ involved in drug metabolism. Nearly all therapeutic agents are subject to hepatic metabolism before they are eliminated from the body. The rate of metabolism will determine the biological half-life of the drug.

Metabolic Polymorphisms and Cancer Susceptibility

Hepatic drug metabolism is carried out by many multigene families of proteins. Of key importance are the ▶ cytochrome P450-dependent monooxygenases that convert lipophilic drugs into more water-soluble products predominantly through hydroxylation reactions. Most anticancer drugs are substrates for one or more of the P450 monooxygenases, including many of the molecularly targeted agents, e.g., cobimetinib and dabrafenib (Table 1). The products of these P450-catalyzed reactions are further conjugated to cofactors such as glucuronic acid or sulfate, further increasing their water solubility and facilitating elimination in the bile or via the kidneys in the urine. The rate of uptake of drugs into liver cells and their rate of metabolism is therefore of central importance in determining therapeutic outcome. Finally, the capacity of a drug to exert its therapeutic effect is determined by its concentration at the target cell and the level of expression and activity of specific receptors and enzymes with which it interacts (Fig. 1). Pharmacogenetic variability resulting in altered drug response can involve any or all of these pathways, and allelic forms (alleles) of many drug-metabolizing enzymes, drug transporters, and drug receptors are now known to exist within the population. In addition to individual genetic variability, for certain types of therapy, genetic variability of the target cell will also be a key determinant of therapeutic efficacy. This is particularly the case in cancer chemotherapy where the genetics of the tumor cell is often a major additional determinant of the effectiveness of drug therapy. This is also of central importance in the chemotherapy of infectious agents and in the use of antibiotics, antiviral agents, etc. In these cases, drug resistance is well characterized and has been ascribed to altered patterns of gene expression in the target organism; many of the factors that determine drug response in these organisms are the same as those that determine circulating drug levels in man, i.e., drug transporters and drug-metabolizing enzymes. Relevance of Pharmacogenetics The importance of pharmacogenetics is well illustrated by studies of genetic variability in the

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Metabolic Polymorphisms and Cancer Susceptibility, Table 1 Anticancer drugs metabolized by P450s Drug Anastrozole Cobimetinib Cyclophosphamide

Target Aromatase MEK1/2 DNA

P450 CYP2C8, CYP3A4 CYP3A4 CYP2B6, CYP2C9, CYP2C19, CYP3A4

Dabrafenib Dasatinib

CYP2C8, CYP2C9, CYP3A4 CYP3A4

Docetaxel

BRAFV600E BCR-ABL/ SRC/c-KIT/ PDGFR Tubulin

Gefitinib

EGFR

Irinotecan Letrozole Osimertinib Paclitaxel Pazopanib

Topoisomerase Aromatase EGFRT790M Tubulin VEGFR/cKIT/PDGFR

Selumetinib Tamoxifen Vemurafenib Vinblastine Vincristine Vinorelbine

BRAFV600E ER BRAFV600E Tubulin

CYP1A1, CYP2D6, CYP3A4, CYP3A5 CYP3A4 CYP2A6, CYP3A4 CYP3A4 CYP2C8 CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 CYP1A2, CYP2C19, CYP3A4 CYP2D6 CYP3A4 CYP3A4

Cancer Breast Melanoma Hodgkin lymphoma; chronic myelocytic leukemia; acute lymphoblastic leukemia; testicular; breast, ovarian; endometrial Melanoma Chronic myelocytic leukemia Acute lymphoblastic leukemia

CYP3A4

Nonsmall cell lung cancer; ovarian breast; prostate; stomach; head and neck Nonsmall cell lung cancer Colorectal Breast Nonsmall cell lung cancer Ovarian; breast; lung; pancreatic Renal cell carcinoma; soft tissue sarcoma

Nonsmall cell lung cancer Breast Melanoma Hodgkin lymphoma; testicular; breast; kidney; nonsmall cell lung cancer; brain

EGFR epidermal growth factor receptor, ER estrogen receptor, MEK MAPK/Erk kinase, PDGFR platelet-derived growth factor receptor, VEGFR vascular endothelial growth factor receptor

Metabolic Polymorphisms and Cancer Susceptibility, Fig. 1 Factors which can limit the therapeutic effectiveness of drugs

Absorption transport Hepatic metabolism

Drug targets

Response

Signal Attenuation

Circulating drug concentration Renal

Metabolism Toxicity to non-target tissues

Elimination

Secondary pharmacological targets

▶ cytochrome P450 system, in particular in cytochrome P450 CYP2D6. In the 1970s, clinical trials identified a group of people who were hypersensitive to certain types of drug including

sparteine, an antihypertensive agent which is prescribed for the treatment of arrhythmia, and debrisoquine. It was subsequently shown that this variability in response was inherited and that

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Metabolic Polymorphisms and Cancer Susceptibility, Table 2 CYP2D6 drugs used in the treatment of psychiatric, neurological, and cardiovascular disease Psychiatric disease

Cardiovascular disease

Amitriptyline, clomipramine, clozapine, desipramine, fluvoxamine, fluoxetine, haloperidol, imipramine, levomepromazine, nortriptyline, olanzapine, paroxetine, perphenazine, thioridazine, tranylcypromine, zuclopenthixol Alprenolol, amiodarone, flecainide, indoramin, mexiletine, nimodipine, oxprenolol, propranolol, timolol

between 5% and 10% of the white population, “poor metabolizers,” do not express a functional form of CYP2D6. In later studies, the molecular basis of this polymorphism was elucidated and shown to be due to gene-inactivating changes in CYP2D6 DNA. This subsequently allowed the development of DNA-based tests to identify individuals with this metabolic defect. Cytochrome P450 CYP2D6 is responsible for the metabolism of up to 25% of therapeutic drugs, including a large number of the drugs used in the treatment of psychiatric illness (Table 2). This polymorphism has now been attributed to aberrant metabolism, and disposition of many of the drugs has been associated with adverse drug reactions induced by many of these agents. In addition to gene-inactivating alleles of CYP2D6, certain individuals contain multiple copies of this gene, which generates an “ultrarapid metabolizer” phenotype. These individuals metabolize and eliminate drugs at a much faster rate than the rest of the population, and as a result the desired therapeutic effect is not achieved. Therefore, pharmacogenetic variability can result in adverse drug reactions, exacerbated drug toxicity, or lack of therapeutic effect. Ethnic Differences The genes and proteins that determine therapeutic outcome following drug treatment evolved to protect us against the effects of toxic environmental chemicals. Part of the genetic variability in response to these agents may be explained by different populations needing a different spectrum of enzymes to cope with their particular

environment and diet. This is exemplified by the large variability in the distribution of pharmacogenetic polymorphisms between different ethnic groups. For example, there is an allele of CYP2D6 that is present in 40% of the Chinese population but is not found in the white population. This allele generates a slower metabolizer phenotype and explains why a high percentage of the Chinese population cannot tolerate doses of drugs prescribed routinely in Europe and the Western world. Future The current aim of research in pharmacogenetics is to exploit the information from the Human Genome Project to identify novel variant forms of genes, to establish their functional significance, and to apply these usefully in the clinical environment. The ability to identify rapidly and test for variant sequences of genes will allow pharmacogenetic testing to be generally applied in medical practice so that optimal drug doses can be used. This is particularly the case in the treatment of diseases such as cancer where inappropriate drug dosing is often life threatening and the success or failure of treatment may depend on the genetics of the tumor cell. It is anticipated that with the identification of pharmacologically important genetic variations within the population, rapid tests will be available to hospitals that will allow clinicians to prescribe drugs at optimal dose regimens or to decide whether specific drug therapies will be effective for individual patients.

References Allison AC (1960) Glucose-6-phosphate dehydrogenase deficiency in red blood cells of east Africans. Nature 186:531–532 Evans DAP, Manley KA, McKusick VA (1960) Genetic control of isoniazid metabolism in man. BMJ 2:485–491 Gibson GG, Skett P (1994) Introduction to drug metabolism, 2nd edn. Blackie Academic & Professional, London Gough AC, Miles JS, Spurr NK et al (1990) Identification of the primary gene defect at the cytochrome P450 CYP2D gene locus. Nature 347:773–776

Metabolic Reprogramming Hayes JD, Wolf CR (eds) (1994) Molecular genetics of drug resistance. Harwood Academic, London Mahgoub A, Idle JR, Dring LG et al (1977) Polymorphic hydroxylation of debrisoquine in man. Lancet 1:584–586 Sadee W, Druebbisch V, Amidon GL (1995) Biology of membrane transporter proteins. Pharm Res 12:1823–1837 Vogel F (1959) Moderne problem der Humangenetik. Ergeb Inn Med U Kinderheilk 12:52–125

Metabolic Reprogramming Bruce A. White Department of Cell Biology, UConn School of Medicine, UConn Health, Farmington, CT, USA

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antitumor therapies (Bobrovnikova-Marjon and Hurov 2014). Metabolic reprogramming is driven primarily by somatic mutations within cell proliferationrelated genes (i.e., ▶ oncogenes and tumor suppressors) that occur relatively early during neoplastic transformation (Ward and Thompson 2012a). In some cases, metabolic enzymes themselves are mutated and drive metabolic changes that are inherently oncogenic (Ward and Thompson 2012b). Cancer cell metabolism remains fluid, so that even after initial “reprogramming,” metabolism continues to respond dynamically to alterations in the immediate microenvironment. Cancer cell metabolism also undergoes significant changes during ▶ epithelial-mesenchymal transition (EMT) and the acquisition of a more proliferative, invasive, and stem-like phenotype.

Definition “Metabolic reprogramming” refers to the collective changes that occur within multiple metabolic pathways in cancer cells. This essay will focus on changes associated with altered metabolism of glucose, lactate, and glutamine (Gln) and will briefly discuss certain side pathways related to glycolysis and the TCA cycle.

Characteristics Introduction Metabolic reprogramming or “deregulating cellular energetics” has been defined as a hallmark of cancer and also contributes to several of the other hallmarks of cancer as defined by Hanahan and Weinberg (Hanahan and Weinberg 2011). Metabolic reprogramming allows for adequate ATP production under stressful conditions but also impacts macromolecular biosynthesis, cellular redox balance and antioxidant defenses, and epigenetic orchestration of gene expression (Boroughs and DeBerardinis 2015; Vander Heiden et al. 2011). The study of metabolic reprogramming in cancer has led to the identification of several differentially utilized enzymes, isozymes, and pathways that have been or are being targeted for the development of new

Metabolic Reprogramming: Some Basic Changes In many normal nonproliferating cell types that are equipped with functional mitochondria and live under normoxic conditions, ATP is generated primarily by mitochondrial oxidative phosphorylation using multiple forms of carbon fuels (Fig. 1a). Glucose, in particular, is metabolized in the cytoplasm through the glycolytic pathway to its end product, pyruvate, producing a relatively small amount of ATP. Pyruvate then enters the mitochondria and is converted to acetyl CoA by mitochondrial pyruvate dehydrogenase (PDH), followed by oxidative decarboxylation of acetyl CoA to CO2 within the TCA cycle. This generates the reduced dinucleotides, NADH and FADH2, which drive O2-dependent oxidative phosphorylation (OXPHOS) and the production of a relatively high amount of ATP (Fig. 1a). In contrast, proliferating cancer cells often depend almost solely on the metabolism of glucose through a substantially upregulated glycolytic pathway in order to meet their needs for ATP synthesis (Fig. 1b). Pyruvate does not become decarboxylated to acetyl CoA, because PDH is inhibited by oncogenically upregulated PDH kinase (PDK; Fig. 1b). Instead, pyruvate is converted to lactic acid by lactate dehydrogenase (LDH). This reaction occurs in the cytoplasm and

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Metabolic Reprogramming, Fig. 1 Generalized comparison of energy metabolism in normal versus cancer cells. This figure emphasizes how many cancer cells switch to the almost exclusive use of glycolysis for ATP production along with the fermentation of pyruvate to lactate. Mitochondrial oxidative phosphorylation is reduced below normal. (a) Normal cell can utilize multiple fuels to generate ATP. Most ATP, including that produced from metabolism of glucose,

is derived from mitochondrial oxidative phosphorylation (OXPHOS). AAs amino acids, FFAs free fatty acids, PDH pyruvate dehydrogenase, ATP adenosine triphosphate, CoA coenzyme A, GLUTs glucose transporters. (b) Aerobic glycolysis and lactate production in cancer cells. GLN glutamine, SLC1A5 solute carrier 1A5 – also called “neutral amino acid transporter”, MCT1,4 monocarboxylate transporters 1 and 4

independently of O2. Lactate is transported out of the cell through monocarboxylate transporters (MCTs), especially MCT1 and MCT4. This shift in energy metabolism occurs even in the presence of normal oxygen levels, and is called “aerobic glycolysis” or the “▶ Warburg effect” named after Nobel Laureate Otto Warburg who first described this curious characteristic of cancer cells in the 1920s. Warburg hypothesized that this metabolic shift compensated for defective mitochondria. However, cancer cells often contain functional mitochondria and depend on mitochondrial metabolism to some extent for energy production. Moreover, the TCA cycle is often robust in cancer but shifts from primarily fulfilling an “energygenerating function” to a “macromolecular biosynthetic function,” in part by synthesizing and exporting citrate for lipid biosynthesis (Fig. 1b). In many cancers, oncogenic signaling upregulates transporters of Gln (e.g., SLC1A5) and mitochondrial enzymes that allow Gln to replace acetyl CoA as the primary carbon source that drives the TCA cycle (Fig. 1b; and see below).

Aerobic Glycolysis A puzzling, major disadvantage of glycolysis concerns the drastically lower yield of ATP produced by glycolysis per glucose molecule (5–10% of the amount of ATP generated by mitochondrial oxidative phosphorylation). So two major questions concerning the switch of cancer cells to aerobic glycolysis are: 1. How do the cells maintain adequate ATP production? 2. What advantage(s) does aerobic glycolysis confer upon a rapidly proliferating cancer cell? To address the first question, glycolysis can generate ATP much faster than mitochondrial respiration, and the primary components of aerobic glycolysis, namely, glucose and lactate transporters along with glycolytic enzymes, are markedly upregulated in cancer cells. This upregulation compensates for the low yield of ATP per glucose molecule. In terms of advantages of utilizing aerobic glycolysis, one advantage relates to the utilization of glucose as a primary

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fuel. The human body has multiple mechanisms to ensure that circulating glucose levels remain above 70 mg/dL (3.9 mM), even during prolonged fasting. Thus, glucose represents a relatively constant and plentiful fuel for tumors that are sufficiently vascularized. Another advantage is that the oncogene-induced increase in glycolytic components confer upon a cancer cell the ability to outcompete neighboring stromal and other cancer cells for glucose and maintain glycolysis within a relatively poorly vascularized (i.e., ischemic) microenvironment. Since glycolysis does not require O2, these cells also survive better in a hypoxic (▶ Hypoxia) microenvironment. These advantages lead to positive selection for cells that have shifted to aerobic glycolysis when faced with the stresses of ischemia and hypoxia. Upregulation of Components of Aerobic Glycolysis The expressions of all glycolytic enzymes are typically increased in glycolytic cancer cells. The components (i.e., transporters and enzymes) of glycolysis and lactate fermentation that are tightly regulated in normal cells by growth-related extracellular signals, nutrients, and metabolites are also regulated by multiple factors in metabolically reprogrammed cancer cells. These upregulated components catalyze “upstream” reactions at the beginning and “downstream” reactions at the end of aerobic glycolysis (Fig. 2). Upregulation of “Upstream” Components in Aerobic Glycolysis

• Glucose Transporters (GLUTs). Cells import glucose through facilitative transporters of the GLUT family (solute carrier protein family 2A). GLUT1 (SLC2A1) is normally expressed in endothelial cells and numerous other cell types. GLUT1 can be upregulated in response to growth factor and cell cycle-related signaling, including insulin and growth factor activation of the phosphatidylinositol-4,5bisphosphate-3-kinase (PI3K)/Akt pathway. Although highly regulated in normal cells, the PI3K/Akt pathway is frequently constitutively activated in numerous tumor types and stimulates GLUT1 gene expression. GLUT1 is also

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increased in cancer by other dysregulated signaling pathways, including activating mutations in Ras (▶ RAS Genes) isoforms and Akt-activated mTORC1 and subsequent stimulation of hypoxia-inducible factor 1 (Hif-1). Another glucose transporter, GLUT3 (SLC2A3), normally displays a more limited expression and has a higher affinity for glucose and a faster transport of glucose than GLUT1. GLUT3 expression is significantly elevated in several cancer types, including some aggressive forms of cancer (e.g., triple-negative breast cancer). GLUT3 is also increased during EMT in breast cancer cells and non-small cell lung cancer. • Hexokinases. GLUT-mediated transport is bidirectional, so that cells must “trap” imported glucose to avoid “leakage” out of the cell. This is accomplished by the first reaction of glycolysis, which involves irreversible, ATP-consuming phosphorylation of glucose to glucose-6-phosphate (G6P) by the family of hexokinases (HKs). This reaction is regulated by PI3K/Akt signaling in several normal cell types. The isoform HK2 (▶ Hexokinase 2) displays a high affinity for glucose and is significantly upregulated by several oncogenic signals in numerous cancer types (Fig. 2). • Phosphofructokinase 1 (PFK1). After G6P is isomerized to fructose-6-P (F6P), a second ATP-consuming phosphorylation takes place, generating F-1,6-bisP. This reaction is catalyzed by phosphofructokinase 1 (PFK1), which is called the “gatekeeper” of glycolysis, as PFK1 is tightly regulated by multiple factors (Fig. 2). PFK1 activity is allosterically inhibited by ATP, citrate and lactate, and allosterically activated by AMP and fructose-2,6bisphosphate (F-2,6-bisP). F-2,6-bisP is produced from F-6-P by the bifunctional enzyme, phosphofructokinase 2 (PFK2)/fructose-2,6bisphosphatase, encoded by one of the four corresponding PFKFB genes. The PFK2 activity generates F-2,6-bP and thus activates PFK1 and glycolysis. In contrast, the fructose-2,6bisphosphatase activity of this same protein catabolizes F-2,6-bisP back to F6P and thus removes activation of PFK1 and slows

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Metabolic Reprogramming, Fig. 2 Aerobic glycolysis with selected regulators. This figure emphasizes how aerobic glycolysis is highly regulated, especially at early and late steps. Arrows indicate stimulation – green promotes glycolysis, red inhibits glycolysis. Red lines with “T” ending indicates inhibition. HK hexokinase, PFK1 phosphofructokinase 1, PFK2/F2,6BP dual activity enzymes phosphofructokinase 2/fructose-2,6-bisphosphatase, TIGAR TP53Inducible Glycolysis and Apoptosis Regulator, GAPDH glyceraldehyde-3-phosphate dehydrogenase, PKM2 pyruvate kinase/muscle isoform/splice variant 2, LDHA lactate dehydrogenase A, G-6-P gucose-6phosphate, F-6-P fructose-6-phosphate, F-1,6-bP fructose-1,6-bisphosphate, F-2,6-bP fructose-2,6bisphosphate, GA3P glyceraldehyde-3-phosphate, DHAP dihydroacetone phosphate, Pi organic phosphate,

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3P GATE 3-phosphoglycerate, 2P GATE 2phosphoglycerate, PEP phosphoenolpyruvate, PYR pyruvate, LAC lactate anion, ATP adenosine triphosphate, ADP adenosine diphosphate, AMP adenosine monophosphate, NAD+ & NADH nicotinamide adenine dinucleotide, oxidized and reduced form, respectively, Ras family of oncogenes originally discovered in Rat sarcoma, Hif-1 hypoxia-inducible factor-1, Akt Akt1 kinase named for oncogene in retrovirus AKT8, activated by several growth factor and growth-related receptors, p53 TP53 tumor suppressor protein – often mutated in cancer, Myc family of cellular derived oncogenes originally named after myelocytomatosis viral oncogene (v-Myc), NF-kB nuclear factor kappa-light-chainenhancer of activated B cells, ERa estrogen receptoralpha – product or ESR1 gene, ROS reactive oxygen species

Metabolic Reprogramming

glycolysis. The PFKBP3 isoform is highly expressed in several cancer types, and the kinase activity is significantly increased by PI3K/Akt and other mitogenic signals. A major inhibitor of these early reactions in glycolysis is the stress sensor and tumor suppressor, p53 (▶ TP53). Loss of wild-type p53 activity is frequently an acquired deficit in numerous types of cancer that leads to enhanced aerobic glycolysis. p53 suppresses GLUT1 expression and glucose uptake (Fig. 2). p53 antagonizes proglycolytic signaling, including the Akt and ▶ Nuclear Factor-kB (NF-kB) pathways. p53 also stimulates the expression of TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR), which acts as a fructose-2,6-bisphosphatase. TIGAR reduces F-2,6-bisP levels, thereby attenuating the activity of PFK1 and slowing glycolytic flux. However, the expression of TIGAR is somewhat independent in some cancer cells. Moreover, TIGAR function is complicated by the fact that by slowing glycolysis, it allows carbons to enter the oxidative arm of the pentose phosphate pathway (PPP) as initiated by glucose-6-phosphate dehydrogenase (see below). Because this pathway generates NADPH that is used for reduction of glutathione, low TIGAR expression can protect cancer cells from oxidative stress induced by some antitumor therapies. Also in contrast to the negative effects of wild-type p53 on glycolysis, one form of mutated p53 actually increases HK2 activity and thus promotes glycolysis. Upregulation of “Downstream” Components in Aerobic Glycolysis

• Pyruvate Kinase Muscle Isoform 2 (PKM2). In the final and rate-limiting reaction of glycolysis, phosphoenolpyruvate (PEP) is converted to pyruvate by the enzyme pyruvate kinase (PK; Fig. 2). Cancers and some normal cells express the embryonic muscle PK isoform/ splicing variant 2 (PKM2). PKM2 is a critically important enzyme in some forms of cancer (Wong et al. 2015; Li et al. 2014). PKM gene expression is stimulated by oncogenic Hif1, and the selective splicing that generates PKM2 as opposed to PKM1 is promoted by

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Myc. PKM2 exists as a dimeric protein kinase and as a tetrameric pyruvate kinase. With respect to aerobic glycolysis, PKM2 is allosterically activated by the binding of the upstream metabolite, F-1,6-bisP, which promotes assembly of the tetrameric form. This positive feedforward regulation promotes efficient conversion of glucose to pyruvate in the presence of an otherwise active glycolytic pathway. Either binding to phosphotyrosine-containing proteins or phosphorylation by various oncogenic tyrosine kinases induces dissociation of F-1,6-bisP and predominance of the dimeric form. This apparent paradox of lowering PKM2 activity under increased growth factor signaling has been explained in terms of allowing upstream glycolytic metabolites to accumulate and be diverted into anabolic side pathways (see below). Similarly, PKM2 is allosterically activated by serine (Ser), and the absence of Ser promotes the redirection of the glycolytic intermediate, 3-phosphoglycerate, into the serine/glycine biosynthetic pathway (Ser/Gly BP; see below). In another adaptive configuration, PKM2 activity is suppressed by an acute increase in ▶ reactive oxygen species (ROS), thus allowing G-6-P to be better diverted into the oxidative arm of the PPP that promotes antioxidant defense (see below). In addition to its metabolic role, dimeric PKM2 enters the nucleus and phosphorylates histone 3 and interacts with several transcription factors. In a positive feedback loop, PKM2 interacts with Hif-1 as a coregulatory protein and promotes the expression of Hif-1 responsive genes, including PKM itself and lactate dehydrogenase isoform A (LDHA). PKM2 also interacts with b-catenin to increase c-Myc expression, which, in turn, promotes PKM2-specific splicing, as well as (LDHA) transcription. Consistent with a pleiotropic nuclear signaling role, along with its critical metabolic role in glycolysis, elevated PKM2 expression may directly contribute to neoplastic transformation in some cancers. • Lactate Dehydrogenase A (LDHA) as opposed to Pyruvate Dehydrogenase (PDH). In cells

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reprogrammed for aerobic glycolysis, reduction of pyruvate to lactate by cytoplasmic LDHA is significantly increased, due to oncogenic induction of LDHA expression. The LDH reaction also rapidly regenerates NAD+ from NADH, enabling the continuation of glycolysis. LDHA is stimulated by several oncogenic signals, including Hif-1, c-Myc, estradiol, and cyclic AMP responsive element-binding (CREB) protein. This conversion of pyruvate to lactate is further enhanced by the coordinated repression of mitochondrial PDH, which decarboxylates pyruvate to acetyl CoA in most normal cells. PDH activity is inhibited primarily by phosphorylation by mitochondrial PDH kinase (PDK) isoforms. PDK expression is induced by several oncogenic signaling pathways, including Hif-1, c-Myc, and NF-kB but is suppressed by p53. • Monocarboxylate Transporters (MCTs). The lactic acid produced by LDHs immediately dissociates into the lactate anion (called simply “lactate”) and a proton (H+). In order to maintain normal intracellular lactate and pH, cancer cells cotransport lactate and H+ out of the cell into the surrounding stroma through one or more isoforms of monocarboxylate transporters (MCTs 1–4; SLC family 16A). Highly glycolytic cancers typically display elevated expression of MCT4 (SLC16A3) or MCT1 (SLC16A1), which export lactate and H+ from the cell. MCT1 transcription is induced by c-Myc in lymphoma cells, and MCT4 expression is stimulated by PI3K/Akt signaling and is highly expressed in HER2positive invasive breast cancer. Lactate as a Signaling Molecule As mentioned above, the fermentation of pyruvate to lactic acid results in an immediate increase in the intracellular concentrations of lactate and H+, which have been linked to inhibitory and stimulatory growth pathways (Luc et al. 2015). Lactate allosterically inhibits PFK1 in muscle, and high lactate in cancer cells could cause G-6-P levels to increase, which, in turn, would negatively feedback on hexokinase. This would increase the probability of glucose moving out of the cell, especially

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in ischemic regions of a tumor with low extracellular glucose. Elevated lactate also compromises the further conversion of pyruvate to lactate, leading to failure to convert NADH to NAD+ and thus paralysis of the glyceraldehyde-phosphate dehydrogenase (GAPDH) reaction. These and other events would lead to the collapse of glycolysis and cell death. In contrast, intracellular lactate has been shown to play a late role in hypoxia in cancer cells by stabilizing the oncogenic signaling protein, ▶ N-Myc Downstream Regulated Gene (NDRG3), and preventing its degradation. Elevated NDRG3 promotes cell proliferation-related signaling and expression of angiogenic (▶ Angiogenesis) interleukins in several cancer cells. Highly glycolytic cancers typically export a significant amount of lactate, which can be imported by neighboring cancer or stromal cells through MCT1 and MCT2 (SLC16A7) transporters, converted to pyruvate by LDHB-rich LDH tetramers, and used in mitochondrial oxidative phosphorylation (Fig. 3). Lactate can act also act as a hypoxia-sensing signaling molecule to endothelial cells, in that MCT1-mediated import of lactate stimulates the NF-kB transcription factor, followed by increased IL8 expression and ultimately endothelial proliferation and tube formation. Additionally, accumulation of exported lactate and H+ inhibits immune surveillance, promotes protumorigenic inflammation, and modifies cellular stroma and cancer cell-stromal interactions that promote motility and invasiveness. Finally, metabolic studies in normal adipocytes have shown that the Gi protein-coupled receptor, GPR81, is an endogenous receptor for lactate. GPR81 expression is elevated in pancreatic cancer specimens, detected in several cancer cell lines, and shown to be necessary for growth and metastasis of pancreatic cancer cells in vivo. GPR81 signaling increases MCT transporter expression and confers the ability of cancer cells to utilize lactate as an energy source in the presence of low glucose (Fig. 3). Aerobic Glycolysis and Anabolic Side Pathways Proliferating cancer cells must produce adequate ATP, but also need to engage a broad range of

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Metabolic Reprogramming, Fig. 3 Actions of extracellular lactate produced by a highly glycolytic cancer cell. This figure emphasizes that lactate can act as a signaling molecule, in some cases through binding to the endogenous lactate receptor, GPR81. This also shows metabolic coupling between a glycolytic cancer cell and an oxidative cancer or normal cell. Lactate produced from the former can be imported and used for energy by the latter. As explained in the text, uptake of

lactate in some cells (e.g., endothelial cells) can also provide a signal for proliferation, etc. For most abbreviations, see legends for Figs. 1 and 2. PDK PDH kinase, Wnt extracellular signaling molecule named after Drosophila “Wingless” gene – ultimately activates b-catenin, which enters nucleus and regulates gene transcription, ECM extracellular matrix, NKCs natural killer cells, GPR81 G protein-coupled receptor 81, LDHB lactate dehydrogenase B

anabolic metabolic pathways in order to double the mass of macromolecules and organelles before cell division. One advantage of markedly upregulating glycolytic flow is to allow several anabolic side pathways to extract carbons out of glycolysis for biosynthetic reactions (Fig. 4). These include the following:

glycosylation for correct folding and directed transport, specifically, to the cell membrane. Thus, the HBP is required for the ability of the cell to respond to changes in the microenvironment. ▶ Glycosylation also modifies intracellular proteins, including histones and metabolic enzymes. The HBP requires contributions from glucose, Gln and acetyl CoA, and thus is responsive to/dependent on nutrient availability. 2. Diversion of G-6-P into the irreversible, oxidative arm of the Pentose Phosphate Pathway (PPP) by the enzyme, glucose-6-phosphate dehydrogenase (G6PDH). G6PDH expression is often enhanced in cancer but is repressed by

1. Diversion of F6P into the Hexosamine Biosynthetic Pathway (HBP) by the enzymes, glutamine/fructose-6-phosphate transaminases 1 and 2 (GFPT1/2). One of the general products of the HBP is membrane glycoproteins, including receptors for extracellular growthpromoting molecules. Such receptors require

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Metabolic Reprogramming, Fig. 4 Anabolic side pathways of glycolysis. This figure emphasizes how a high flux of carbons through glycolysis can be redirected to multiple anabolic pathways that synthesize macromolecules required for proliferation. For most abbreviations, see legends for Figs. 1 and 2. G6PDH glucose-6-phosphate dehydrogenase, TA transaldolase, TK transketolase, GFPT

glutamine/fructose-6-phosphate transaminase, G3PDH glycerol-3-phosphate dehydrogenase, PHGDH 3phosphoglycerate dehydrogenase, 6-PGT 6phosphoglunonate, R-5-P ribose-5-phosphate, GM-6-P glucosamine-6-phosphate, SER serine, NADP+/NADPH nicotinamide adenine dinucleotide phosphate, oxidized and reduced forms, respectively

p53. This pathway generates a pentose phosphate, ribulose-5-P, as a precursor for ribose-5P, for the generation of nucleotides and nucleic acids. The oxidative arm of the PPP also generates NADPH, which is required for phospholipid, cholesterol, and nucleotide synthesis. NADPH also generates reduced glutathione that neutralizes ROS. 3. Diversion of F-6-P and/or glyceraldehyde-3-P into the reversible, nonoxidative arm of the PPP through the activities of transaldolase and transketolase, which are increased in some cancers. This pathway also generates ribose-5-P for nucleotide and nucleic acid biosynthesis but not NADPH.

4. Diversion of 3-phosphoglycerate into the Serine/Glycine Biosynthetic Pathway (Ser/Gly BP) by the enzyme, 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH expression is enhanced through gene duplication in some cancers. Serine (Ser) is a quasi-nonessential amino acid that is used for the synthesis of other amino acids, proteins, and complex lipids (e.g., ceramide), as well as nucleotides and nucleic acids via contribution of a one-carbon group to the tetrahydrofolate system. In addition, folate-related one-carbon metabolism generates a significant amount of NADPH. Low Ser results in inactivation of PKM2, allowing accumulation of glycolytic

Metabolic Reprogramming

intermediates that become available for the Ser/Gly BP. 5. Diversion of dihydroxyacetone phosphate within the glycerol-3-P pathway by the enzyme glycerol-3-P dehydrogenase. This reaction consumes NADH and generates NAD+, and thus is driven in part by active glycolysis and high glucose. Glycerol-3phosphate is utilized for the synthesis of membrane phospholipids. Glutamine (Gln) and the Switch of the TCA Cycle to Subserving De Novo Lipogenesis Normally, most cells import lipids. In contrast, under nutrient-replete conditions, numerous cancer cell types synthesize a significant amount of free fatty acids, monoacyl-, diacyl-, and triacyl glycerides, and phospholipids to meet the demands of new membrane synthesis (Corbet and Feron 2015). This process is called De Novo Lipogenesis (DNL) and utilizes carbons that exit the TCA cycle (this is called cataplerosis) as citrate, which exits the mitochondria. The carbons that exit the TCA cycle as citrate must be replaced (this is called anaplerosis). A major anaplerotic source of carbon in several cancers is glutamine (Gln), which is the most abundant amino acid in the circulation (0.5 mM). Gln provides a source of nitrogen and is used for synthesis of several products in rapidly dividing cancer cells, including the antioxidant glutathione and nucleotides. However, Gln also plays a major role in TCA cycle function (Fig. 5). Oncogenic signaling, especially through c-Myc, induces expression of the high affinity membrane transporter, SLC1A5 (also called “neutral amino acid transporter”), along with mitochondrial glutaminase that converts Gln to glutamate (Glu). Glu can then be converted to the TCA intermediate a-ketoglutarate (aKG; also called 2-oxoglutarate) by glutamate dehydrogenase (GDH) or by the mitochondrial transaminases glutamic oxaloacetate transaminase 2 (GOT2) and glutamic pyruvate transaminase 2 (GPT2). aKG can be oxidatively decarboxylated to eventually produce oxaloacetate through the “forward” TCA cycle. Oxalocaetate can then condense with acetylCoA to form citrate. Under hypoxic conditions, in which aKG

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dehydrogenase (aKGDH) is inhibited by HIf-1/ Hif-2 and the aKG/citrate ratio is increased, aKG undergoes reduction carboxylation by isocitrate dehydrogenase (IDH) to form isocitrate, which can then be converted to citrate. Once citrate enters the cytoplasm, it is converted to acetyl CoA and oxaloacetate by ATP-citrate lyase (ACLY; Fig. 5). It should be noted that ACLY-produced acetyl CoA is a major source for the acetylation of histones and other proteins, including many metabolic enzymes. Thus, DNL can have global side effects on cancer cell metabolism and function. Cytoplasmic acetyl CoA is converted to malonyl CoA by acetyl CoA carboxylase (ACC). In normal hepatocytes, malonyl CoA is an endogenous inhibitor of the mitochondrial import of FFAs for b-oxidation, thereby avoiding a futile cycle of synthesis and degradation of fatty acids. The role of malonyl CoA in regulating b-oxidation is less clear in cancer cells. Malonyl CoA is then converted to palmatoyl CoA (a fatty acid) by fatty acyl/acid synthase (FASN). Subsequent steps including desaturation and esterification to glycerol-3-P lead to the production of membrane phospholipids. Overall, DNL consumes a significant amount of ATP, NADPH, and carbons. ACLY, ACC1/2, and FASN are all induced at the transcriptional level by carbohydrate-responsive element binding protein (ChREBP) and sterol-responsive element binding protein 1c (SREBP1c; Fig. 4). ChREBP is activated by glucose metabolites, and thus is a signal of glucose abundance. SREBP1c is activated by mTORC1, and thus is another indicator of nutrient availability. Under nutrient-depleted conditions, DNL is reduced and malonyl CoA levels decrease, thus allowing the import of FFAs into mitochondria. Malonyl CoA is also regulated by malonyl CoA decarboxylase (MCD), which converts malonyl CoA back to acetyl CoA. MCD itself is normally under tight regulation, as it: (1) controls the pool of acetyl CoA for protein acetylation and (2) commits the cell further to fatty acyl synthesis, which consumes significant ATP and NADPH. Other factors activated under nutrient/energy stress, such as AMP kinase (AMPK), promote the oxidation of mitochondrial FFAs for ATP production.

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Metabolic Reprogramming, Fig. 5 The role of the amino acid, glutamine, Gln, in anaplerosis. This slide emphasizes that in some cancer cells, the mitochondrial TCA cycle provides carbons for anabolic pathways. A major example of this is the exit of citrate from the mitochondria into the cytoplasm to be used for De Novo Lipogenesis (DNL). This removal of carbons from the TCA cycle (called cataplerosis) must be matched by entry of carbons at another step (called cataplerosis). Acetyl CoA production from pyruvate is often diminished due to inhibition of pyruvate dehydrogenase (PDH) by oncogenic signaling, and thus cannot provide adequate anaplerotic feed into the TCA cycle. Instead, some cancer cells upregulate uptake of Gln through several transporters, especially SLC1A5. Oncogenes also upregulates glutaminase, which catalyzes the conversion of Gln to Glu (with release of ammonia- not shown). Glu can be converted to a-ketoglutarate (aKG; an intermediate of the TCA cycle) by glutamate dehydrogenase (GDH), with release of ammonia (not shown), or by two transaminases (not shown – see text). aKG can be oxidized, starting with its conversion of succinyl CoA (SUC CoA) by the enzyme aKG dehydrogenase (aKGDH). Hypoxia, acting through Hif, inhibits aKGDH. This ultimately promotes the reduction of aKG to isocitrate by isocitrate dehydrogenase

Metabolic Reprogramming

(IDH), which can then be converted to citrate. Once in the cytoplasm, citrate is converted to: (1) acetyl CoA by ATP/citrate lyase (ACLY); (2) to malonyl CoA by acetyl CoA carboxylase (ACC); and (3) to palmitoyl CoA by fatty acyl synthase (FASN). Ultimately, palmitoyl CoA is further modified and used for synthesis of membrane phospholipids after esterification to glycerol-3-phosphate. Growth factor signaling through Akt and certain amino acids activate “mechanistic Target of Rapamycin Complex 1” (mTORC1), which in turn phosphorylates and activates the transcription factor, sterol-responsive element binding protein 1c (SREBP1c). Additionally, several glycolysisrelated metabolites directly or indirectly activate the transcription factor, carbohydrate-responsive element binding protein (ChREBP). Both SREBP1c and ChREBP increase the transcription of the genes encoding ACLY, ACC, and FASN. Note that in normal hepatocytes, malonyl CoA, an intermediate of the DNL pathway, inhibits carnitine/palmitoyl transferase 1 (CPT1), which transfers fatty acyl CoAs through the outer mitochondrial membrane (followed by CPT2-mediated transport across the inner mitochndrial membrane – not shown). This prevents the futile cycle of fatty acid synthesis and degradation. The extent of this negative regulation in cancer is variable

Metabolic Reprogramming

Glossary Novo Lipogenesis The production of palmitoyl CoA from acetyl CoA is normally performed by the liver as a means of converting excess glucose into triglycerides, which can ultimately be packaged into very low density lipoproteins (VLDL) and transferred to adipocytes for storage. The first reaction converts cytoplasmic citrate to acetyl CoA and oxaloacetate by the enzyme, ATP/citrate lyase (ACLY). Cytoplasmic acetyl CoA is converted to malonyl CoA by acetyl CoA carboxylase (ACC1, ACC2). Malonyl CoA is an inhibitor of the carnitine/ palmitoyl transferase 1 (CPT1) enzyme, which transports fatty acyl CoA across the outer mitochondrial membrane on the way to undergoing b-oxidation. This avoids the futile cycle of making and oxidizing fatty acids at the same time. This inhibitory function of malonyl CoA is reduced at least in some cancers. Malonyl CoA is then converted to palmitoyl CoA through several steps as catalyzed by fatty acyl synthase (or fatty acid synthase). These three enzymes of DNL (ACLY, ACC, FASN) are upregulated at the transcriptional level by SREBP-1c and ChREBP. ACC activity is inhibited by phosphorylation by AMP kinase (AMPK) under low energy conditions. Malonyl CoA can be converted back to acetyl CoA by malonyl CoA decarboxylase (MCD). MCD is highly regulated (e.g., by mTORC1/ SIRT4), and inhibition of MCD induces apoptosis in MCF7 breast cancer cells, possibly due to elevated consumption of ATP and NADPH by FASN or by inhibition of the TCA cycle and electron transport system by the metabolite, malonate. GLUT transporters (Solute Carrier 2A Family – SLC2A) Bidirectional facilitative transporters of glucose (GLUTs 1–14). GLUT1 and GLUT3 are high affinity transporters. GLUT2 is a low affinity, high capacity transporter normally expressed in pancreatic b cells, De

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hepatocytes, and the basolateral membranes GI and renal tubular epithelia. GLUT4 is a high affinity transporter that is normally expressed in skeletal muscle and adipocytes and is dependent on insulin/Akt signaling to move into the cell membrane. Expression of GLUTs, especially GLUT1 and GLUT3, is frequently upregulated in cancer. GPR81 Member of the seven-transmembrane, G protein-coupled receptor superfamily. Receptor for lactate. Expressed at high levels in adipocytes. Under FED conditions with a high insulin/glucagon ratio, adipocytes actively import glucose and metabolize it through glycolysis (and through the glycerol-3-phosphate side pathway to make the backbone of triglycerides [TG]). Adipocytes produce a significant amount of lactate, which in turn binds to and activates GPR81. GPR81 is coupled to Gi, and thereby inhibits adenylate cyclase. Thus, during a time when the adipocyte is making TGs, it also inhibits cAMP-protein kinase A in order to inhibit the lipolysis of TG via activation of hormone-sensitive lipase. GPR81 is expressed in several cancer cell lines and shown to be important for the expression of MCT transporters and the ability of cancer cells to adjust to low glucose/high lactate conditions in pancreatic cancer cells. Hexokinase (HK) ATP-dependent kinases of six carbon sugars, especially glucose. Conversion of glucose to glucose-6-phosphate traps it inside the cell, as glucose-6-phosphate is not recognized by GLUTs. Most isoforms of hexokinases are high affinity, low Vmax enzymes that are product inhibited by glucose-6-phosphate, making their activity closely linked to the need of cells for more glucose-6-phosphate. Hexosamine Biosynthetic Pathway (HBP) Glutamine/fructose-6-P transamidase (GFPT 1 and/or 2) converts the glycolytic intermediate, fructose-6-P, to glucosamine-6-phosphate. An important use of the HBP is glycosylation of proteins to produce glycoproteins.

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Glycoproteins include cell membrane proteins such as growth factor receptors, etc. Isocitrate Dehydrogenase (IDH) Catalyzes reversible conversion of isocitrate and 2oxoglutarate (2-OG; also called a-ketoglutarate). 2-OG is an essential cofactor for several enzymes, including ones that alter chromatin structure and gene expression. IDH1 is a cytoplasmic isoform that can generate NADPH from conversion of isocitrate to 2OG, or consume NADPH from conversion of 2OG to isocitrate. IDH2 and 3 are mitochondrial isoforms. IDH2 reversibly catalyzes the reaction and generates NADPH from isocitrate conversion to 2-OG. IDH3 can only catalyze this reaction in the direction of isocitrate to 2-OG, producing NADH. Gliomas and other cancers are caused by mutations in IDH isoforms. Mutations reduce the amount of 2-OG produced but also make a structurally similar metabolite called 2hydroxyglutarate (2-HG). High levels of 2-HG induce neoplastic transformation. Thus, 2-HG is called an oncometabolite. Mitochondrial Pyruvate Dehydrogenase (PDH) Catalyzes the decarboxylation of mitochondrial pyruvate to form acetyl CoA. A multienzyme complex composed of pyruvate decarboxylase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). PDH requires the following cofactors: thiamine pyrophosphate, lipoic acid, coenzyme A (CoA), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD +). PDH is directly allosterically inactivated by NADH and acetyl CoA. PDH is inactivated by PDH kinase (PDK), which is activated by ATP, acetyl CoA, and NADH but inactivated by pyruvate. PDH is activated by PDH phosphatase (PDPH), which is activated by Ca2+ and insulin signaling via Akt. Deficiency in PDH E1 is the most common cause of congenital lactic acidosis (overall a rare disorder).

Metabolic Reprogramming

Monocarboxylate Transporters (MCTs; Solute Carrier 16A Family – SLC16A) Cotransporters of lactate anion and H+. MCT1 and MCT2 are typically involved in the import of lactate in oxidative cells – or in cells in which lactate serves as a signaling molecule (e.g., endothelial cells) – and both can also allow the export of lactate from the cell in certain contexts. MCT1 expression is induced by c-myc and is upregulated in several cancers. MCT4 is typically expressed by highly glycolytic cancers and is primarily involved in the export of lactate and H+. Glucokinase (Hexokinase IV) is a low affinity, high capacity enzyme expressed in pancreatic b cells and hepatocytes that only acts on glucose when it is highly abundant (i.e., after a meal). Hexokinases (especially HK2) are upregulated in several cancers. Serine/Gycine Biosynthetic Pathway (Ser/Gly BP) The Ser/Gly BP is composed of four enzymatic steps. The first step converts the glycolytic intermediate, 3-phosphoglycerate, into phosphohydroxypyruvate (PHP). This reaction is catalyzed by the enzyme, phosphoglycerate dehydrogenase (PHGDH), and consumes NAD+. The PHGDH gene is amplified in some cancers. The second reaction converts PHP into phosphoserine. This reaction is catalyzed by phosphoserine aminotransferase 1 (PSAT1). This reaction converts glutamic acid to a-ketoglutarate, which can enter the mitochondria and the TCA cycle. The third reaction converts phosphoserine to Ser by the enzyme phosphoserine phosphatase. Ser is used for the synthesis of other amino acids, proteins, complex lipids, and nucleotides. Ser is converted to glycine by the enzyme, Serine Hydroxymethyltransferase (SHMT1 or 2). This reaction is linked to single-carbon folate metabolism, generating 5,10-methylene tetratrahydrofolate (5,10-methyleneTHF) from THF. 5,10-methylene THF is used for deoxythymidine triphosphate (dTTP) synthesis or can be converted to other forms of THF for use in pyrimidine synthesis.

Metabolic Reprogramming

Cross-References ▶ Angiogenesis ▶ Epithelial-to-Mesenchymal Transition ▶ G Proteins ▶ Glycosylation ▶ Hexokinase 2 ▶ Hypoxia ▶ Mammalian Target of Rapamycin ▶ N-myc Downstream-Regulated Gene ▶ Nuclear Factor-kB ▶ Oncogene ▶ RAS Genes ▶ Reactive Oxygen Species ▶ TP53 ▶ Warburg Effect

References Bobrovnikova-Marjon E, Hurov JB (2014) Targeting metabolic changes in cancer: novel therapeutic approaches. Annu Rev Med 65:157–170 Boroughs LK, DeBerardinis RJ (2015) Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol 17:351–359 Corbet C, Feron O (2015) Metabolic and mind shifts: from glucose to glutamine and acetate addictions in cancer. Curr Opin Clin Nutr Metab Care 18:346–353 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 Li Z, Yang P, Li Z (2014) The multifaceted regulation and functions of PKM2 in tumor progression. Biochim Biophys Acta 1846:285–296 Luc R, Tortorella SM, Ververis K, Karagiannis TC (2015) Lactate as an insidious metabolite due to the Warburg effect. Mol Biol Rep 42:835–840 Vander Heiden MG, Lunt SY, Dayton TL, Fiske BP, Israelsen WJ, Mattaini KR, Vokes NI, Stephanopoulos G, Cantley LC, Metallo CM, Locasale JW (2011) Metabolic pathway alterations that support cell proliferation. Cold Spring Harb Symp Quant Biol 76:325–334 Ward PS, Thompson CB (2012a) Signaling in control of cell growth and metabolism. Cold Spring Harb Perspect Biol 4:a006783 Ward PS, Thompson CB (2012b) Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell 21:297–308 Wong N, Ojo D, Yan J, Tang D (2015) PKM2 contributes to cancer metabolism. Cancer Lett 356:184–191

See Also (2007) SLC1A5 (Neutral Amino Acid Transporter). Landowski CP, Suzuki Y, Hediger MA Transporters

2781 for excitatory and neutral amino acids. Neural membranes and transport. In: Lajtha A, Reith MEA (ed) Handbook of neurochemistry and molecular neurobiology. Springer, Boston, pp 305–323. doi:10.1007/ 978-0-387-30380-2_15 (2008) Acetyl-CoA (acetyl coenzyme A, ACoA). In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, Netherlands, pp 11–12. doi:10.1007/978-1-4020-6754-9_119 (2008) Fructose-2,6-bisphosphatase. In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, Netherlands, p 716. doi:10.1007/978-1-4020-6754-9_6262 (2008) Glutamate dehydrogenase (Mr 330,000). In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, Netherlands, p 806. doi:10.1007/978-1-4020-6754-9_6961 (2008) Glutamate. In: Offermanns S, Rosenthal W (eds) Encyclopedia of molecular pharmacology. Springer, Berlin/Heidelberg, p 552. doi:10.1007/978-3-54038918-7_5802 (2008) Glutaminase (GLS). In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, Netherlands, p 807. doi:10.1007/978-1-4020-6754-9_6970 (2008) Pentose phosphate pathway. In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, Netherlands, pp 1463–1464. doi:10.1007/978-1-4020-6754-9_12511 (2008) Phosphofructose Kinase 1 (PFK-1, PFKL, phosphofructokinase). In: Rédei GP (ed) Encyclopedia of Genetics, Genomics, Proteomics and Informatics, 3rd edn. Springer Netherlands, p 1483. doi:10.1007/ 978-1-4020-6754-9_12739 (2012) Adipocytes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 67. doi:10.1007/978-3-642-16483-5_100 (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163 (2012) Antioxidant defenses. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 216. doi:10.1007/978-3-642-16483-5_330 (2012) ATP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 302. doi:10.1007/978-3-642-16483-5_440 (2012) Beta-catenin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 385. doi:10.1007/978-3-642-16483-5_889 (2012) Biosynthesis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 415. doi:10.1007/978-3-642-16483-5_647 (2012) cAMP response element binding protein. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 603. doi:10.1007/9783-642-16483-5_789 (2012) C-Myc. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 886. doi:10.1007/978-3-642-16483-5_1232

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2782 (2012) Glucose. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1558. doi:10.1007/978-3-642-16483-5_2430 (2012) G-protein couple receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1587. doi:10.1007/978-3-642-16483-5_2294 (2012) HER2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1678. doi:10.1007/978-3-642-16483-5_2676 (2012) Ischemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1917. doi:10.1007/978-3-642-16483-5_3151 (2012) Lactate dehydrogenase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1967–1968. doi:10.1007/978-3-642-164835_3260 (2012) Lactate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1967. doi:10.1007/978-3-642-16483-5_3259 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720 (2012) Neoplastic cell transformation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2474. doi:10.1007/978-3-642-16483-5_4013 (2012) Non-essential amino acid. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4108 (2012) Oxidative phosphorylation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2730. doi:10.1007/978-3-642-164835_4308 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) Phospholipids. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2869–2870. doi:10.1007/978-3-642-164835_4542 (2012) SREBP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3499. doi:10.1007/978-3-642-16483-5_5473 (2012) Tumor suppressor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3803. doi:10.1007/978-3-642-16483-5_6056 (2013) Glutamate. In: Gebhart GF, Schmidt RF (eds) Encyclopedia of pain, 2nd edn. Springer, Berlin/Heidelberg, p 1383. doi:10.1007/978-3-642-28753-4_200889 Arese P, Gallo V (2014) Pentose phosphate pathway. In: Hommel M, Kremsner PG (eds) Encyclopedia of malaria, live reference. Springer, New York, pp 1–14. doi:10.1007/978-1-4614-8757-9_5-1

Metal Complex ▶ Metal Drugs

Metal Complex

Metal Compound ▶ Metal Drugs

Metal Drugs Petra Heffeter1 and Christian Kowol2 1 Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria 2 Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria

Synonyms Coordination compound; Metal complex; Metal compound; Metal-based drug; Metallodrug; Organometallic drug

Definition A chemical substance containing a metal ion used in the treatment, cure, prevention, or diagnosis of a disease.

Characteristics With regard to the different subtypes of metal drugs, we can distinguish between simple metal salts (e.g., CuCl2) and metal complexes (e.g., [PtCl4]2). Metal salts are characterized by ionic bonds between charged atoms, while for metal complexes the metal ion is bound by ligands (molecules, which are bound to the central metal ion) via so-called coordinative bonds. To distinguish between both species, a compound which dissociates in aqueous solution like CuCl2 ! Cu2 + + 2Cl is defined as simple metal salt. In contrast, in metal complexes the coordinated ligands do not dissociate in solution. Most metal drugs belong to the class of metal complexes. In addition, a metal complex is called organometallic

Metal Drugs

when a direct metal-carbon bond is present (e.g., ferrocen; Fe(C5H5)2). However, no organometallic drug is currently used as anticancer agent in clinical routine. In general, metal complexes offer a much more diverse chemistry than purely organic compounds, which contain mainly carbon, hydrogen, nitrogen, oxygen, phosphor, sulfur, and chlorine. In addition, the number of ligands in a metal complex can distinctly vary between different oxidation states of the metal center. Thus, e.g., platinum(II) drugs have only four coordination sites, while platinum (IV) compounds typically have six. In case of platinum(IV), these additional ligands can also be used to attach known bioactive compounds to generate multitargeted anticancer drugs. One of the important chemical characteristics of metals (with regard to their biological activity) is their potential to undergo redox processes. Especially transition metal ions (e.g., iron, copper, etc.) are usually able to switch between several oxidation states. However, not all oxidation states are observed under physiological conditions in the living organism. Due to the redox activity of many metals, the sensitive cellular redox homeostasis can be easily disturbed (CROSSREF: Reactive Oxygen Species). Thus, a tight regulation of the cellular metal and redox balance is crucial for health and survival. Cancer cells are known to differ distinctly in their redox metabolism from healthy tissues and are very sensitive to interferences with the cellular redox homeostasis. Thus, there are several attempts to specifically target the altered redox conditions in cancer cells, and metal-based anticancer drugs are ideal candidates as most of them show a distinct interaction with the cellular redox system. History of Metals in Anticancer Medicine Since ancient times, metal compounds have been successfully used for the treatment of a variety of diseases. For example, already in ancient Egypt the therapeutic potential of gold salts was known. Moreover, in traditional Chinese medicine, arsenic drugs were used as antiseptic agents or in the treatment of rheumatoid diseases, syphilis, as well as psoriasis. Indeed, arsenic trioxide (ATO) was one of the first compounds that was suggested for

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anticancer therapy, and during the eighteenth and nineteenth century this drug represented the main treatment for leukemia. The modern era of metalbased anticancer drugs began with the discovery of the platinum(II) complex cisplatin by Barnett Rosenberg in the 1960s. Nowadays, cisplatin and its successors carboplatin and oxaliplatin are among the most important chemotherapeutics (CROSSREF: Chemotherapy) used against a wide variety of different cancers. Stimulated by the success of cisplatin, also other metal complexes were tested for their anticancer activity, and several promising candidates (e.g., ruthenium complexes) are currently in (pre)clinical evaluation. Platinum(II) Drugs: Best Sellers in Cancer Therapy As already mentioned above, the modern clinical use of metal-based anticancer drugs began with the discovery of the anticancer properties of the platinum(II) compound cisplatin (CROSSREF: Cisplatin, Platinum Complexes) by Barnett Rosenberg in the 1960s. With the aim to reduce the therapy-related side effects, the less toxic carboplatin was developed in the 1980s. Nowadays, platinum-based drugs are among the most important chemotherapeutics and are clinically used against a wide variety of different solid tumors, including testicular, bladder, ovarian, as well as head and neck cancer. In addition, oxaliplatin is used as standard therapeutic for colorectal cancer (typically together with folinic acid and 5-fluorouracil in the so-called FOLFOX scheme). With regard to the underlying mode of action, it is widely accepted that at least one part of the anticancer activity of cisplatin is based on the formation of platinum-DNA adducts. Subsequently, this leads to distortion of the DNA structure resulting in inhibition of DNA replication and transcription. However, for example, the outstanding curative potential of cisplatin in case of testicular cancer cannot be explained so far, and the exact mechanism(s) of platinum(II) drugs are still under lively discussion. Platinum(IV) Drugs: A Prodrug Strategy Interestingly, the anticancer activity of platinum (IV) complexes was already discovered together

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with cisplatin in the 1960s. Nevertheless, these platinum complexes have been less well studied and developed than platinum(II) compounds. The main difference between platinum(II) and platinum(IV) drugs is that due to the higher coordination number platinum(IV) complexes have six in contrast to four coordination sites attached to the platinum core. This allows not only the introduction of additional functional ligands but also distinctly impacts on their pharmacological properties. Consequently, platinum(IV) drugs are usually characterized by higher lipophilicity, higher stability, and altered redox properties. In addition, platinum(IV) complexes are chemically more inert than their platinum(II) counterparts. Based on this lower reactivity with biomolecules, platinum(IV) drugs are considered as prodrugs, which are characterized by reduced adverse effects as well as possible oral application. The most important platinum(IV) drug candidate is satraplatin (JM-216), which has been considered for approval by the FDA for the treatment of hormone-refractory prostate cancer in a combination regimen with prednisone, a synthetic corticosteroid. However, the first phase III study did not achieve the anticipated endpoint of overall survival. Consequently, several further clinical trials with satraplatin in combination regimes (e.g., with Erlotinib or Abraxane) are ongoing. Arsenic Trioxide: Rediscovery of an Old Drug Arsenic and cancer is a two-faced story (CROSSREF: Arsenic): On the one hand, arsenic is a known human cancerogen, which has been repeatedly affecting people due to exposure via drinking water over the last centuries (there are currently millions of people affected in Bangladesh and neighboring countries). Based on these poisonings, it is now well established that longterm exposure to arsenic is linked to cancer development in skin, liver, prostate, lungs, as well as bladder and kidney. On the other hand, some of the oldest remedies of human medicine are known to contain arsenic. For example, in traditional Chinese medicine, arsenic acid and arsenic trioxide (ATO) were used as antiseptic agents or in the treatment of rheumatoid diseases, syphilis, and psoriasis. In the Western world, the potassium

Metal Drugs

bicarbonate-based Fowler’s solution of ATO (developed for oral use in 1788) was frequently applied against eczema, asthma, and psoriasis but also against diverse malignant diseases including leukemias like CML and Hodgkin disease. In fact, already Celsus in the first century AD had suggested activity of arsenic against solid tumors. During the eighteenth and nineteenth centuries, ATO represented the main treatment for leukemia, and its importance remained until the development of modern radio- and chemotherapy during the twentieth century. Then ATO was replaced by novel chemotherapeutic regimens and in part was abandoned based on chronic toxicity observed in treated patients. Surprisingly, during the 1990s a Chinese group reported an exceptionally high rate of complete, long-lasting remissions in a small cohort of patients with acute promyelocytic leukemia (APL), a specific subtype of acute myeloid leukemia (AML), after ATO treatment. These promising initial data finally led to the approval of ATO for the treatment of APL in 2000. Ruthenium: Anticancer Agents with Reduced Side Effects Ruthenium complexes are among the best-studied nonplatinum anticancer metal complexes, and two drug candidates (KP1019/NKP-1339 and NAMI-A) have already been tested in clinical phase I trials. Notably, KP1019 was developed for solid tumors, whereas NAMI-A was developed as a drug to prevent (further) metastasis development. Especially KP1019/NKP-1339 was proven to be very tolerable with only minor side effects in these clinical trials. With regard to their modes of action, ruthenium(III) compounds have a very high affinity to (serum) proteins (e.g., albumin). As the solid tumor tissue is characterized by enhanced accumulation (the so-called enhanced permeability and retention effect) (CROSSREF: Drug delivery systems for Cancer Treatment; Targeted Drug Delivery) this feature makes ruthenium(III) compounds intrinsically tumor targeted. After delivery into the malignant tissue, ruthenium(III) complexes are (comparable to platinum(IV) drugs) reduced to their highly reactive ruthenium(II) metabolites, which then lead to cell damage and apoptosis induction via

Metastasis

the mitochondria. In addition, GRP78 has been supposed to be crucial in the anticancer activity of NKP-1339. Overall, due to their special characteristics ruthenium complexes are currently the most promising nonplatinum compounds in the field of metal-based drug development. Conclusions (Heavy) metals are often considered as “toxic,” which is probably linked to several intoxications due to environmental contaminations, as described for arsenic. However, the clinical routine demonstrates that metal complexes can be highly effective chemotherapeutics, which is reflected by the huge importance of platinumbased drugs in the treatment of multiple cancer types. In addition, the specific characteristics of the metal coordination chemistry make this kind of drugs very multifaceted tools as it intrinsically allows tumor-specific activation or drug delivery. Consequently, metal complexes can be designed as highly active and at the same time very tolerable anticancer drugs, which is an optimal requirement for modern drug development.

2785 Trondl R, Heffeter P, Kowol CR, Jakupec MA, Berger W, Keppler BK (2014) NKP-1339, the first rutheniumbased anticancer drug on the edge to clinical application. Chem Sci 5(8):2925–2932 Wheate NJ, Walker S, Craig GE, Oun R (2010) The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans 39:8113–8127

Metal-Based Drug ▶ Metal Drugs

Metallodrug ▶ Metal Drugs

Metalloprotease Disintegrin Cysteine Rich ▶ ADAM Molecules

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Cross-References

Metaplasia

▶ Arsenic ▶ Chemotherapy ▶ Cisplatin ▶ Drug Delivery Systems for Cancer Treatment ▶ Platinum Complexes ▶ Reactive Oxygen Species ▶ Targeted Drug Delivery

▶ Stem Cell Plasticity

References Heffeter P, Jungwirth U, Jakupec M, Hartinger C, Galanski M, Elbling L, Micksche M, Keppler B, Berger W (2008) Resistance against novel anticancer metal compounds: differences and similarities. Drug Resist Updat 11(1–2):1–16 Jungwirth U, Kowol CR, Hartinger C, Keppler BK, Berger W, Heffeter P (2011) Anticancer activity of metal complexes: involvement of redox processes. Antioxid Redox Signal 5(4):1085–1127 Ott I, Gust R (2007) Non platinum metal complexes as anti-cancer drugs. Arch Pharm Chem Life Sci 340:117–126

Metastasis Ruth J. Muschel Radiation Oncology and Biology, University of Oxford, Oxford, UK

Synonyms Secondary tumor; Tumor spread

Definition Metastasis is the growth of a tumor at a site not physically connected to the primary tumor.

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Characteristics The presence of metastasis is a key feature in determining the prognosis and the treatment strategy for any cancer patient. The probability that a patient will have a metastasis varies widely depending upon the type of cancer. Squamous cell carcinoma of the skin rarely metastasizes, but squamous cell carcinoma of the lung frequently does. An important part of the clinical workup for every patient who is diagnosed with cancer is an evaluation for metastasis. The presence and extent of metastases are a critical component of clinical staging systems that clinicians use to choose appropriate therapy and to judge prognosis. Metastases follow different routes that can be broadly classified into three. One is metastasis to the regional lymph nodes or lymphatic metastasis. This involves colonization of draining lymph nodes. The second is travel via open cavities such as the pleural cavity or abdominal cavity (peritoneal metastases). The third is travel though the blood stream or hematogenous metastasis. Metastasis also has organ specificity. Different types of cancer have characteristic tendencies to metastasize to particular sites. For example, adenocarcinoma of the proximal colon tends to metastasize to the regional lymph nodes within the colon and distantly to the liver. In contrast, prostatic adenocarcinoma tends to metastasize to its regional lymph nodes and to the bone. Other cancer types are more promiscuous – for example, breast cancer spreading frequently to the regional lymph nodes, lung, liver, brain, bone, ovaries, and adrenals or carcinomas of the lung metastasizing to the liver, brain, bone, skin, and adrenals. While metastases are often identified at the time of initial diagnosis, they can also become manifest many years after therapy. This feature of tumor dormancy is particularly notable in breast cancer where metastases can emerge after 10 or 20 years. In many cancer types, this long dormancy is less likely. Mechanisms Cancer is now recognized to be a disease not only consisting of the tumor but involving the whole

Metastasis

organism. Metastasis depends upon properties of the host as well as alterations in the tumor cells. The notion that either the host or the tumor might play a role was first expressed as the soil and seed hypothesis. This frequently cited expression posits that metastasis might be due to alterations in the tumor cell or alternatively to adaptations of the host that now allow the tumor cell to migrate and colonize a distant site. Many years of research now reveal that both mechanisms operate. The study of the molecular changes underlying metastasis is only in its beginnings so that much of the information lacks certainty and detail. In keeping with the seed hypothesis, however, are findings showing that the patterns of gene expression in metastatic tumor cells differ from those in nonmetastatic cells. The expression of these genes may help explain organ specificity and may potentially be targets for therapeutic intervention. One question that has been addressed is to ask whether the gene expression profile of a tumor might predict whether that tumor was likely to have formed metastases. If so, the genes involved might also be identified from such a screen. In fact in many studies such gene profiles could be identified, but in general the actual genes identified were not the same in different studies. These profiles are now being refined and additional components such as hypoxia and wound healing profiles are being integrated into the original findings. In some cases, the genes found were those made by the host stromal response to the tumor rather than by the tumor itself. Furthermore, in comparing metastatic to nonmetastatic tumors in experimental models, the acquisition of new gene expression patterns were clearly seen. Thus the actual situation is more complicated than the original hypothesis that the tumor might express all of the genes needed for metastasis very early in its development. It seems most likely that some of the necessary genes are present early on, but that others are acquired during progression. Finally, some of the identified gene products appear to have been synthesized by host responses. Thus, host responses may have several roles some of which impede tumor growth but others that stimulate metastasis.

Metastasis Suppressor Gene

Invasion is a necessary precursor to metastasis. For the tumor cell to reach a distant lymph node or other organ or cavity, it must first separate from the primary tumor mass itself, either as a single cell or as a clump. In vivo imaging in mice has revealed such tumor cell migration through tissue adjacent to the tumor itself. Tumor cells in the blood or lymph then can disseminate. In some systems, host cells such as macrophages, fibroblasts, and the coagulation system are required for efficient invasion and metastasis, findings in keeping with the soil concept of tumor metastasis. The microenvironment of a tumor also appears to play a role in metastatic potential. For example, hypoxic tumors are more likely to metastasize than tumors with more normal oxygen levels. The infiltrating cells in a tumor may also affect its metastatic potential including fibroblasts, macrophages, and NK cells. For metastasis to the lymph nodes there is some debate about the importance of new host lymphatic channels within the tumor. Data raise the exciting hypothesis that the new channels are generated in the lymph nodes themselves. The contribution by host cells is perhaps most well understood in the formation of bone metastasis in which tumor cells that are successful in establishing themselves in bone synthesize factors such as parathyroid hormone related protein that activate osteoblasts to produce factors such as RANKL that stimulate osteoclasts that release tumor growth factors setting up a complex positive feedback loop. The elements of this loop then form targets for bone metastasis specific therapy. Thus potential targets for therapy are being identified, many of which will be organ specific. Less clear are the factors that cause tumors to become metastatic in the first place. While genetic mutations clearly drive tumor progression and malignancy, which changes affect metastasis are less clear. Both tumor cell factors and external factors appear to play a role.

Cross-References ▶ Gastrointestinal Stromal Tumor ▶ Metastatic Colonization ▶ Trefoil Factors

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References Gupta GP, Minn AJ, Kang Y et al (2005) Identifying sitespecific metastasis genes and functions. Cold Spring Harb Symp Quant Biol 70:149–158 Kopfstein L, Christofori G (2006) Metastasis: cellautonomous mechanisms versus contributions by the tumor microenvironment. Cell Mol Life Sci 63:449–468 Sohara Y, Shimada H, DeClerck YA (2005) Mechanisms of bone invasion and metastasis in human neuroblastoma. Cancer Lett 228:203–209

Metastasis Suppressor Gene Danny R. Welch, Ken Sasaki and Gada Al-Ani Department of Cancer Biology, University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, USA

Definition Metastasis suppressors are a family of molecules functionally defined by their ability to block metastasis without blocking primary tumor growth. The suppressing activity could be the result of alterations at any of the steps in the multistep metastatic cascade.

Characteristics To fully understand metastasis suppressors, it is first important to define a number of terms associated with carcinogenesis, tumor progression, invasion, and metastasis. Tumorigenesis refers to

Modified version of Welch DR, Vaidya KS, Hurst DR, Silveira AC (2012) Metastasis Suppressor Gene. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 2265-2267. doi:10.1007/978-3-64216483-5_3673 The entry “Metastasis Suppressor Gene” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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the acquired capacity of cells to proliferate in the absence of persistent stimulation by triggering carcinogenic agent(s). Tumor progression is the evolution of tumorigenic cells toward increasing malignancy. Invasion involves migration away from the primary tumor mass, involves breakdown of basement membranes, and usually involves secretion of proteinases. Metastasis is the process by which a malignant neoplastic cell(s) spreads to discontiguous sites and establishes secondary masses. In order to metastasize, cells must complete a series of selective steps termed the metastatic cascade. Conceptually, metastasis is comprised of two components: positive and negative regulators. Metastasis-promoting genes drive metastasis formation. However, most metastasis promoters are neither necessary (because of redundancy) nor sufficient (because multiple steps are involved in the metastatic cascade). In contrast, metastasis suppressors block metastases by inhibiting one or more steps in the metastatic cascade. Since metastasis requires cells to complete every step in the metastatic cascade, these genes can block any step(s) in the metastatic cascade. The first metastasis suppressor gene, Nm23 (or NME1, NDKB), was discovered by Patricia Steeg in 1989. Since that time several others have been identified and functionally validated (note: there are other candidates, but they are not included in this list due to lack of functional validation). To date, more than 30 have been shown to suppress metastasis in vivo (aliases are in parentheses): BRMS1, cadherin-11, CADM1 (TSLC1, IgSF4, Necl2, Syncam), caspase-8, CD44 (CDW44, SCPG8, ECMR-III), claudin-1 (CLDN1), claudin-4 (CLDN4), CRSP3 (MED23), CRMP4 (DRP-3, ULIP-1), connective tissue growth factor (CTGF), DCC (CRC18, CRCR1, MRMV1), DLC-1 (STARD12, p122, RhoGAP), Drg-1, E-cadherin (CD324), FXR (NR1H4), GAS1, GSN (ADF, AGEL), gelsolin, HUNK (MAK-V), KAI1 (CD82, SAR2, TSPAN27), KISS1, LIFR (CD118, STWS), LSD1 (KDM1), MTBP (ACTFS, HDMX, HDM2), microRNA (metastamiR are metastasis-

Metastasis Suppressor Gene

regulatory microRNA; miR-6a,b; 9; 19a-3p; 31; 34a; 126; 139; 145; l46a,b; 196a2/b; 206; 335), N-cadherin, RECK, RhoGDI2, Src-suppressed C kinase substrate (SSeCKs), JNKK1/MKK4 (MAPKK4), MKK6 (MAPKK6), MKK7 (MAPKK7), P38 (MAPKK14), NDRG1 (DRG1, Cap43, Rit42, RTP, PROXY-1), OGR1 (GPR68, GPR12A), RhoGDI2 (ARHGDIB), RKIP (’), RRM1 (RIR1, RR1), SSeCKs (AKAP12, GRAVIN), stefin A (CST6, EPM1, STFB), TIMP1, TIMP2, TIMP3, TIMP4, and TXNIP. How are metastasis suppressor genes identified? Three general approaches have been used to identify metastasis-controlling genes. The first involves comparison of gene expression in poorly or nonmetastatic cells with matched metastasis-competent cells. The specific techniques employed are differential display and subtractive hybridization. The second takes advantage of clinical observations that identified nonrandom chromosomal changes that occur during tumor progression. This information localized the gene(s) from which cloning could commence. Based upon karyotypic patterns observed in human cancers, additional metastasis suppressor genes to those listed above are hypothesized to exist. However, the identities of the specific genes have yet to be determined. Third, insertional mutagenesis or library knockdown of selective molecules in an unbiased manner (e.g., transduction of shRNA libraries into cells to observe acquisition of metastatic capability) has been used. The role of metastasis suppressors is context dependent: the ability to suppress varies by cancer type and mode of action. Each varies in the step(s) of the metastatic cascade inhibited. Each metastasis suppressor regulates by manipulating specific biochemical functions. Several affect signaling cascades that are involved in (1) cell growth, angiogenesis, and local invasion, (2) cell transit and adhesion, and (3) colonization. Readers are referred to individual entries for metastasis suppressors to learn more about their discovery and their specific roles in regulating metastasis.

Metastasis Suppressor Gene

Clinical Relevance In general, metastasis suppressor expression is inversely correlated with recurrence and directly correlated with patient survival. Mutations of metastasis suppressors have been relatively uncommon. Rather, downregulation of expression is more commonly seen. Theoretically, restoration of metastasis suppressor expression or function has therapeutic potential, but to date the therapeutic potential of metastasis suppressors has not yet been realized.

Cross-References ▶ ADAM17 ▶ Adherens Junctions ▶ Adhesion ▶ Adiponectin ▶ Adriamycin ▶ Aggressive Fibromatosis in Children ▶ Allogeneic Cell Therapy ▶ Anoikis ▶ Anoxia ▶ AP-1 ▶ APC/b-Catenin Pathway ▶ Apoptosis ▶ Aryl Hydrocarbon Receptor ▶ BCL2 ▶ Betulinic Acid ▶ Bid ▶ Bile Duct Neoplasms ▶ Bladder Cancer ▶ Bone Loss Cancer Mediated ▶ BRAF Somatic Alterations ▶ Breast Cancer ▶ Breast Cancer Carcinogenesis ▶ Breast Cancer Familial Risk ▶ Breast Cancer Multistep Development ▶ Breast Cancer Prognostic and Predictive Biomarkers ▶ Breast Cancer Prognostic Biomarkers ▶ Breast Cancer Stem Cells ▶ Breast Cancer Susceptibility Genes ▶ BRMS1 ▶ Calcitonin

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▶ Calreticulin ▶ Cancer Epigenetics ▶ Cancer Stem-Like Cells ▶ Carcinoid Tumors ▶ Caspase-8 ▶ Caveolins ▶ CD44 ▶ CDKN2A ▶ Celecoxib ▶ Cell Adhesion Molecules ▶ Chelators as Anti-Cancer Drugs ▶ Circulating Tumor Cells ▶ Class II Tumor Suppressor Genes ▶ Colorectal Cancer Clinical Oncology ▶ Colorectal Cancer Premalignant Lesions ▶ Cortactin ▶ Cripto-1 ▶ Curcumin ▶ Desmoglein-2 ▶ Desmoplasia ▶ Diabody ▶ DNA Damage-Induced Apoptosis ▶ Dormancy ▶ Doublecortin ▶ Duffy Antigen Receptor for Chemokines ▶ E-cadherin ▶ Elongin BC Complex ▶ Endocytosis ▶ Endosomal Compartments ▶ EpCAM ▶ Epithelial-to-Mesenchymal Transition ▶ Epstein-Barr Virus ▶ ERM Proteins ▶ E-Selectin-Mediated Adhesion and Extravasation in Cancer ▶ Exfoliation of Cells ▶ Extracellular Matrix Remodeling ▶ FLICE-Inhibitory Protein ▶ Furin ▶ G Proteins ▶ GAGE Proteins ▶ Gastric Cancer ▶ Gelsolin ▶ GLI Proteins ▶ Glycosylation ▶ Grape Seed Extract

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▶ Hematological Malignancies, Leukemias, and Lymphomas ▶ Hepatitis B Virus x Antigen-Associated Hepatocellular Carcinoma ▶ Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention ▶ Hyaluronan Synthases ▶ Inflammatory Breast Cancer ▶ Inositol Lipids ▶ Insulin-Like Growth Factors ▶ Integrin Signaling ▶ Intrinsically Unstructured Proteins ▶ Invasion ▶ IQGAP1 Protein ▶ JNK Subfamily ▶ KISS1 ▶ Laminin Signaling ▶ Lats in Organ Size Regulation and Cancer ▶ Linoleic Acid ▶ Lobular Involution of the Breast ▶ Lung Cancer Staging ▶ Lymphangiogenesis ▶ MAP Kinase ▶ Mast Cells ▶ Matrix Metalloproteinases ▶ MDM Genes ▶ Melanocytic Tumors ▶ Mesoblastic Nephroma ▶ Mesothelin ▶ MET ▶ Metastasis ▶ Metastasis Suppressor KAI1/CD82 ▶ Metastatic Colonization ▶ Methoxyestradiol ▶ Micrometastasis ▶ MicroRNA ▶ Migration ▶ Mitotic Arrest-Deficient Protein 1 ▶ Motility ▶ Neuroendocrine Neoplasms ▶ Neurofibromatosis 2 ▶ Neuromedin ▶ Neutrophil Elastase ▶ N-myc Downstream-Regulated Gene ▶ Oncostatin M ▶ Osteopontin ▶ Ovarian Cancer ▶ Ovarian Cancer Chemoresistance

Metastasis Suppressor Gene

▶ Ovarian Cancer Pathology ▶ Pancreatic Cancer Stem Cells ▶ Particle-induced Cancer ▶ Pheochromocytoma ▶ Platelet-Derived Growth Factor ▶ Platinum Complexes ▶ Pleiotrophin ▶ Podoplanin ▶ Pre-mRNA Splicing ▶ Progression ▶ Prostate Cancer Clinical Oncology ▶ Prostate Cancer Stem Cells ▶ Proteasome Inhibitors ▶ Prune ▶ Pseudomyxoma Peritonei ▶ Raf Kinase ▶ RAS Transformation Targets ▶ RECK Glycoprotein ▶ Rhabdomyosarcoma ▶ Rho Family Proteins ▶ Ron Receptor ▶ Scatter Factor ▶ Secreted Protein Acidic and Rich in Cysteine ▶ “Seed and Soil” Theory of Metastasis ▶ Sjögren Syndrome ▶ Slit ▶ Snail Transcription Factors ▶ Sphingolipid Metabolism ▶ Src ▶ Stefins ▶ Stem Cell Markers ▶ Stress Response ▶ Stromagenesis ▶ Stromelysin-1 ▶ Sulforaphane ▶ Supervised Classification ▶ Surgical Trauma and Cancer Recurrence ▶ Taxotere ▶ Testicular Cancer ▶ Thioredoxin System ▶ Three-Dimensional Tissue Cultures ▶ Tiam1 ▶ Tight Junction ▶ Tissue Inhibitors of Metalloproteinases ▶ TNF-Related Apoptosis-Inducing Ligand ▶ Tongue Cancer ▶ TP53 ▶ TRAIL Receptor Antibodies

Metastasis Suppressor KAI1/CD82

▶ Transcoelomic Metastasis ▶ Transforming Growth Factor-Beta ▶ Trefoil Factors ▶ Tumor Cell-Induced Platelet Aggregation ▶ Tumor Suppression ▶ Tumor Suppressor Genes ▶ Tumor-Associated Macrophages ▶ Ubiquitination ▶ Urothelial Carcinoma, Clinical Oncology ▶ Valproic Acid ▶ Vascular Stabilization ▶ Vasculogenic Mimicry ▶ Viral Oncology Epigenetics ▶ von Hippel-Lindau Tumor Suppressor Gene ▶ Warburg Effect ▶ Withaferin A ▶ Zonula Occludens Protein-1

2791 (2012) Macrometastasis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2130. doi:10.1007/978-3-642-16483-5_3483 (2012) Malignant tumor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2150. doi:10.1007/978-3-642-16483-5_3519

Metastasis Suppressor KAI1/CD82 Xin A. Zhang and Rafijul Bari Departments of Medicine and Molecular Sciences, Vascular Biology Center, Cancer Institute, University of Tennessee Health Science Center, Memphis, TN, USA

Synonyms References Bohl CR, Harihar S, Denning WL, Sharma R, Welch DR (2014) Metastasis suppressors in breast cancer: mechanistic insights and clinical potential. J Mol Med (Berl) 92(1):13–30 Eccles SA, Welch DR (2007) Metastasis: recent discoveries and novel therapeutic strategies. Lancet 369:1742–1757. doi:10.1016/S0140-6736(07)60781-8 Liu W, Vivian CJ, Brinker AE, Hampton KR, Lianidou E, Welch DR (2014) Microenvironmental influences on metastasis suppressor expression and function during a metastatic cell’s journey. Cancer Microenviron. doi:10.1007/s12307-014-0148-4954-63/s12307-0140148-4

See Also (2012) Beta-catenin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 385. doi:10.1007/978-3-642-16483-5_889 (2012) BH3-interacting death domain agonist. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 389. doi:10.1007/9783-642-16483-5_601 (2012) Drg-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1160. doi:10.1007/978-3-642-16483-5_1730 (2012) Experimental metastasis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1361. doi:10.1007/978-3-642-164835_2062 (2012) Lung colony formation assay. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2115. doi:10.1007/978-3-642-164835_3436

4F9; C33; CD82; IA4; KAI1; R2; Tetraspanin; Transmembrane 4 superfamily protein

Definition KAI1/CD82 is a ubiquitously expressed type III transmembrane protein or tetraspanin that functions as a ▶ metastasis suppressor for a variety of solid malignant tumors, an inhibitor of cell movement, and a costimulator in immune cells.

Characteristics KAI1/CD82 glycoprotein contains 267 amino acid residues and belongs to the tetraspanin superfamily. As a member of the tetraspanin family, it has an 11-residue cytoplasmic tail at the N-terminus and a 12-residue tail at the C-terminus region consisting of 11 and 12 amino acids, respectively, and a 4-residue intercellular loop between the second and third transmembrane domains (Fig. 1). The tails and loop form the intracellular face of KAI1/CD82. It also contains two extracellular loops, known as large extracellular loop (LEL) and small extracellular loop (SEL), and four transmembrane domains (Fig. 1). Based on the sequence homology, the

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Metastasis Suppressor KAI1/CD82

Metastasis Suppressor KAI1/CD82, Fig. 1 Schematic presentation of KAI1/CD82. KAI1/CD82 protein contains an N- and a C-cytoplasmic domain, two extracellular domains, one cytoplasmic intercellular loop, and four

transmembrane domains. As depicted, the highly conserved CCG motif is located in the large extracellular domain, and three highly conserved polar residues are embedded in the transmembrane regions

LEL is divided into two regions: variable and constant. The variable region usually mediates the heterogenous protein–protein interaction, while the constant region is predicted to be responsible for the dimerization of tetraspanins. The variable region of LEL of KAI/CD82 contains six cysteine residues that form three pairs of disulfide bonds crucial for the correct folding of the protein. This variable region also contains three N-glycosylation sites, and the glycosylation was reported to be important for KAI1/CD82’s ▶ motility-inhibitory activity. The constant region of KAI1/CD82 LEL likely contains the structural elements needed for the function of KAI1/CD82, since lack of this region KAI1/CD82 can no longer suppress cell ▶ migration. Little is known about the function of SEL of KAI1/CD82. Evidence from the studies of other members of tetraspanin superfamily suggests that the SEL may be important for the optimal folding of LEL. KAI1/CD82 contains four highly conserved transmembrane domains. The transmembrane domains are needed for the proper posttranslational maturation and the cell surface targeting of KAI1/CD82. For example, it was reported that a truncated KAI1/CD82 which lacks transmembrane domain 1 cannot reach to the cell surface and remains in endoplasmic reticulum. In

addition, studies on other tetraspanins suggest that the transmembrane domains are needed for the intramolecular hydrophobic interactions and are likely to be important for the intermolecular associations between tetraspanins in the tetraspanin web or tetraspanin-enriched microdomain. In addition, KAI/CD82 contains three polar residues in its transmembrane domains. The exact biochemical role of these polar residues remains unknown. There are multiple cysteine residues located at or near the intracellular portions of KAI1/CD82. These intracellular cysteine residues are constitutively palmitoylated, and the palmitoylation of KAI1/ CD82 is needed for the motility- and invasiveness-inhibitory activity of KAI1/CD82. The YRSV sequence at the C-terminal tail of KAI1/CD82 falls into the category of tyrosinebased endocytosis and sorting motif. The functional relevance of this motif remains to be determined though KAI1/CD82 is found to be internalized and trafficking between different endosomal and lysosomal compartments. KAI1/CD82 is a component of tetraspanin web or tetraspanin-enriched microdomain, in which tetraspanins associate with other transmembrane proteins such as integrins and immunoglobulin superfamily proteins and intracellular signaling

Metastasis Suppressor KAI1/CD82

proteins such as kinases and G proteins. Studies also demonstrated that KAI1/CD82 interplays with lipid rafts or ganglioside/cholesterol-enriched membrane microdomains, probably through the gangliosides enriched in lipid rafts or caveolae. KAI1/CD82 was initially identified as a metastasis suppressor of prostate ▶ cancer based on the fact that it only suppresses the formation of metastatic lesion but not the growth of primary tumors. Later studies indicated that KAI1/CD82 actually suppresses the ▶ progression of a variety of solid tumors. In normal tissues, KAI1/CD82 is ubiquitously expressed, while in invasive or metastatic cancer lesions, KAI1/CD82 expression is constantly reduced or lost. In prostate, gastric, colon, cervix, breast, skin, bladder, lung, pancreas, liver, and thyroid cancers, an inverse correlation between KAI1/CD82 expression and the invasive and metastatic potentials of cancer has been frequently observed. In cancer patients, the presence of KAI1/CD82 expression predicts a better prognosis, whereas the diminution or loss of KAI1/CD82 expression is constantly found in the clinically advanced cancers. Consistent with these observations, reintroduction of KAI1/CD82 into cancer cells inhibits cell migration and cancer invasion in vitro and suppresses cancer metastasis in animal model. In addition to the suppression of cancer progression, it has been reported that the overexpression of KAI1/CD82 in cancer cells causes ▶ apoptosis. Mechanism The exact mechanism responsible for KAI1/ CD82 as a tumor metastasis suppressor is unknown. Several lines of evidence support the notion that KAI1/CD82 may suppress cancer metastasis by primarily inhibiting cancer cell migration and ▶ invasion. There are several hypotheses regarding the mechanism by which KAI1/CD82 inhibits cell movement. Firstly, KAI1/CD82 may inhibit cell movement by regulating the functions of its associated proteins. The associated proteins of KAI1/CD82 include tetraspanins, integrins, immunoglobulin superfamily proteins, growth factor receptors, intracellular signaling proteins, etc. KAI1/CD82 negatively regulates integrin-dependent cell

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signaling and cell–extracellular matrix ▶ adhesion. For example, KAI1/CD82 overexpression has been demonstrated to diminish the cell adhesion primarily mediated by laminin-binding integrins. KAI1/CD82 also downregulates the activity of Src family kinases and the formation of p130CAS–Crk complex, both important for cell motility. By associating with epidermal growth factor receptor (EGFR), KAI1/CD82 desensitizes the EGF signaling possibly by accelerating the endocytosis of EGFR and recompartmentalizing the plasma membrane EGFR. KAI1/CD82 expression also reduced the activation of c-Met. The second possible mechanism for suppression of cell movement by KAI1/CD82 is that it directly initiates signal to inhibit cell motility. This hypothesis is based on the observation that the engagement of KAI1/CD82 with its antibody triggers intracellular signaling in immune cells. In contrast to the notion that KAI1/CD82 suppresses metastasis by directly inhibiting cell motility, a discovery opens a completely different avenue to understand KAI1/CD82’s metastasis suppression mechanism. In that study, the extracellular domains of KAI1/CD82 were found to directly bind to ▶ Duffy antigen receptor for chemokines (DARC), a cell surface protein expressed on the vascular endothelium. The receptor–counter receptor engagement leads to the inhibition of tumor cell proliferation and the induction of cellular senescence. In DARC knockout mice or in the absence of DARC, the metastasis suppression activity of KAI1/CD82 was compromised, whereas in DARC wild type and heterozygous littermate mice or in the presence of DARC, KAI1/CD82 inhibits the pulmonary metastasis of the implanted tumor. The proposed mechanism was that, when tumor cells enter into the blood vessels after dislodge from primary tumors, only the cells that expressed KAI1/CD82 interact with DARC expressed on the surface of endothelium. This KAI1/ CD82–DARC interaction transmits a senescence signal to the tumor cells, resulting in growth arrest and subsequently the suppression of metastasis. The cells that do not express KAI1/CD82 will escape and enter the circulation and subsequently establish metastasis.

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Metastatic Breast Cancer Experimental Therapeutics

Cross-References

Definition

▶ Adhesion ▶ Apoptosis ▶ Cancer ▶ Duffy Antigen Receptor for Chemokines ▶ Invasion ▶ Metastasis ▶ Migration ▶ Motility ▶ Progression

Experimental therapies for metastatic ▶ breast cancer can be defined as novel interventions directed toward the more effective treatment of secondary, progressively growing tumors distant from the primary site of the breast. Experimental therapies may include newly developed drug agents or may be different modalities for existing agents, such as the novel indication of a drug in the setting of breast cancer or the combination of approved single agents.

References Bandyopadhyay S, Zhan R, Chaudhuri A et al (2006) Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression. Nat Med 12(8):933–938 Dong JT, Lamb PW, Rinker-Schaeffer CW et al (1995) KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 268:884–886 Jackson P, Marreiros A, Russell PJ (2005) KAI1 tetraspanin and metastasis suppressor. Int J Biochem Cell Biol 37(3):530–534 Liu WM, Zhang XA (2006) KAI1/CD82, a tumor metastasis suppressor. Cancer Lett 240(2):183–194 Tonoli H, Barrett JC (2005) CD82 metastasis suppressor gene: a potential target for new therapeutics? Trends Mol Med 11(12):563–570

See Also (2012) Senescence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3370. doi:10.1007/978-3-642-16483-5_5236

Metastatic Breast Cancer Experimental Therapeutics Alexandra Silveira Ocular Molecular Genetics Institute, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, USA

Synonyms Breast adenocarcinoma; Breast carcinoma; Malignant breast carcinoma

Characteristics Breast cancer affects one in four women and is the second leading cause of cancer mortality among women in the United States. Globally, breast cancer is the leading cause of cancer mortality in women. Approximately 1% of all cases of breast cancer occur in men. The majority of mortality associated with the disease is due to the advanced, metastatic form of breast cancer, characterized by progressively growing tumors distant from the primary site. Approximately 40% of patients that initially present with localized disease develop metastatic disease. In those cases where the breast cancer has spread to distant sites, the 5-year survival rate is 27% in sharp contrast to 98% and 84% survival rates for localized and regional breast cancer, respectively. Risk factors for breast cancer reflect the influence of genetics, hormonal status, and environment on the development of the disease. These risk factors include age, gender, a personal or familial history of breast cancer, known genetic risk factors such as ▶ BRCA1 and BRCA2 mutations, radiation exposure, ▶ obesity, ▶ alcohol consumption, postmenopausal hormone therapy, and age at menarche, menopause onset, and having a first child. Once a primary tumor has been surgically removed, risk can be stratified into three categories (low, intermediate, and high) determined by the calculated risk of recurrence, tumor size, histopathological grade, ▶ invasion into the vasculature, lymph node involvement, presence of ▶ estrogen receptor/progesterone receptor, and status of the

Metastatic Breast Cancer Experimental Therapeutics

epidermal growth factor receptor ▶ HER2. Treatment for metastastic breast cancer typically involves a combination of surgical resection, radiotherapy, ▶ chemotherapy, and, depending on hormonal sensitivity and ▶ HER2 status, ▶ endocrine therapy and biological agents, such as ▶ trastuzumab and lapatinib. As much of metastatic breast cancer is incurable and there exist few proven standards of care, novel treatment agents and strategies are currently under experimental and clinical investigation to improve clinical outcome. Gene Therapy A promising avenue for the use of ▶ gene therapy is to elicit a more potent immune response, either by being directly targeted to an individual’s cancer cells or as part of a vaccine therapy. Although there are a variety of gene therapy vectors available, ▶ adenoviruses are the preferred gene delivery vectors due to their high ability to transduce both dividing and nondividing cells and an almost negligible integration potential. A phase I ▶ clinical trial is being conducted to evaluate the safety and efficacy of an adenoviral vector delivery of the immune system stimulating ▶ cytokine, interleukin-12 cDNA, intratumorally (NCT008494590). Vaccine Therapy Vaccine therapies are intended to bolster the immune system response against specific breast cancer antigens. Immune cells can be stimulated with tumor-specific cell-surface antigens, such as ▶ HER2 or MUC-1, delivered by allogeneic cell lines engineered by ▶ gene therapy to express the antigen or via direct injection of an immunogenic portion. In other trials, allogeneic tumor cells lines are engineered to secrete granulocyte-macrophage colony-stimulating factor, an immune stimulating cytokine that is currently being used in vaccines against a variety of cancers. Most often, these therapies are combined with other anticancer agents such as ▶ interferon-alpha, ▶ cyclophosphamide, and/or ▶ trastuzumab. Vaccine-based therapies against metastatic breast cancer are currently in stage I or II clinical trials to test dose, safety, and efficacy.

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Chemotherapeutic and Biological Agents A large number of clinical trials are currently investigating the use of FDA-approved drugs in combination to more effectively treat the secondary tumor by targeting multiple pathways involved in cancer. Combinations often include the pairing of an anti-angiogenic (e.g., ▶ bevacizumab) or antiproliferative (e.g., ▶ everolimus, exemestane, ▶ trastuzumab, sunitinib, or ▶ imatinib) agent with a second antimitotic drug that directly results in cell death (e.g., ▶ paclitaxel, capecitabine, vinorelbine, and ▶ gemcitabine). ▶ Bevacizumab is a humanized monoclonal antibody that binds and inhibits ▶ vascular endothelial growth factor A, thereby inhibiting ▶ angiogenesis. Phase III ▶ clinical trials have combined bevacizumab with a variety of ▶ chemotherapy agents in both first-line and second-line settings. A previous phase III clinical trial showed the combination of bevacizumab and capecitabine to result in significantly increased response rates when compared to capecitabine alone, although there was no difference in the endpoints of longer progression-free or overall survival. Based on these and other data, a phase II study is being conducted to investigate this combination as first-line therapy and in the adjuvant setting. Several phase III trials are also examining the combination of ▶ bevacizumab and ▶ paclitaxel, a mitosis inhibitor. These studies (NCT00028990, NCT00600340, and NCT00600340) differ in comparators, paclitaxel alone or combination bevacizumab and capecitabine, the restriction of study groups to HER2-negative metastatic breast cancer patients, and the primary endpoint of either time to progression or overall survival. Nab-paclitaxel (Abraxane; Abraxis Oncology, Los Angeles, CA) is a newly developed solventfree formulation of paclitaxel that yields higher levels of paclitaxel and an improved adverse-event profile. Trials are underway to examine the combination of nab-paclitaxel and bevacizumab and nab-paclitaxel and ▶ gemcitabine. ▶ Everolimus is an orally active ▶ mammalian target of rapamycin (mTOR) inhibitor that is being combined with a variety of agents in advanced clinical trials including the ▶ aromatase

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inhibitor exemestane (Phase III, NCT00863655), both ▶ trastuzumab and ▶ paclitaxel (Phase III, NCT00876395), and ▶ trastuzumab with vinorelbine (Phase III, NCT01007942). ▶ Gefitinib, an epidermal growth factor inhibitor (EGF), is similarly being used in combination with chemotherapeutic or hormone therapy to target cancer cell proliferation. Sunitinib is a multitargeted ▶ receptor tyrosine kinase that has been shown to have both antiangiogenic and antiproliferative effects. Early clinical trials are underway to evaluate the efficacy of sunitinib in the setting of metastatic breast cancer either in comparison to chemotherapy or in addition to either hormonal or chemotherapeutic agents for advanced disease. ▶ Imatinib, another tyrosine kinase inhibitor, is also under evaluation in combination therapy with vinorelbine (Phase II/Phase III, NCT00372476). Histone Deacetylase (HDAC) Inhibitors Unlike chemotherapeutic, hormonal therapy, and biological agents, histone deacetylase inhibitors are not thought to directly cause cell death or inhibit ▶ angiogenesis or proliferation. Rather, inhibitors such as ▶ vorinostat (Zolinza, Merck & Co., Inc., White House Station, NJ) are proposed to act by relieving epigenetic silencing of ▶ tumor suppressor genes. Vorinostat is currently in early clinical trials for the treatment of advanced breast cancer in combination with chemotherapeutic agents. Conclusion Because of the difficulty in treating metastatic disease, experimental therapies are using multipronged approaches to enhance an individual’s immune response, to directly cause cell death, and to target signaling cascades and cellular processes involved in tumor growth. The use of combination therapy and the development of novel gene therapy and vaccine approaches are yielding promising results. However, continued research of the underlying molecular mechanisms of cancer is required to suggest novel targets and new avenues of treatment.

Metastatic Breast Cancer Experimental Therapeutics

Cross-References ▶ Adenovirus ▶ Alcohol Consumption ▶ Angiogenesis ▶ Antiangiogenesis ▶ Antimitotic Drugs ▶ Aromatase and its Inhibitors ▶ Bevacizumab ▶ BRCA1/BRCA2 Germline Mutations Breast Cancer Risk ▶ Breast Cancer ▶ Chemotherapy ▶ Clinical Trial ▶ Cyclophosphamide ▶ Cytokine ▶ Endocrine Therapy ▶ Epidermal Growth Factor Inhibitors ▶ Epigenetic ▶ Epigenetic Gene Silencing ▶ Estrogen Receptor ▶ Everolimus ▶ Gefitinib ▶ Gemcitabine ▶ Gene Therapy ▶ Grading of Tumors ▶ HER-2/neu ▶ Imatinib ▶ Interferon-Alpha ▶ Invasion ▶ Mammalian Target of Rapamycin ▶ Obesity and Cancer Risk ▶ Paclitaxel ▶ Receptor Tyrosine Kinases ▶ Trastuzumab ▶ Tumor Suppressor Genes ▶ Vascular Endothelial Growth Factor ▶ Vorinostat

and

References Cardoso F, Castiglione M (2009) Locally recurrent or metastatic breast cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol 20(Suppl 4):15–18

Metastatic Colonization Kataja V, Castiglione M (2009) Primary breast cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol 20(Suppl 4):10–14 Tkaczuk KH (2009) Review of the contemporary cytotoxic and biologic combinations available for the treatment of metastatic breast cancer. Clin Ther 31(Pt 2):2273–2289 Wong ST (2009) Emerging treatment combinations: integrating therapy into clinical practice. Am J Health Syst Pharm 66:S9–S14

See Also (2012) Adjuvant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 75. doi:10.1007/978-3-642-16483-5_107 (2012) Allogeneic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 138. doi:10.1007/978-3-642-16483-5_194 (2012) EGF. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1211. doi:10.1007/978-3-642-16483-5_1824 (2012) Exemestane. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1357. doi:10.1007/978-3-642-16483-5_6752 (2012) Interleukin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1892. doi:10.1007/978-3-642-16483-5_3094 (2012) Lapatinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1980. doi:10.1007/978-3-642-16483-5_3277 (2012) Monoclonal Antibody. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-164835_6842 (2012) Proliferation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4766 (2012) Progesterone Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2990. doi:10.1007/978-3-642-164835_4754 (2012) Sunitinib. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3562. doi:10.1007/978-3-642-16483-5_5575 (2012) Vaccine Therapy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3879. doi:10.1007/978-3-642-16483-5_6147

Metastatic Cancer ▶ Metastatic Colonization

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Metastatic Colonization Russell Szmulewitz1, Jennifer Taylor2 and Carrie Rinker-Schaffer3 1 The University of Chicago Medicine, Chicago, IL, USA 2 Committee on Cancer Biology, The University of Chicago, Chicago, IL, USA 3 Department of Surgery, Section of Urology, The University of Chicago, Chicago, IL, USA

Synonyms Metastasis; Metastatic cancer; Systemic spread of cancer

Definition The term metastatic colonization refers to the final biological events required for cancer cells to form a clinically relevant ▶ metastasis at a secondary cancer site(s). It is a distinct process in which disseminated cells survive and subsequently proliferate to form overt metastases within this site.

Characteristics In order to successfully form overt metastases in a secondary organ/site, cancer cells must successfully complete all the steps of the metastatic process, as summarized in Fig. 1. A cancer cell must first survive, grow, and eventually break free from its primary cancer site. Figure 1 shows a single layer of cells upon a basement membrane (thick gray line), with a single tumorigenic cell. During progressive growth, tumor cells can acquire malignant properties which enable them to invade through the basement membrane and escape the primary site. Cells must then move to a discontinuous secondary site, often via blood vessels or lymphatics, in order to travel to other places within the body. After surviving this process,

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Metastatic Colonization, Fig. 1 Simplified schematic of the metastatic process. Metastatic disease begins with the formation of a primary tumor and its subsequent growth and invasion into the surrounding environment. In this example, a single cell becomes cancerous and subsequently replicates and is not subject to normal growth control mechanisms. To successfully metastasize, a cancer cell (or group of cells) must disassociate from the primary

tumor and then travel to a discontinuous site via (a) blood supply, (b) regional lymphatics, or (c) the peritoneal/pleural space (in cases such as intraperitoneal/pleural metastasis). In the final process of metastatic disease, termed metastatic colonization, the cancer enters its secondary site, where it must survive and proliferate to eventually form overt metastatic disease (such as in the bone, lung, and peritoneum)

cancer cells arriving at favorable secondary sites can survive and grow into overt metastases. In some instances, such as in the lining of the abdominal cavity and the chest cavity (peritoneum and pleura, respectively), cancer cells can be shed into those spaces and spread to other sites within those spaces without blood vessel or lymphatic transport (Fig. 1). Once cancer cells disseminate to and lodge within an end organ, they must survive and proliferate within their new environment, often referred to as microenvironment. Historically, earlier events in the metastatic process, such as invasion, were considered the more critical steps in cancer metastases, setting the timing and efficiency of metastasis formation. However, work

over the last decade has identified metastatic colonization as a biological phenomenon that plays a key role in the regulation and clinical progression of metastatic disease. Cancer cells can be detected in secondary sites as single disseminated cells or as small clusters of cancer cells, termed ▶ micrometastases, throughout the course of clinical disease. However, in many instances, these cells either remain functionally dormant (▶ dormancy) at their secondary sites or are unable to survive and complete the process of metastatic colonization to become clinically apparent metastases. Metastatic colonization therefore often begins well before symptom presentation or radiographic evidence (by CT scan, MRI, bone scan, etc.) of

Metastatic Colonization

metastasis is apparent. A clear clinical example of this latency is seen in prostate cancer. Many men with surgically removed prostate cancer are found to have an elevated ▶ prostate-specific antigen (PSA) levels, which is a protein only made by prostate tissue that can be monitored by a blood test, sometimes years after their surgery, despite having no symptoms or evidence of metastatic disease. In most of these instances, the elevated PSA is a marker of microscopic prostate cancer metastases colonizing the bone that have yet to present as clinically significant metastatic disease. Distinct molecular, cellular, and biological mechanisms regulating metastatic colonization are only now being examined experimentally. There is mounting evidence that interactions between the colonizing cancer cells and their new microenvironment play a key role in regulating the process of metastatic colonization. In the 1880s, the physician Stephen Paget used the analogy of a seed growing in favorable soil to propose a role for interactions between dispersed cancer cells (the seeds) within the microenvironment of the target organ (the soil) to explain why cells from certain types of cancers have a predilection for particular secondary organ, for example, why prostate cancer cells often metastasize to bone. Molecular insights into the process of metastatic colonization are being elaborated by studies of metastasis suppressor proteins. These proteins can specifically inhibit cancer metastasis, while having no effect on primary tumor formation or growth. Functional studies of several of these regulatory proteins, including MAP kinase kinase 4 (MKK4), demonstrate that they can regulate and inhibit cancer metastasis by directly targeting the process of metastatic colonization. Metastatic colonization is clearly a clinically relevant phenomenon. In the United States alone, there are projected to be 1.4 million new cancer diagnoses and 559,000 cancer deaths in 2007. Cancer is second to heart disease in overall mortality in the United States and, in fact, is the primary cause of death in persons under 85 years of age. The majority of patients who die from cancer succumb to metastatic disease. In addition to mortality, cancer metastasis, and metastatic

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colonization specifically, is a major cause of cancer-related morbidity, including pain, fatigue, shortness of breath, etc. Frequently, as is the case in prostate cancer, metastatic colonization is suspected often well before the detection of overt metastatic disease. Keeping disseminated cancer cells dormant or quiescent as long as possible is a key goal of cancer control strategies. As the biology of metastatic colonization becomes clearer, it will likely become a major target for cancer therapeutics.

Cross-References ▶ Dormancy ▶ Metastasis ▶ Micrometastasis ▶ Prostate-Specific Antigen ▶ Prostate-Specific Membrane Antigen

References Berger JC et al (2002) Metastasis suppressor genes: from gene identification to protein function and regulation. Cancer Biol Ther 4(8):805–812 Chambers AF, Groom AC, MacDonald IC (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2(8):563–572 Jemal A et al (2007) Annual report to the nation on the status of cancer, 1975–2001, with a special feature regarding survival. Cancer 101(1):3–27 Poste G, Fidler IJ (1980) The pathogenesis of cancer metastasis. Nature 283(5743):139–146 Steeg PS (2006) Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 12(8):895–904

See Also (2012) Basement Membrane. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 349. doi:10.1007/978-3-642-16483-5_537 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720 (2012) Primary Cancer Site. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2985. doi:10.1007/978-3-642-16483-5_4732 (2012) Secondary Cancer Site. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3347. doi:10.1007/978-3-642-164835_5199

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Metastatic Pancreatic Cancer ▶ Pancreatic Cancer Metastasis

Metastatic PDAC ▶ Pancreatic Cancer Metastasis

Metastatic Prostate Cancer ▶ Castrate-Resistant Prostate Cancer

Methazolastone

Metastatic Pancreatic Cancer

▶ estradiol is processed (metabolized) in the body. 2ME2 prevents both tumor growth (antiproliferative) and the formation of new blood vessels that tumors require to grow (antiangiogenic). Although 2ME2 is derived from estradiol, it binds poorly to ▶ estrogen receptors. On the contrary, it binds directly to a protein called tubulin, which is involved in cell division. This binding not only interferes with cell division (cell growth) but also inhibits hypoxia inducible factor-1 (HIF-1), an important factor for tumor cell survival. Additionally, 2ME2 has been reported to preferentially kill tumor cells, sparing normal cells, by causing ▶ reactive oxygen species (ROS) accumulation in cancer cells. 2ME2 under the trademark Panzem ® (EntreMed Inc., Rockville, MD) is currently in Phase I/II clinical studies to investigate its safety and efficacy and evaluate its drug-like properties in patients with advanced cancers.

▶ Temozolomide

Characteristics

2-Methoxyestradiol ▶ Methoxyestradiol

Methoxyestradiol Hyunsuk Shim Department of Hematology/Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA

Synonyms 2ME; 2ME2; 2-Methoxyestradiol; Panzem

Definition 2-Methoxyestradiol (2ME2) is a naturally occurring substance that is produced when the hormone

Mechanisms 2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite that has a low binding affinity for estrogen receptors (ER) a and b, consistent with its low estrogenic activity. In addition, the antiproliferative and proapoptotic effects of 2ME2 have been shown to be independent of ERs, since cell lines devoid of ER were sensitive to 2ME2 and ER antagonists did not attenuate 2ME2’s inhibitory effects. The biological activity of 2ME2 was first reported by Seegers et al. who found it to cause mitotic accumulation and the formation of abnormal mitotic spindles in cells, regardless of ER status. These effects were confirmed by others in diverse cancer cell types and in endothelial cells. The antiangiogenic activity of 2ME2 was reported over a decade ago. 2ME2 inhibits the proliferation, ▶ migration, and ▶ invasion of endothelial cells in vitro and has antiangiogenic effects in several in vivo models. Unlike most antiangiogenic agents, 2ME2 targets both proliferating endothelial cells and tumor cells, leading ultimately to the initiation of ▶ apoptosis. 2ME2

Methoxyestradiol

also has indirect antiangiogenesis effects mediated through inhibition of HIF-1 expression in tumor and endothelial cells. HIF-1 is a proangiogenic transcription factor that interacts with ▶ hypoxia response elements (HREs) to stimulate transcription of multiple proangiogenic genes including, most notably, ▶ vascular endothelial growth factor (VEGF). In addition, HIF-1targeted genes also include genes involved in cell proliferation, survival, invasion, and drug resistance. Due to its role in several key events of cancer progression, HIF-1 is an attractive molecular target for cancer therapy. 2ME2 inhibits the expression of HIF-1 in cancer cells lines. The effects of 2ME2 are dose dependent and occur in cells exposed to both normoxic and hypoxic conditions. The 2ME2-mediated decrease in cellular HIF-1 levels leads to inhibition of its nuclear translocation, inhibition of VEGF transcription, and a decrease in VEGF secretion. The activity of 2ME2 on HIF-1 is expected to inhibit the transcription of over 60 genes that have been identified as HIF-1 target genes. 2ME2 does not inhibit HIF-1 transcription or protein stability but rather appears to act by inhibition at the level of translation. The effects of 2ME2 on HIF-1 are not limited to cancer cells: under hypoxic conditions, 2ME2 also downregulates HIF-1 protein levels in human umbilical vein endothelial cells (HUVEC). Thus, 2ME2 not only directly inhibits endothelial and tumor cell proliferation leading to ▶ apoptosis, it also inhibits HIF-1-mediated HREs that are directly tied to ▶ angiogenesis, proliferation, survival, and ▶ metastasis. Important studies link the ability of 2ME2 to inhibit HIF-1 with its microtubule-depolymerizing effects. The effects of 2ME2 on microtubule disruption and inhibition of HIF-1 are not unique. Other microtubule-disrupting drugs, such as ▶ paclitaxel and vincristine, also cause similar effects and these data are consistent with the reported antiangiogenic effects of microtubule disruption, either stabilization or depolymerization. Cell lines with mutations in the paclitaxel-binding site remained sensitive to 2ME2, demonstrating that the two agents do not bind to the same site on tubulin. Rather, 2ME2 binds directly to the colchicine binding site of the tubulin protein, thereby inhibiting

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polymerization. This disruption not only interferes with the ▶ mitotic spindle apparatus but also inhibits HIF-1 translation and its nuclear translocation. The inhibitory effects of 2ME2 on both cancer cells and endothelial cells involve the activation of apoptotic cascades. In multiple cancer cell lines and in endothelial cells, 2ME2 increases the expression of death receptor 5 (DR5), a member of the tumor necrosis factor (▶ TNF) death receptor family. Activation of the extracellular domain of DR5 by its ligand, TNF-related apoptosis inducing ligand (▶ TRAIL), initiates signals through the death adapter protein FADD in the intracellular death domain to activate ▶ caspase-8, an effector caspase. The effects of 2ME2 on DR5 upregulation were confirmed in vivo in an orthotopic xenograft model using therapeutic doses of 2ME2. The expression of dominant-negative FADD significantly inhibited 2ME2-induced apoptosis, suggesting that this apoptotic pathway plays an important role in 2ME2-mediated apoptosis. In addition to the DR5 pathway of apoptosis, 2ME2 has been reported to mediate apoptosis through activation of the c-Jun NH2-terminal kinase (▶ JNK). In myeloma cells, 2ME2 causes JNK phosphorylation and translocation to mitochondria, where it initiates a decrease in mitochondrial membrane potential and the release of two key apoptotic modulators, cytochrome c and second mitochondria-derived activator of caspase (Smac). The ability of 2ME2 to activate JNK is not limited to myeloma cells. Breast, prostate, hepatic, and colorectal cancer cell lines respond to 2ME2 by rapid JNK activation. In 2ME2sensitive ▶ pancreatic cancer cell lines, 2ME2 causes cytochrome c release and Bax translocation from cytosol to mitochondria. Thus, in diverse cell types, 2ME2-mediated cytotoxicity involves activation of the mitochondrial apoptotic pathway. Furthermore, the effects of 2ME2 have been shown to be independent of ▶ p53 status because 2ME2 was equally effective against cell lines with wild type or mutant p53. 2ME2 was also shown to preferentially kill tumor cells, while sparing normal cells, by causing the accumulation of ▶ reactive oxygen species

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(ROS) in cancer cells. Inhibition of the mitochondrial electron transport chain by arsenic trioxide, an antineoplastic medicine, enhanced ROS generation and increased its anticancer effect when combined with 2ME2. Clinical Aspects EntreMed, Inc. (Rockville, MD) has formulated 2ME2 as an orally-administered liquid suspension (Panzem ® NCD) using NanoCrystal ® Colloidal Dispersion (NCD) technology to enhance the bioavailability of 2ME2 in patients. NCD is a proprietary technology of Elan Drug Delivery (Elan) that is currently used in multiple marketed pharmaceuticals. The NCD technology produces nanometer-sized particles, which are up to 500 times smaller than particles manufactured by conventional milling techniques. While Phase I and II clinical trials in 171 patients with a prior capsule formulation showed evidence of biological activity, the newer formulation increased bioavailability 5–10 fold to levels at which optimum antitumor activity was observed in preclinical studies. Studies with xenograft and metastatic cancer animal models indicate that 2ME2 targets both the tumor and the tumor vasculature in sarcomas and melanomas. In an orthotopic breast tumor model, 2ME2 caused microtubule depolymerization and the formation of aberrant mitotic spindles in both tumor cells and endothelial cells. Using noninvasive imaging and histological evaluation in a preclinical orthotopic glioma tumor model, 2ME2 treatment resulted in a dose-dependent reduction in tumor size. Tumor burden in the brain was significantly reduced following 2ME2 treatment, as determined by magnetic resonance imaging (MRI). Further histological examination of the 2ME2-treated tumors demonstrated a dosedependent decrease in acetylated tubulin, a marker of microtubule disruption, consistent with 2ME2 causing tumor inhibition through interfering with microtubules. In this preclinical study, 2ME2 also led to a significant improvement in tissue oxygenation, which may result in improved responsiveness to therapy and a decrease in HIF-1. This drug is potentially well suited to glioma treatment because of the

Methoxyestradiol

abundance of neovasculature and the positive association between high expression of HIF-1 and tumor grade in glioma. The ability of 2ME2 to initiate microtubule changes in vivo at therapeutic concentrations suggests that these effects may play critical roles in its antiangiogenic and antitumor activities. Additionally, preclinical models show that 2ME2 has potential therapeutic applications in inflammatory diseases such as rheumatoid arthritis. Panzem ® is currently in Phase I/II clinical studies to investigate its safety, efficacy, and ▶ pharmacokinetics. The results from two Phase Ib studies of Panzem ® NCD in patients with advanced cancer conducted at the University of Wisconsin Comprehensive Cancer Center and the Indiana University Cancer Center identified a maximum tolerated dose at one site with fatigue as the dose limiting toxicity and determined a recommended Phase II dose. Since January 2006, the Company has commenced five additional clinical studies with Panzem ® NCD. These include: (i) a Phase II clinical trial in glioblastoma multiforme (GBM) patients at the Duke University Medical Center’s Brain Tumor Center; (ii) a Phase Ib study of Panzem ® NCD in combination with paclitaxel (Taxol ®) in patients with metastatic breast cancer, also being conducted at the Duke University Medical Center; (iii) a Phase II multisite study in combination with Avastin ® in metastatic carcinoid tumor patients; (iv) a multicenter Phase II study in patients with hormone refractory ▶ prostate cancer; and (v) a multicenter Phase II study in patients with recurrent or resistant epithelial ▶ ovarian cancer. Additional Phase II studies under consideration include breast cancer, renal cell cancers, and ▶ multiple myeloma.

References Kang SH, Cho HT, Devi S et al (2006) Antitumor effect of 2-methoxyestradiol in a rat orthotopic brain tumor model. Cancer Res 66:11991–11997 LaVallee TM, Zhan XH, Johnson MS et al (2003) 2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway. Cancer Res 63:468–475

Methylation Mabjeesh NJ, Escuin D, LaVallee TM et al (2003) 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 3:363–375 Pelicano H, Feng L, Zhou Y et al (2003) Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J Biol Chem 278:37832–37839 Seegers JC, Aveling ML, Van Aswegen CH et al (1989) The cytotoxic effects of estradiol-17 beta, catecholestradiols and methoxyestradiols on dividing MCF-7 and HeLa cells. J Steroid Biochem 32:797–809

Methylase ▶ Methyltransferases

Methylation Christoph Plass German Cancer Research Center (DKFZ), Heidelberg, Germany

Definition An ▶ epigenetic modification of DNA is the addition of a methyl (CH3) group to position 5 of a cytosine residue. The majority of methylation events in humans occur on cytosines that are located next to a guanine (50 -CpG-30 dinucleotides).

Characteristics DNA methylation results from the addition of a methyl (CH3) group to position 5 of a cytosine (Fig. 1). The addition of a methyl group by DNA methyltransferases is an ▶ epigenetic modification to DNA that is maintained after cell division and does not change the DNA sequence. 50 -CpG-30 dinucleotides are not uniformly distributed in the human genome. ▶ CpG islands are short stretches of DNA, usually located in

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promoter regions of genes, with an unusually high GC content and a significantly higher frequency of 50 -CpG-30 dinucleotides compared to the rest of the genome. It is well accepted that DNA methylation is involved in gene regulation. Inhibition of transcription factor binding by methylation of the target sequence was the first mechanism identified. This mechanism is limited to a subset of transcription factors that contain 50 -CpG-30 dinucleotides in their recognition sequence. A second mechanism is the binding of methyl-CpG-binding protein complexes on proteins (MeCP1 and MeCP2) to methylated DNA. Transcription can then be repressed by either the inhibition of transcription factor binding or the recruitment of histone deacetylases (HDACs). HDACs mediate the deacetylation of lysine residues in the N-terminal tails of histones and thus cause an increase in compaction of the chromatin that makes the DNA less accessible for the transcriptional machinery. The establishment of normal DNA methylation patterns is a tightly regulated process of significant importance in many developmentally regulated pathways. • Early embryonic development in mice is characterized by a genome wide demethylation in the early cell divisions (8-cell stage) to the blastocyst stage. The methylation pattern is reestablished during implantation by a wave of de novo methylation. • X-chromosome inactivation is a developmentally regulated process in which DNA methylation plays an active role. In females, most genes on one of the X-chromosomes are silenced. This mechanism assures the same

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expression levels in male and female cells. Dense methylation of only the inactive X-chromosome is correlated with transcriptional silencing of the genes located on this X-chromosome. • While most genes are expressed from both the maternal and the paternal alleles, a small number of imprinted genes are expressed in a parent-of-origin dependent manner.

targets hemimethylated DNA, is ubiquitously expressed in somatic tissue, and interacts with the replication machinery at the replication fork. Furthermore, data suggests that DNMT1 can establish a repressive transcription complex that includes HDAC2, DMAP1, and the transcriptional co-repressor TSG101.

The underlying regulatory mechanisms of genomic ▶ imprinting are unclear but allelespecific methylation in CpG islands associated with imprinted genes seems to be involved. Since methylation patterns are established in selected sequences of the genome, a sequence specificity for DNA methyltransferases has been postulated. A first indication how this could be achieved came from a report describing a protein complex of DNMT1 with RB, E2F1, and HDAC1. This complex has the ability to target specifically those genes involved in growth regulation that contain E2F1 binding sites.

Hypomethylation

Cellular and Molecular Aspects Several steps are required for the establishment of DNA methylation patterns. The methylation of an unmethylated sequence requires an enzyme with de novo methylase activity. This enzyme recognizes a potential target sequence and methylates both DNA strands. During replication of the genome, a new DNA strand is synthesized that is unmethylated creating a hemimethylated state. A different enzyme that can detect and methylate the hemimethylated DNA is required for the maintenance of the methylation pattern (DNA maintenance methyltransferase). Since certain developmental processes require the erasure of the methylation pattern, an enzyme with demethylating activity was postulated. However, several rounds of DNA replication without maintaining the original methylation pattern could also accomplish demethylation. Two methyltransferases, Dnmt3a and Dnmt3b, were shown to have de novo methyltransferase activity in vivo. The DNA maintenance methyltransferase, Dnmt1, was identified several years ago and fulfills the expected criteria. This enzyme

Aberrant DNA Methylation in Cancer

Both extensive aberrant hypo- and hypermethylation have been described for several human cancers. Global hypomethylation in human cancers was one of the earliest changes described to be associated with tumor progression. For example, some reports describe the activation of the ▶ MYC oncogene in correlation with decreased methylation of CpG dinucleotides in the third exon of the gene. The underlying mechanisms however are unclear. In addition, there is convincing evidence that links hypomethylation with chromosomal breakage. Patients with ▶ ICF syndrome are characterized by instability of the pericentromeric heterochromatin and decondensation in chromosomes 1, 9, and 16, resulting in multibranched chromosomes. The chromosomes involved show hypomethylation of satellite DNA, and several groups reported mutations in the de novo methyltransferase DNMT3B in ICF patients, consistent with the idea of a defect in the methylation pathway. Hypermethylation

Silencing of tumor suppressor genes either by mutations of both alleles, homozygous deletion, or deletion of one allele (LOH) combined with mutation of the remaining allele are welldocumented mechanisms. Homozygous DNA hypermethylation of promoter sequences has been identified as an additional mechanism of inactivation of tumor suppressor genes. The development of a methylation sensitive PCR (MSP; PCR) allowed the rapid identification of methylated promoter sequences in various tumor samples. The list of genes that become hypermethylated in human cancers is growing

Methylation-Controlled J Protein

rapidly and includes genes involved in growth/ development, repair, and apoptosis. Hypermethylation of CpG islands located in promoter region of genes is correlated with the transcriptional silencing of the adjacent genes. Using ▶ restriction landmark genome scanning (RLGS) for genome wide assessment of methylation patterns in CpG islands, it was shown that up to 10% of the total 29,000 CpG islands in a tumor genome could be methylated. The methylation patterns are not random, suggesting a selective pressure for either the methylation of certain susceptible CpG islands or the selection of cells with a certain methylation pattern and a growth promoting transcriptional profile. In addition, by comparing different tumor types, it was shown that some targets of methylation are shared between tumor types and others are specific for one tumor type.

References Baylin SB, Herman JG, Graff JR et al (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196 Costello JF, Fruhwald MC, Smiraglia DJ et al (2000) Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet 24:132–138 Herman JG, Graff JR, Myohanen S et al (1996) Methylation-specific PCR: a noval PCR assay for methylation status of CpG island. Proc Natl Acad Sci U S A 93:9821–9826 Okano M, Bell DW, Haber DA et al (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257 Rountree MR, Bachman KE, Baylin SB (2000) DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet 25:269–277

Methylation-Controlled J Protein Janet C. Lindsey and Steven C. Clifford Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

Synonyms DnaJ (Hsp40) homolog subfamily C member 15; DNAJC15; DNAJD1; HSD18; MCJ; MCJ

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Definition MCJ is a member of the DNAJ family of co-chaperone proteins, whose expression is controlled by methylation of its associated CpG island. Reduced MCJ expression increases resistance to several commonly used cancer therapeutics by inducing expression of the ABCB1 drugtransport pump.

Characteristics The MCJ gene maps at 13q14.1 and codes for a protein of 150 amino acids and 16–17 kDa. MCJ contains a highly conserved 70 amino acid DNAJ domain (or J-domain) located at the C terminus. J-domain proteins interact with the heat shock protein 70 (Hsp70) family of chaperone proteins and act as co-chaperones recruiting Hsp70 members to specific substrate proteins. MCJ has been shown to be a type II transmembrane protein localized in the Golgi network, with a cytoplasmic N terminus and a C terminus lying within the Golgi lumen. The expression of MCJ is epigenetically (▶ Epigenetics) regulated by ▶ methylation of a ▶ CpG island located in the 50 transcribed region of the gene. Methylation is present in some normal cell types in a tissue-specific manner and changes in methylation patterns occur during cancer development. In ovarian tissue, MCJ is methylated and not expressed in normal ovarian cells of epithelial origin but is unmethylated and expressed in cells of lymphocyte or fibroblast origin. MCJ is methylated in approximately 93% of primary ▶ ovarian tumors (derived from the ovarian surface epithelium) but only approximately 17% show levels of methylation comparable to the normal tissue of origin, suggesting that 83% of tumors have become hypomethylated (▶ Hypomethylation). In contrast, MCJ is unmethylated in normal kidney and brain tissues but becomes hypermethylated in a proportion of ▶ Wilms tumors (90%) and several different malignant brain tumor types, medulloblastomas (7%), supratentorial PNETs (30%), and ependymomas (10%). In addition, 13q14 is a

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common region of genetic loss in a wide range of human cancers. The disruption of MCJ expression in tumorigenesis suggests it may play a role in tumor development; however, the mechanisms underlying this role are not understood at present; studies have failed to demonstrate a difference in the rate of proliferation of MCJ  cells in culture. To date, the major significance of MCJ in cancer appears to lie in its role in resistance to chemotherapy. Initial studies from MCJ-transfected ovarian cancer cell lines implicated loss of MCJ expression in chemotherapeutic resistance to a diversity of agents commonly used in cancer chemotherapy including ▶ paclitaxel, topotecan, and ▶ cisplatin. Complementary studies showed that silencing of MCJ expression by ▶ RNA interference in a drug sensitive breast cancer cell line conferred resistance to doxorubicin and paclitaxel. Furthermore, high levels of MCJ methylation in ovarian cancer patients are associated with both a poor response to chemotherapy (cisplatin/ carboplatin  a taxoid) and poor overall survival. Insights into the functional mechanism whereby loss of MCJ expression confers drug resistance have been made. MCJ associates with c-Jun, a component of the transcription factor complex ▶ AP-1, leading to its degradation. In the absence of MCJ, levels of c-Jun rise resulting in increased AP-1 mediated transcription of ABCB1 (▶ P-Glycoprotein), one of a family of drug exporters characterized by an ATP-binding cassette motif. ABCB1 is a large transmembrane protein which functions as an ATP-dependent pump resulting in the efflux of a variety of drugs from the cell, preventing their intracellular accumulation and cytotoxic effects. The development of simultaneous resistance to multiple structurally unrelated drugs is a major problem in cancer chemotherapy. Over expression of ABCB1 has been associated with drug resistance in tumors. Previously, it was known that this upregulation could occur in cell line models by amplification of its DNA locus. The discovery that it can also be

4-Methylsulfinylbutyl Isothiocyanate

upregulated by loss of MCJ expression has important therapeutic implications as MCJ silencing by DNA hypermethylation is a feature of certain tumor types and is reversible by DNA methyltransferase inhibitors or demethylating agents. This means that tumors proven to be resistant to chemotherapy as a result of MCJ methylation could potentially be resensitized by pharmacological reactivation of MCJ prior to chemotherapy.

Cross-References ▶ Drug Resistance ▶ Epigenetic Gene Silencing ▶ Methylation ▶ Molecular Chaperones ▶ Ovarian Cancer Drug Resistance

References Hatle KM, Neveu W, Dienz O et al (2007) MCJ promotes c-Jun degradation to prevent ABCB1 transporter expression. Mol Cell Biol 27:2952–2966 Lindsey JC, Lusher ME, Strathdee G et al (2006) Epigenetic inactivation of MCJ (DNAJD1) in malignant paediatric brain tumours. Int J Cancer 118: 346–352 Shridhar V, Bible KC, Staub J et al (2001) Loss of expression of a new member of the DNAJ protein family confers resistance to chemotherapeutic agents used in the treatment of ovarian cancer. Cancer Res 61:4258–4265 Strathdee G, Vass JK, Oien KA et al (2005) Demethylation of the MCJ gene in stage III/IVepithelial ovarian cancer and response to chemotherapy. Gynecol Oncol 97:898–903

4-Methylsulfinylbutyl Isothiocyanate ▶ Sulforaphane

Methyltransferases

Methyltransferases Kamaleshwar Singh The Institute of Environmental and Human Health (TIEHH), Texas Tech University, Lubbock, TX, USA

Synonyms Methylase; MTase

Definition Methyltransferases (MTases) are a family of enzymes that catalyze the transfer of a methyl group from a donor to an acceptor. Methyltransferases are found in both prokaryotes and eukaryotes; however, their structure and function are different. Based on the substrate for methylation, the two well-known types of methyltransferases in vertebrates are DNA methyltransferase (DNMTases) and histone methyltransferase (HMTases). Besides the DNMTases and HMTases, there is another class of MTases that catalyzes the transfer of methyl group to the 5-prime-terminal guanosine of pre-mRNA, known as RNA MTases.

Characteristics DNMTases Depending on bases and the position of its carbon in DNA on which the DNMTases catalyze the transfer of methyl group, DNMTases can be divided into three different groups: N-6 adenine-specific DNA methylase (A-MTase): This class of enzymes known as m6A methyltransferases, specifically methylates the amino group at the C-6 position of adenines in DNA. They are found mainly in bacteria and participate in bacterial restriction-modification

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systems. These methylases have the same DNA recognition sequence specificity as their corresponding restriction enzymes and their function is to methylate these specific DNA sequences in order to prevent the host restriction enzymes from digesting its own genome. N-4 cytosine-specific DNA methylases (C4MTase): This class of enzymes known as m4C methyltransferases, specifically methylates the amino group at the C-4 position of cytosine in DNA. They are found mainly in bacteria and participate in another type (type II) of restriction-modification systems. They recognize a specific DNA sequence and methylate a cytosine in that sequence in order to protect DNA from digestion by type II restriction enzymes that recognize the same sequence. C-5 cytosine-specific DNA methylase (C5MTase): This class of enzymes known m5C methyltransferases, specifically methylate the C-5 carbon of cytosines in DNA to produce C5-methylcytosine. They are mainly found in mammalian cells. Brief descriptions and characteristic features of mammalian DNMTases are as follows: Mammalian DNA methyltransferases (DNMTases) are a family of enzymes that are involved in establishment and maintenance of DNA methylation (Robertson 2005). DNMTases use S-adenosylmethionine as donor for an activated methyl group. In vertebrate DNA, the cytosine residue located 50 to a guanosine residue is commonly known as CpG dinucleotide. Genomic regions, about 0.5–4 kb in length, that are rich in CpG content are known as CpG islands. The modification of cytosine residue of CpG by addition of methyl group resulting in methylated cytosine (cytosine-C5 methylation) is commonly known as DNA methylation. DNA methylation does not change the sequence or order of bases, and therefore, it is known as epigenetic modification of DNA. DNA methylation is the only known epigenetic modification of DNA in mammalian genome. The mammalian DNA methylation involves mainly two components: the first

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one is DNA methyltransferases (DNMTases) that are responsible for establishing and maintaining DNA methylation patterns, and the second one is methyl-CpG-binding proteins (MBDs) that “recognize” the methylation marks. DNA Methylation Patterns in Normal and Cancer Cells

Generally, in normal cells, the DNA methylation occurs predominantly in repetitive sequences in the genome. Most of the CpG islands-containing promoter regions of the genes in the mammalian genome are generally remain unmethylated in normal cells. Introns of most of the genes contain multiple transposons, and transcriptional activation of these transposons may interfere in the regulation of gene expression. Additionally, the transposons can also cause genomic instability by insertional mutagenesis and rearrangements through recombination between nonallelic repeat sequences. DNA methylation of these transposon sequences do not allow the transcriptional activation of these sequences and therefore helps in maintaining the genomic instability. Demethylation in DNA methyltransferase-deficient mouse embryos caused transcription of transposons further support the role of DNA methylation in transcriptional repression of transposons. In cancer cells, however, global genomic hypomethylation and gene loci-specific hypermethylation of promoter regions are wellknown epigenetic changes in DNA. The hypermethylation of gene promoter causes loss of expression of tumor suppressor genes and DNA repair genes resulting in tumorigenesis. Types of Mammalian DNMTases

There are five different types of DNMTases known so far in mammalian cells. These are DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. However, three (DNMT 1, DNMT3A, and DNMT3B) of them are well characterized and known to play critical role in establishing and maintaining DNA methylation in mammalian genome (Bestor 2000). Structure and Function of Mammalian DNMTases

After fertilization, during embryonic development, particularly at early embryogenesis stage,

Methyltransferases

most of the DNA is devoid of methylation. At this stage, the DNA methylation pattern has to be created, and this pattern of methylation has to be maintained depending on the tissue type. The primary establishment of methylation patterns on the genome is performed mainly by DNMT3A and DNMT3B. These two enzymes are also known as de novo methyltransferases because they have high affinity for non-methylated DNA. The DNMT1 faithfully maintains the DNA methylation patterns in somatic cells after each replication cycle. After DNA replication during cell division, newly synthesized DNA strand is unmethylated. DNMT1 scans this new DNA strand, recognizes the site which is methylated in old DNA strand, and adds the methyl groups on these sites in new DNA strand. DNMT1 is the most abundant form of DNMT in mammalian cells and is highly expressed during the S-phase of cell cycle. The function of DNMT2 is unclear. DNMT3L (DNMT3 like) has partial sequence similarity with DNMT3A and DNMT 3B but lacks critical catalytic motifs for activation of target cytosine, binding of methyl donor S-adenosyl L-methionine, and sequence recognition. Because of the lack of these motifs, the function of DNMT 3L is not fully understood. However, the study suggest that DNMT 3L functions as a regulator of methylation at imprinted loci rather than a DNA methyltransferase (Bourc’his et al. 2001). Besides these functional differences, there are some structural differences among the DNMTases. DNMT1 requires N-terminal domain for its catalytic activity, whereas the C-terminal catalytic domains of DNMT 3A and DNMT3B are enzymatically active, and therefore, these two enzymes do not require N-terminal domain for their catalytic activity. Besides the DNMTases, there are also other types of methyltransferases, such as RNA methyltransferases that catalyzes the addition of methyl group to RNA and histone methyltransferases that catalyzes the addition of methyl group to histone proteins. RNA MTases In mammalian cells, the processing of nascent pre-mRNA involves addition of 5-prime-terminal methylated caps. This process requires two

Metronomic Chemotherapy

enzymes namely RNA guanylyltransferase and 50 -phosphatase (RNGTT) and RNA methyltransferase. RNGTT catalyzes the removal of the gamma-phosphate of the initiating nucleotide and transfers GMP from GTP to the resulting diphosphate end. RNA methyltransferase catalyzes the subsequent N7 methylation of the newly formed termini. The terminal 7methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression. Histone Methyltransferases (HMTases) Histone methyltransferases are a family of enzymes that methylates histones. Histones are proteins involved in chromatin structure and organization, and histone methylation is an important posttranslational epigenetic mark that controls gene expression (Jenuwein and Allis 2001). Histone methyltransferases adds methyl group to the lysine and arginine amino acid residues at N-terminal tails of histone proteins. Lysine amino acid residue is known to be mono-, di-, or trimethylated, whereas the arginine amino acid residue is known to be mono- or dimethylated in histone H3. Depending on the specific amino acid affected by methylation, histone methylation can be associated either transcriptional repression or activation. For example, trimethylation of lysine 4, methylation of lysine 36, and methylation of lysine 79 of histone 3 are known to be associated with active chromatin and transcriptional activation, whereas trimethylation of lysine 9 and methylation of lysine 27 of histone H3 are associated with transcriptional repression and gene silencing. HMTases play an important role in the regulation of gene expression associated with cell proliferation, and aberrations in HMTases expression and function are implicated in development of cancer (Esteller 2008).

References Bestor TH (2000) The DNA methyltransferases of mammals. Hum Mol Genet 9:2395–2402 Bourc’his D, Xu G, Lin C, Bollman B, Bestor T (2001) DNMT3L and the establishment of maternal genomic imprints. Science 294:2536–2539

2809 Esteller M (2008) Epigenetics in cancer. N Engl J Med 358:1148–1159 Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080 Robertson KD (2005) DNA methylation and human disease. Nat Rev Genet 6:597–610

Metronomic Chemotherapy Eddy Pasquier1,2,3 and Nicolas André1,2,4 1 Centre for Research in Oncobiology and Oncopharmacology, INSERM U911, Marseille, France 2 Metronomics Global Health Initiative, Marseille, France 3 Children’s Cancer Institute, Randwick, NSW, Australia 4 Department of Pediatric Hematology and Oncology, La Timone Children’s Hospital, Marseille, France

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Synonyms Anti-angiogenic chemotherapy; Metronomic scheduling; MSAT – metronomic scheduling of anticancer treatment; Protracted low-dose chemotherapy

Keywords Chemotherapy; Immunity

Angiogenesis;

Resistance;

Definition Metronomic chemotherapy was originally defined as the chronic administration of chemotherapeutic drugs at relatively low, minimally toxic doses and with no prolonged drug-free breaks. It is by essence opposed to conventional chemotherapy, which is based on the administration of chemotherapy drugs slightly below or at the maximumtolerated dose every 2 or 3 weeks.

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Characteristics Origin Given the high sensitivity of vascular endothelial cells to chemotherapeutic drugs, it was hypothesized that more frequent administration of these drugs at lower doses would result in a potent inhibition of tumor angiogenesis and would prevent the rapid vascular rebound that often occurs during prolonged drug-free breaks following conventional chemotherapy. This gave rise to the concept of anti-angiogenic chemotherapy, which was then termed metronomic chemotherapy (Hanahan et al. 2000). Two pioneering studies using animal models of neuroblastoma, leukemia, and lung and breast cancers (Browder et al. 2000; Klement et al. 2000) thus showed that the frequent administration of relatively low doses of chemotherapy agents (e.g., cyclophosphamide or vinblastine) induced potent antitumor effects, even when tumor cells were resistant to chemotherapy. The antitumor activity was associated with a significant inhibition of tumor angiogenesis and was further enhanced by combining metronomic chemotherapy with anti-angiogenic agents. This showed that a change of dose and schedule resulted in a switch of the primary target of chemotherapy: from tumor cells to the tumor vasculature. Mechanisms of Action Based on these pioneering studies, metronomic chemotherapy was initially thought to act mainly through direct anti-angiogenic mechanisms. This was evidenced by (1) reduced vascular density within the tumor, (2) decreased mobilization of circulating endothelial progenitors from the bone marrow and homing to the tumor site, and (3) increase in expression of anti-angiogenic factors, such as thrombospondin-1. Studies however have demonstrated the multi-targeted nature of metronomic chemotherapy (André et al. 2014). The proven mechanisms of action now include several immunostimulatory effects (e.g., depletion of regulatory T cells, modulation of myeloid-derived suppressor cells, activation of innate immune response, immunogenic cell

Metronomic Chemotherapy

death, and dendritic cell maturation) as well as targeted inhibition of hypoxia-inducible factor 1a (HIF-1a) and reduction of cancer stem cell maintenance. It is important to note that these different mechanisms are both model and drug dependent. For instance, depletion of regulatory T cells appears to be a key mechanism of action of metronomic cyclophosphamide, gemcitabine, and temozolomide, while HIF-1a inhibition is specifically induced by type I topoisomerase inhibitors (e.g., topotecan) and anthracyclines (e.g., doxorubicin). Clinical Settings Initially developed in animal models, metronomic chemotherapy has been rapidly translated into the clinic both in adult and pediatric oncology. Numerous phase I and II clinical trials prospectively investigating the safety and efficacy of metronomic chemotherapy have been completed in a variety of human cancers. Increased response rates and improved patient survival have been reported in some tumor types, such as low-grade gliomas, sarcomas, and breast and ovarian cancers, while other cancers were found to be poor responders (e.g., osteosarcomas). Currently, metronomic chemotherapy is mainly used as palliative care in relapsed patients or as maintenance therapy. It has also shown benefit in frail patients and those with poor performance status (e.g., breast cancer in elderly patients and locally advanced hepatic carcinoma) where it can be used as first-line treatment. Furthermore, metronomic chemotherapy was found to be superior (i) to observation in the maintenance setting of leukemia treatment, (ii) to radiotherapy alone in high-grade glioma, and (iii) to conventional chemotherapy in lung, breast, and head and neck cancers. Several randomized phase III trials are currently underway to further validate the benefits of metronomic chemotherapy, especially in the maintenance setting. Advantages Metronomic chemotherapy displays several advantages when compared with conventional chemotherapy. First of all, the use of low doses

Metronomic Chemotherapy

results in a significant reduction in toxic side effects (e.g., alopecia, neutropenia, vomiting) and little or no need for supportive care. This allows metronomic chemotherapy to be easily combined with other therapeutic strategies such as anti-angiogenic agents, repurposed drugs, and targeted therapy. Metronomic regimens often rely on the use of oral formulations of drugs, which allows patients to be treated from the comfort of their home. Reduced toxicity and oral treatment result in a major improvement of quality of life, which is particularly important in the context of palliative care and maintenance therapy. Finally, metronomic regimens are often based on off-patent chemotherapeutic drugs, which considerably decreases treatment cost. Low cost, oral formulation, and acceptable toxicity make metronomic chemotherapy a suitable therapeutic strategy in low- and middle-income countries, where cancer treatments should be constraint-adapted (André et al. 2013). Drawbacks Metronomic chemotherapy also has a number of specific limitations and challenges. There is currently no reliable biomarker that can be used for treatment monitoring or for patient selection. Most metronomic chemotherapy regimens are therefore largely empirical, and schedule optimization requires further preclinical and clinical testing and the development of new experimental tools, such as mathematical modeling. Relying on oral medications and long-term treatment can affect compliance and, thus, treatment response. Furthermore, the use of off-patent drugs makes it unattractive for the pharmaceutical industry, and large clinical trials are hard to fund. Finally, metronomic chemotherapy often leads to disease stabilization rather than partial or complete responses, making standard assessment methods such as RECIST criteria inappropriate. Conclusions More than a change in the primary target of chemotherapy – from tumor cells to the tumor vasculature – the emergence of metronomic chemotherapy has led to a paradigm shift in cancer treatment. While conventional chemotherapy aims

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at eliminating all cancer cells at all cost, metronomic chemotherapy mostly aims at disease stabilization rather than eradication. The use of minimally toxic doses offers the possibility to develop treatment regimens that are tolerated by patients for a long period, thus treating cancer as a chronic disease. Future studies, and especially randomized clinical trials, will need to address whether treatment for cancer control can improve patient survival as compared with treatment for cure.

Cross-References ▶ Chemotherapy ▶ Hypoxia-Inducible Factor-1

References André N, Banavali S, Snihur Y, Pasquier E (2013) Has the time come for metronomics in low-income and middleincome countries? Lancet Oncol 14:e239–e248 André N, Carré M, Pasquier E (2014) Metronomics: towards personalized chemotherapy? Nat Rev Clin Oncol 11:413–431 Browder T, Butterfield CE, Kräling BM, Shi B, Marshall B, O’Reilly MS, Folkman J (2000) Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878–1886 Hanahan D, Bergers G, Bergsland E (2000) Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Investig 105:1045–1047 Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, Bohlen P, Kerbel RS (2000) Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Investig 105:R15–R24

See Also (2012) Maintenance chemotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin, p 2137. doi:10.1007/978-3-642-16483-5_6974 (2012) Maximum tolerated dose. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin, p 2188. doi:10.1007/978-3-642-16483-5_3567 (2012) Palliative care. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2759. doi:10.1007/978-3-642-16483-5_4351 (2012) Tumor-associated angiogenesis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin, p 3807. doi:10.1007/978-3-642-16483-5_6016

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Metronomic Scheduling

Metronomic Scheduling

MIC Molecules

▶ Metronomic Chemotherapy

Stefan Holdenrieder1, Helmut Rainer Salih2 and Alexander Steinle3 1 Institute of Clinical Chemistry and Clinical Pharmacology, Universitatsklinikum Bonn, Bonn, Germany 2 Department of Internal Medicine II, University Hospital of Tübingen, Eberhard-Karls-University, Tübingen, Germany 3 Institute for Molecular Medicine, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany

MGC126245 ▶ Daxx

MGC126246 ▶ Daxx

Synonyms Major histocompatibility complex (MHC) class I-related chain molecules

MGC126806 ▶ BRaf-Signaling

MGC138284 ▶ BRaf-Signaling

MGC34538 ▶ Aurora Kinases

MGF Receptor ▶ Kit/Stem Cell Factor Receptor in Oncogenesis

Mib-2 ▶ Skeletrophin

Definition MICA and MICB are human inducible MHC class I-related glycoproteins expressed by stressed and malignant cells and serve as ligands for the NKG2D receptor (NKG2D receptor) on natural killer (NK) cells (natural killer cell activation) and T cells thereby stimulating innate and adaptive immune responses.

Characteristics Human MIC molecules are encoded within the MHC on the short arm of chromosome 6. The MIC gene family comprises six genes (MICA to MICF) of which only MICA and MICB are functional, whereas MICC to MICF constitutes pseudogenes. MICA and MICB are tandem genes located between the HLA-B gene and the BAT1 locus at the transition from the MHC class I to the class III region. The MICA locus is highly polymorphic with 86 alleles known to date. MICA*08 is by far the most frequent allele encoding for a MICA protein with a truncated cytoplasmic domain due to a frameshift mutation. MICA and

MIC Molecules

MICB molecules are type I transmembrane glycoproteins of 70 kDa and 58 kDa, respectively, consisting of three extracellular domains (a1, a2, a3), a hydrophobic membrane-spanning domain, and a cytoplasmic domain. The a1 and a2 domains form an MHC class I-like fold followed by an immunoglobulin-like a3 domain much alike an MHC class I heavy chain. However, at difference to classical MHC class I molecules, MIC molecules neither present antigenic peptides nor associate with b2-microglobulin and are not ubiquitously or constitutively expressed. Rather, expression of MIC glycoproteins is inducible by various forms of cellular stress (▶ Stress response), including oxidative and genotoxic stress which is associated with malignant transformation. Accordingly, MICA and MICB are broadly expressed by malignant cell lines, epithelial tumors, and leukemias. In healthy tissue, MICA has solely been detected on epithelial cells of the gastrointestinal tract which has been attributed to stimulation by the intestinal flora. MICA and MICB are engaged by the activating NKG2D (natural killer group 2 member D) receptor expressed by almost all human cytotoxic lymphocytes including NK cells, CD8 T cells, and gd T cells. The C-type lectin-like, homodimeric NKG2D interacts with the MHC class I-like a1a2 superdomain of MIC molecules in a manner reminiscent of the interaction of ab T-cell receptors with MHC class I molecules. NKG2D is associated with the adaptor DAP10 that transduces activating signals upon NKG2D engagement leading to stimulation of NK and T-cell effector functions. Besides MIC molecules, there is a second family of NKG2D ligands (NKG2DL) in humans, the UL16-binding proteins (ULBP). ULBP are encoded by a gene cluster on the long arm of chromosome 6 comprising six functional members (ULBP1–4, RAET1G, RAET1L). The MHC class I-like a1a2 superdomain of the ULBP is only distantly related to MIC molecules (amino acid sequence identity (25%)). A hallmark of ULBP is the lack of an immunoglobulin-like a3 domain and the GPI-anchorage of some family members (ULBP1–3). Altogether, there are eight functional human NKG2DL known to date, comprising two MIC and six ULBP molecules, raising

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questions about the selective force behind this redundancy. There is emerging evidence that NKG2DL differ in their affinity for NKG2D or in their expression pattern and are differentially targeted by viral immunoevasins. For example, there are reports that MICA allelic variants vary broadly in their binding to NKG2D, that MIC molecules are primarily expressed by epithelial tumors, whereas ULBP preferentially are expressed on hematopoietic cells, and that MICA and MICB are differentially targeted by immunoevasins (US18, US20, UL16, UL142) of human cytomegalovirus (HCMV). Expression of MIC molecules is upregulated by cells infected with viruses (e.g., HCMV or adenovirus) or in the course of a ▶ DNA damage response. The latter has been linked to the frequent expression of MIC molecules on tumor cells. In fact, NKG2DL expression was upregulated during tumorigenesis (▶ Carcinogenesis) in mice and led to the rejection of tumor cells by stimulating NK and CD8 T cells. Further, NKG2D was shown to protect the host from spontaneous tumors in chemically induced mouse tumor models. These findings led to the hypothesis that the NKG2D/NKG2DL system acts as a novel immunosurveillance mechanism aiding the elimination of transformed, infected, or other potentially harmful cells. A strong expression of NKG2DL can render tumor cells susceptible to NK cell cytotoxicity, even in the presence of MHC class I, and thus NKG2D-mediated tumor immunosurveillance may counteract the development of neoplasms already at an early stage. In this context it is noteworthy that patients with the rare autosomal recessive Chediak-Higashi syndrome characterized by abnormal cytotoxic function of NK cells have a 200-fold higher risk of developing cancer than other individuals supporting the important role of NK cells in antitumor immunity. During carcinogenesis, however, antitumor immunity also results in the selection of cancer cells capable of evading the immune response (▶ Esophageal Cancer). Escape from NKG2Dmediated tumor immunosurveillance can be achieved either by silencing NKG2D signaling or by abrogation/reduction of NKG2DL

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expression. Proteolytic shedding of MIC molecules from tumor cells impairs NKG2D-mediated immunosurveillance in both ways by locally reducing MIC expression levels on tumor cells and by systemically downregulating NKG2D receptors on NK cells and CD8 T cells through soluble MIC (sMIC) molecules. Similarly, TGFb which is frequently produced by tumors downregulates both MICA and NKG2D surface expression. A mouse study indicated that soluble tumor-derived NKG2DL may also promote tumor immunity by competing for NKG2D occupancy with membrane-bound NKG2DL of macrophages in the tumor microenvironment, thus preventing NKG2DL-induced anergy of NKG2D-bearing effector lymphocytes. Future studies will have to address the relevance of such effects in human cancer patients as mouse NKG2DL differ from human NKG2DL with regard to several critical parameters such as affinity for NKG2D. Clinical Aspects of MIC Expression by Tumors Enhanced expression of MIC molecules has been observed in many epithelial tumors as well as in hematological neoplasias. Several studies have shown a beneficial role of MIC expression on tumor cells such as in colorectal cancer or ▶ multiple myeloma for patient survival. In tumors with low MIC molecule expression or pronounced MICA shedding, novel agents such as ▶ imatinib mesylate and histone deacetylation (HDAC) inhibitors were found to induce the expression of MIC molecules and subsequently to enhance the tumoricidal activity of natural killer cells. It remains to be shown to what extent activation of the NKG2D/NKG2DL system contributes to the clinical success of these agents in antitumor therapy. Clinical Aspects of Soluble MIC Molecules in Blood The process of shedding MIC molecules from tumor cell surfaces during ▶ progression of cancer disease opens the possibility to measure concentrations of sMIC molecules in blood for diagnostic and prognostic purposes. If benign diseases potentially interfering with diagnosis can be excluded by clinical and laboratory means, MIC

MIC Molecules

levels in peripheral blood may represent valuable indicators of tumor development. Enzyme-linked immunosorbent assays (ELISAs) have been developed with specific antibodies against MICA and MICB which allow the quantification of such sMIC molecules in serum, plasma, and other body fluids. Several studies have addressed these clinical questions, particularly the potential of MIC molecules for diagnostic, staging, and prognostic purposes. Soluble MIC in Diagnosis of Cancer Disease Most studies found higher sMIC levels in individuals with cancer diseases, particularly in those with advanced stages of cancer as compared to healthy controls. However, the potential impact of most studies on the clinical practice was limited due to the small patient numbers investigated. The currently most comprehensive studies analyzed sMICA and sMICB levels in sera of more than 500 individuals with cancer diseases (including colorectal (colon cancer) and various other gastrointestinal cancers (▶ gastrointestinal stromal tumors), ▶ lung cancer, ▶ breast cancer, ▶ ovarian cancer and other gynecologic cancers, renal and ▶ prostate cancer) and with benign diseases and in healthy individuals. Levels of sMICA and sMICB in sera of healthy donors are generally fairly low. In contrast, pretherapeutic serum levels in various malignancies were significantly higher. However, various benign diseases tended to have elevated serum levels of sMIC molecules limiting the diagnostic capacity for cancer disease. Particularly infectious diseases as well as hepatic and renal diseases potently affecting the marker metabolism were associated with increased levels of MIC molecules. Nevertheless, sMICA levels significantly distinguished between benign and malignant diseases in general but also in subgroups of lung and gynecological cancers. In contrast, soluble MICB molecules failed to discriminate effectively between cancers and the relevant organ-specific benign diseases. This failure was also due to the considerable number of individuals with “MICBnegative” cancer disease. In about 70% of cancer patients, there was no sMICB detectable while only about 20% of cancer patients were

MIC Molecules

“sMICA-negative.” These data correspond with immunohistochemical analyses which revealed a significant number of tumors lacking MIC molecule expression. Further, there was only a weak correlation found between sMICA and sMICB levels suggesting differences in expression, shedding, or metabolism of MICA versus MICB. No substantial addition in clinical sensitivity was observed by the combined use of both biomarkers (▶ Serum biomarkers). Soluble MIC in Staging of Cancer Disease After diagnosis, pretherapeutic staging (▶ Staging of Tumors) of cancer disease is essential for therapy stratification and estimation of prognosis. Interestingly, elevated levels of both sMICA and sMICB significantly correlated with the extent of disease reflected by UICC classification: While there was no association between sMICA and sMICB levels and tumor size, cell differentiation (▶ Grading of Tumors), or lymph node involvement, both markers showed a clear correlation with the presence of distant metastasis. Detailed subgroup analysis confirmed the correlation of both markers with the presence of distant metastasis for various cancer entities, particularly for gastrointestinal tumors. In contrast, high levels of sMICA and sMICB were already observed in early stages of lung cancer, and there was no association with tumor stage. Because sMICA and sMICB levels were particularly elevated in patients with distant metastases, the presence of sMICA and sMICB in serum appears to be an indicator for systemic manifestation of malignancy. Soluble MIC in Cancer Prognosis and Monitoring of Cancer Disease As sMICA and sMICB correlate with cancer stage and as staging systems are developed to enable an efficient stratification of patients for alternative therapy options and to estimate prognosis, it can be expected that sMICA and sMICB will provide prognostic information in various tumor entities. A first study revealed a correlation between sMICA serum levels and disease progression in 23 patients with prostate cancer. In addition, a further study on 97 patients with multiple

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myeloma showed a clear correlation between elevated levels of sMICA and poor overall and progression-free survival in univariate and multivariate analyses. Analysis of sMICA and sMICB in sera enables longitudinal measurements, e.g., for the estimation of therapy response of cancer disease and for the early detection of cancer recurrence. This may turn out to be valuable in advanced cancers with elevated sMICA levels and in therapies affecting the NKG2DL expression such as treatments with imatinib mesylate and HDAC inhibitors. Altogether, levels of sMICA and sMICB are elevated in sera of cancer patients when compared to healthy controls. However, due to elevations of sMIC levels in patients with certain benign diseases and due to the considerable number of MIC-negative cancer patients, both markers seem not to be suitable for cancer diagnosis. Because MIC levels are particularly elevated in advanced cancer stages, they may add information in cancer staging and estimation of prognosis. Future studies may address the prognostic relevance of soluble MIC molecules as well as their value in longitudinal observations for the estimation of systemic therapy response and the early detection of cancer recurrence.

Cross-References ▶ Breast Cancer ▶ Carcinogenesis ▶ Colorectal Cancer ▶ DNA Damage Response ▶ Esophageal Cancer ▶ Gastrointestinal Stromal Tumor ▶ HDAC Inhibitors ▶ Imatinib ▶ Lung Cancer ▶ Multiple Myeloma ▶ Ovarian Cancer ▶ Progression ▶ Prostate Cancer ▶ Serum Biomarkers ▶ Staging of Tumors ▶ Stress Response

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References Holdenrieder S, Stieber P, Peterfi A et al (2006) Soluble MICA in malignant diseases. Int J Cancer 118:684–687 Raulet DH, Gasser S, Gowen BG, Deng W, Jung H (2013) Regulation of ligands for the NKG2D activating receptor. Annu Rev Immunol 31:413–441 Salih HR, Rammensee HG, Steinle A (2002) Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. J Immunol 169:4098–4102 Salih HR, Holdenrieder S, Steinle A (2008) Soluble NKG2D ligands: prevalence, release, and functional impact. Front Biosci 13:3448–3456 Steinle A, Cerwenka A (2015) Immunology. MULT1plying cancer immunity. Science 348:45–46 Ullrich E, Koch J, Cerwenka A, Steinle A (2013) New prospects on the NKG2D/NKG2DL system for oncology. Oncoimmunology 2:e26097

MIC-1 David A. Brown1, Asne R. Bauskin2 and Samuel N. Breit3 1 St. Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital, University of New South Wales, Sydney, NSW, Australia 2 Department of Medicine, Centre for Immunology, St. Vincent’s Hospital, University of New South Wales, Sydney, NSW, Australia 3 Cytokine Biology and Inflammation Research Program, St Vincent’s Centre for Applied Medical Research (AMR), St Vincent’s Hospital, Sydney, NSW, Australia

Synonyms GDF15; Growth differentiation factor 15; Macrophage inhibitory cytokine 1; NAG-1; PDF; PL74; PLAB; PTGF-b

Definition Macrophage inhibitory cytokine 1, also known as growth differentiation factor 15 (MIC-1/GDF15), is a divergent member of the human TGF-b superfamily (▶ Transforming Growth Factor Beta), which was originally cloned on the basis of

MIC-1

increased expression with macrophage activation but has subsequently been found to be strongly linked to cancer.

Characteristics The MIC-1/GDF15 gene, localized on chromosome 19p12-13.1, consists of two exons separated by an intronic sequence of about 1,800 bp. Exon I is 309 bp in length and contains 71 bp of 50 untranslated sequence. Exon II is 891 bp in length with a 30 untranslated region of 244 bp. A single nucleotide polymorphism in the MIC-1/GDF15 coding region results in the change of a histidine (H) to an aspartic acid (D) residue at position 6 of the mature MIC-1/GDF15 protein (Fig. 1). While other polymorphisms have been documented, this polymorphism significantly impacts on cancer predisposition and survival. Other polymorphisms may impact serum levels and/ or protein function, but have yet to be fully characterized. The transcribed MIC-1/GDF15 mRNA is 1,239 bp, with a coding sequence of 924 bp. MIC-1/GDF15 is synthesized as a 62 kD intracellular protein, which then undergoes disulfidelinked dimerization. This dimeric precursor is variably cleaved by a furin-like protease (▶ furin), separating the propeptide domain from the mature MIC-1/GDF15 peptide. The processed, 24.5 kD, and sometimes the unprocessed 62.2kD disulphide-linked dimeric proteins are then secreted (Fig. 1). The receptor for MIC-1/GDF15 is unknown and its signaling pathways are as yet to be delineated. However, as a TGF-b superfamily cytokine, it probably utilizes elements of the conserved TGF-b superfamily receptors and Smad signaling pathway (▶ Smad proteins in TGF-b signaling). There is suggestion that MIC-1/GDF15 also induces activation of Akt (▶ AKT Signal Transduction Pathway in oncogenesis) and ERK pathways. Regulation of MIC-1/GDF15 Gene Expression Under physiological conditions, in humans, the placenta is the major tissue that expresses large

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MIC-1, Fig. 1 Genomic organization, mRNA and protein synthesis, and posttranslational processing of MIC-1/GDF15

amounts of MIC-1/GDF15. Low levels of MIC-1/ GDF15 mRNA are found in liver, lungs, kidneys, and a variety of epithelial cells, however it has been difficult to identify MIC-1/GDF15 protein at these sites. However, MIC-1/GDF15 mRNA and protein is usually substantially increased in injury, inflammation, and cancer in most if not all cells and tissues. Anti-tumorigenic Stimuli Induce MIC-1/ GDF15 Gene Expression. MIC-1/GDF15 is one of the major secreted proteins induced by the p53 transcription factor (p53 Protein, Biological and Clinical Aspects) and may mediate some of the p53 tumor suppressor activity. Indeed, several studies show MIC-1/GDF15 induction being associated with cell cycle arrest and ▶ apoptosis. However, other transcription factors such as Egr-1 (early growth response-1) are also likely to play a role in inducing MIC-1/GDF15 expression.

A variety of anti-tumorigenic agents that activate p53 and/or Egr-1-related pathways can induce MIC-1/GDF15 expression in cancer cell lines. These include many cytotoxic drugs, NSAIDS (▶ Nonsteroidal Anti-Inflammatory Drugs), as well as dietary compounds such as cruciferous vegetable indole-3-carbinol as well as green tea catechins (Fig. 2). In normal epithelial cells, there is little or no detectable expression of MIC-1/GDF15. However, neoplastic transformation causes a major increase in MIC-1/GDF15 expression, and this is further increased by the tumor response to a variety of anti-tumorigenic stimuli, such as gamma irradiation, cytotoxic drugs, NSAIDs, etc. In early-stage cancer, this could lead to tumor cell apoptosis, inhibition of blood vessel formation, and tumor cell cycle arrest. However, in later stage cancers, MIC-1/GDF15 may help tumor

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MIC-1, Fig. 2 Model for the proposed effects of MIC-1/GDF15 on tumor biology

spread, and high serum levels of MIC-1/GDF15 may lead to other systemic effects such as cancerassociated anorexia and weight loss (see below). The level of active MIC-1/GDF15 in the tumor microenvironment is further regulated by the creation of stromal stores of unprocessed proMIC-1/ GDF15, which is often secreted in varying degrees from different tumors. Only unprocessed proMIC-1/GDF15 binds extracellular matrix (ECM) via the propeptide, while processed mature MIC-1/GDF15 is freely soluble and moves from the tumor into blood vessels. Therefore, both intra- and extracellular processing will determine levels of available active MIC-1/ GDF15 (Fig. 2). Indeed, stromal MIC-1/GDF15 storage varies in ▶ prostate cancer patients, and those having low stromal stores are more

susceptible to relapse following removal of the affected prostate gland. MIC-1/GDF15 serum levels in advanced prostate cancer patients predict survival. There is also a direct relationship between MIC-1/GDF15 serum levels and cancer-associated weight loss. Serum MIC-1/GDF15 Measurement as a Clinical Tool Increased tumor tissue expression of MIC-1/ GDF15 is often associated with serum levels outside the normal range of about 200–1,200 pg/ml. Serum MIC-1/GDF15 levels in several cohorts of patients have revealed potential clinical utility in the diagnosis and/or monitoring of cancers such as those of the prostate, breast, ovary, pancreas and colon.

MIC-1

Colon Cancer. MIC-1/GDF15 serum levels are increased in patients with colonic adenomatous polyps and may predict their presence. Levels increase further with colon cancer development in proportion to its stage and extent. MIC-1/ GDF15 serum level at presentation is an independent predictor of metastasis and overall survival. Furthermore, the MIC-1/GDF15 D allele is associated with earlier relapse, but significantly better survival. Studies indicate that serum MIC-1/ GDF15 level is a potential diagnostic tool for the detection of adenomatous colonic polyps. Additionally, MIC-1/GDF15 serum levels combined with colonoscopy might be used to significantly increase the detection of premalignant lesions, while lengthening the intervals between screening colonoscopies. Pancreatic Cancer. Pancreatic cancer (▶ Pancreatic Cancer Basic and Clinical Parameters) is often diagnosed late and there is poor survival. Tumor markers have largely been unhelpful. However, measuring serum levels of MIC-1/GDF15 in combination with the known tumor marker (biomarkers), CA19-9, significantly improved the diagnosis of pancreatic cancer providing a sensitivity of 70% and specificity of 85%. Patients with pancreatic ductal adenocarcinoma had significantly higher levels of serum MIC-1/ GDF15 than those with chronic pancreatitis or healthy controls, while patients with early disease had the highest serum MIC-1/GDF15 levels. This could help in monitoring patients at higher risk of pancreatic cancer development. Prostate Cancer. In prostate cancer (▶ prostate cancer, clinical oncology), when serum MIC-1/ GDF15 levels are combined with total and free PSA (▶ prostate-specific antigen) measurement, diagnosis is significantly improved and is potentially useful for the monitoring of disease progression. Furthermore, the presence of the MIC-1/ GDF15 H allele leads to an increased risk of developing prostate cancer. In established prostate cancer, serum MIC-1/GDF15 level increases with the development of bone metastasis and anorexia/ cachexia, and measurement early in disease is potentially predictive of future metastatic disease and an independent predictor of overall survival. Additionally, the level of stromal bound MIC-1/

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GDF15 protein in prostate tumors is the single best predictor of disease progression in earlystage tumors with a Gleason score less than 7 (▶ Gleason grading). During treatment, elevated serum MIC-1/GDF15 predicts Docetaxil treatment resistance and might be used to rationalize this treatment approach. All-Cause Mortality. Several studies have now demonstrated that serum MIC-1/GDF15 levels predict all-cause mortality. The major underlying causes of mortality in these populations were cancer and cardiovascular disease. These data indicate that MIC-1/GDF15 levels may be involved in or respond to fundamental processes leading to a variety of diseases, as well as cancer. Role of MIC-1/GDF15 in Tumor Biology There is important published, largely indirect evidence, linking tumor MIC-1/GDF15 expression to the biology of cancer. MIC-1/GDF15 expression is clearly linked to the outcome, but direct and convincing evidence causally linking its expression to altered cancer behavior is more limited. As MIC-1/GDF15 is constitutively overexpressed in the majority of patients with common cancers and it is induced with most, if not all, cancer therapies this is a critical question which needs to be addressed. Most, largely in vitro studies, suggest an anti-tumorigenic role: firstly, reports suggest MIC-1/GDF15 induces apoptosis of tumor cells via both p53-dependent and p53-independent mechanisms. Secondly, several in vivo studies using colon and glioblastoma cell lines genetically engineered to express very high levels of MIC-1/GDF15 inhibited tumor growth when transplanted into immunodeficient nude mice (▶ Mouse models). Thirdly, MIC-1/ GDF15 is thought to inhibit blood vessel formation (▶ Angiogenesis), a process essential to feeding the growing tumor. However, there are also contradictory studies suggesting that MIC-1/GDF15 can promote tumorigenesis. High MIC-1/GDF15 expression in a gastric tumor cell line has been shown to correlate with a higher invasive potential, and in melanoma cell lines, which constitutively overexpress MIC-1/GDF15, its knockdown decreased the growth of tumor xenografts. Such

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contradictory effects can be due to a variety of factors including the nature of the tumor and its environment (Fig. 2). The most compelling evidence for an overall protective role for MIC-1/GDF15 in early cancer is suggested by studies using more realistic mouse transgenic cancer models, all of which give a consistent message. Adenomatous polyposis coli (APC) gene mutant mice (APCmin) that develop colonic neoplasia, when crossed with mice constitutively overexpressing MIC-1/GDF15, are protected from tumor formation. Further, MIC-1/ GDF15 gene deleted APCmin mice lose the protection from development of colonic tumors normally afforded by NSAIDS. Transgenic prostate cancer prone TRAMP mice, also genetically overexpressing MIC-1/GDF15, have reduced cancer growth and histological grade, which leads to substantially increased survival. Conversely, TRAMP mice also bearing a germline deletion in MIC-1/GDF15 display faster tumor growth and reduced survival. Whilst these studies of early cancer suggest MIC-1/GDF15 plays a protective, this may not be the case with advanced disease. TRAMP mice also overexpressing MIC1/GDF15, whilst having smaller primary tumors and better survival, have more metastatic disease. This suggests that in advanced prostate cancer, like its relative TGF-b, MIC-1/GDF15 may speed patient demise by increasing metastases and, as discussed below, causing cancer anorexia/cachexia In advanced cancers, serum levels of MIC-1/ GDF15 can commonly rise by 10–100 or more from a normal mean of about 450 pg/ml. Elevated serum MIC-1/GDF15 levels above 3,000–5,000 pg/ml can lead to the onset of a cancer anorexia/cachexia syndrome. This complication can also be seen in other conditions where there is a markedly elevated serum level of MIC-1/GDF15 such as chronic renal and cardiac failure. In advanced prostate cancer patients, MIC-1/GDF15 serum levels show a direct relationship between MIC-1/GDF15 abundance and cancer-associated weight loss. High levels of MIC-1/GDF15 act on brain feeding centers to markedly decrease appetite and hence food intake and body weight. In mice, this process could be

MIC-1

reversed by antibodies to MIC-1/GDF15, suggesting this may be a useful approach to therapy of this largely untreatable and devastating complication of advanced cancer. Conclusion Much still needs to be discovered regarding the precise role of MIC-1/GDF15 in cancer development. However, there is very strong data to support the measurement of MIC-1/GDF15 levels in tumor tissue and blood as a means to detect and monitor cancer. Substantial data also indicates it is the cause of anorexia/cachexia in a proportion of patients with late stage cancer. The data on its role in cancer biology suggests that at least in earlystage cancer, expression of MIC-1/GDF15 is likely to be protective.

Cross-References ▶ Akt Signal Transduction Pathway ▶ Angiogenesis ▶ Apoptosis ▶ Furin ▶ Gleason Grading ▶ Mouse Models ▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Pancreatic Cancer Basic and Clinical Parameters ▶ Prostate Cancer ▶ Prostate Cancer Clinical Oncology ▶ Prostate-Specific Antigen ▶ Smad Proteins in TGF-Beta Signaling ▶ Transforming Growth Factor-Beta

References Baek SJ, Eling TE (2006) Changes in gene expression contribute to cancer prevention by COX inhibitors. Prog Lipid Res 45:1–16 Baek SJ, Okazaki R, Lee SH et al. (2006) Nonsteroidal anti-inflammatory drug-activated gene-1 over expression in transgenic mice suppresses intestinal neoplasia. Gastroenterology 131:1553–1560 Bauskin AR, Brown DA, Junankar S, et al. (2005) The propeptide mediates formation of stromal stores of proMIC-1: Role in determining prostate cancer outcome. Cancer Res. 65:2330-6

Microarray (cDNA) Technology Bootcov, MR, Bauskin AR, Valenzuela, SM et al. (1997) MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci USA 94:11514–11519 Husaini Y, Qiu MR, Lockwood GP et al. (2012) Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice. PLoS One 7: e43833 Husaini Y, Lockwood GP, Nguyen T, et al. (2015) Macrophage Inhibitory Cytokine-1 (MIC-1/GDF15) Gene Deletion Promotes Cancer Growth in TRAMP Prostate Cancer Prone Mice. PLoS one 10: e0115189 Johnen H, Lin S, Kuffner T et al (2007) Tumour-induced anorexia/cachexia is mediated by the TGF-b superfamily cytokine MIC-1. Nat Med 13:1333–1340 Strelau J, Bottner M, Lingor P et al (2000) GDF-15/MIC-1 a novel member of the TGF-beta superfamily. J Neural Transm Suppl 60:273–276 Welsh JB, Sapinoso LM, Kern SG, et al. (2003) LargeScale Delineation of Secreted Protein Biomarkers Overexpressed in Cancer Tissue and Serum. Proc Natl Acad Sci USA. 100:3410–3415 Wiklund FE, Bennet AM, Magnusson PK et al (2010) Macrophage inhibitory cytokine-1 (MIC-1/GDF15): a new marker of all-cause mortality. Aging Cell 9:1057–1064

See Also (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 408– 409. doi:10.1007/978-3-642-16483-5_6601 (2012) Egr-1. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1212. doi:10.1007/978-3-642-16483-5_1832 (2012) ERK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1307–1308. doi:10.1007/978-3-642-16483-5_1987 (2012) Extracellular matrix. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Immunodeficient nude mice. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1816. doi:10.1007/978-3-642-164835_2986 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331 (2012) PSA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3111–3112. doi:10.1007/978-3-642-16483-5_6738 (2012) Single nucleotide polymorphism. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3412. doi:10.1007/978-3-642-164835_5316 (2012) TGF-β superfamily. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3662. doi:10.1007/978-3-642-16483-5_5756

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Microadenomas ▶ Colorectal Cancer Premalignant Lesions

Microarray ▶ Microarray (cDNA) Technology

Microarray (cDNA) Technology Spyro Mousses Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA

Synonyms cDNA chips; Microarray; Parallel gene expression analysis

Definition cDNA microarray technology is one of several developing functional genomics approaches to comparatively analyze genome-wide patterns of mRNA expression. Parallel gene expression profiling of cancer genomes with cDNA microarrays has revolutionized many aspects of cancer research. Gene expression “fingerprinting” has been used for tumor classification, prediction of therapeutic response and resistance, and elucidating genetic pathways associated with particular cancer phenotypes. First cDNA arrays were used during early 1980 (▶ OncoArray) for identifying overexpressed oncogenes. A major discovery, by using the OncoArray, has been the overexpression of the MYCN gene, as the consequence of genomic amplification, in a subgroup of ▶ neuroblastoma.

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Microarray (cDNA) Technology

Characteristics cDNA microarray technology entails the development and standardization of various hardware, analytical software, statistical methodology, biological resources, and biochemical methodologies. The diagram in Fig. 1 demonstrates the various stages of cDNA microarray analysis. cDNA Microarray Construction Construction of very high-density cDNA microarrays of specific and distinct DNA hybridization “targets,” each one representing a single gene, is spotted in an arrayed format on a polyL-lysine-coated glass microscope slide. The printing process involves the sequential transfer of individual purified PCR-amplified fragments (200 bp to 2 kb) from a 96-well microtiter tray to an exact predefined location on glass slides. The “arrayer” encompasses a robotic arm that moves (x, y, z axis) the “quill pen probes” into position to either pick up or spot down DNA (SDS solution), a manifold to hold the slides, a wash station to

rinse the “quill pens” when a different DNA is to be picked up, a place to house the microtiter plates, an airflow cabinet to keep everything in, and a computer which orchestrates the operation of the components. Multiple glass slides, each containing thousands of spots of DNA, can be synthesized and used in subsequent experiments. Hybridization with Labeled cDNA For each experiment, complex “probes” are made that consist of a pool of fluorescently labeled cDNAs. This step begins with the extraction and preparation of mRNAs from two populations of cells. Then each of the two mRNAs is reverse transcribed separately with the incorporation of different fluorescently tagged nucleotides, producing two populations of differentially labeled cDNA probes. Typically Cy3 and Cy5 dyes (which emit light at distinct wavelengths after laser excitation) are used to differentially label cDNA pools from different sources. The two complex labeled probes are combined and then simultaneously hybridized to the cDNA targets on the microarray.

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Microarray (cDNA) Technology, Fig. 1 Schema of microarray technology (Adapted from Duggan et al. (1999))

Microarray (cDNA) Technology

Scanning the cDNA Microarray Using a “Reader” To quantitatively measure the fluorescence of the hybridized probes, the cDNA microarray is scanned by a “reader.” This device is a computercontrolled inverted scanning fluorescent confocal microscope which detects the emitted fluorescence of the dyes after excitation with a double laser illumination system (a 532 nm, 100 mw NdYag laser is used for Cy3 and a 633 nm, 35 mw HeNe laser for Cy5). Digital images of fluorescence intensity data are generated by the “readers” confocal laser scanning microscope. Image Analysis and Generation of cDNA Microarray Data Analysis of the image data generated by the “reader” is done with a software for array target segmentation, target detection, background intensity extraction, target intensity extraction, ratio calculation, and ratio normalization. Typically, cDNA microarray data is transferred to a relational database so they are available for analysis by query-based data mining. Analysis of Large-Scale cDNA Microarray Data Management and analysis of the large volume of data that results from multiple cDNA microarray experiments represents a formidable challenge. Fortunately, new and effective bioinformatic methods have been developed to effectively analyze large-scale expression data in ways conducive to extracting biologically relevant conclusions. Bioinformatic approaches using statistical tools can compare gene expression profiles from multiple experiments. One bioinformatic approach involves clustering, whereby the genes with similar expression profiles across multiple experiments are statistically clustered together. In some cases, the samples can also be clustered based on how similar the global gene expression patterns are. There are a wide variety of statistical methods to cluster microarray data. The most commonly used is unsupervised, hierarchical clustering that, unlike supervised clustering, is not based on fitting the data to a model. Plotting of hierarchical gene clustering results is aided by color representations of the relative gene

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expression ratios and branching dendrograms. Other visualization methods, such as multidimensional scaling (MDS), have been developed to visually illustrate (by distance in a three-dimensional graph) the degree of correlation among multiple samples based on global-scale gene expression profiles. In other words, MDS can cluster samples together in 3-D space based on the similarity of their gene expression profiles. More detailed information on cDNA microarray technology and related links is available in http:// www.nhgri.nih.gov/DIR/LCG/15K/HTML/. Application DNA microarrays facilitate highly parallel analysis of gene expression. Analysis of cDNA microarray data using statistical clustering has revealed groups of genes that have very similar gene expression profiles associated with a phenotype or a response to a physiological condition. Also, genes sometimes cluster together tightly across a large number of conditions revealing co-regulated genes. Furthermore, investigation of genes that cluster together often reveals that they have related structures and functions. As a consequence, clues about the function and regulation of unknown genes can be hypothesized based on other genes that cluster with it. An added advantage is that cellular phenotypes such as migration of cancer cells can be correlated with specific clusters and gene expression fingerprints. Clinical Relevance Gene clusters from cDNA microarray data analysis have been used as cancer biomarkers for diagnosis, progression, and prognosis. For example, the use of global gene expression profiles has successfully identified a gene cluster that distinguishes various classifications of malignancies. This type of classification by gene expression fingerprinting is useful in identifying subtypes of hematological malignancies that were histopathologically not distinguishable but had different clinical outcomes. These profiles can also identify genes that are associated with therapy response and resistance. For example, a gene expression program was identified in prostate cancer that is associated with androgen ablation therapy and

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resistance to hormonal therapy. As a consequence, such genes can be used to predict individual responses to therapy. Furthermore, identification of genes that mediate therapy response also permits rational drug design to target these genes and gene products. cDNA microarray analysis of a small set of cancers can identify hundreds of gene expression alterations. Conventional molecular pathology techniques are relatively slow, thus creating a “bottleneck effect” in the validation and translation of such alterations to a large population of clinical specimens. To alleviate this problem, tissue microarray technology has been developed for parallel high-throughput molecular pathology (immunohistochemistry, mRNA in situ, and fluorescence in situ hybridization) on hundreds of clinical tissue sections on a single glass microscope slide. The prevalence of candidate gene expression alterations can therefore be assayed in hundreds to thousands of clinical samples in a single experiment. These two types of microarray technology are highly complementary, allowing for rapid identification and translation of genes associated with cancer.

References Chen Y, Dougherty E, Bittner M (1997) Ratio-based decisions and the quantitative analysis of cDNA microarray images. J Biomed Opt 2:364–376 DeRisi J, Penland L, Brown PO et al (1996) Use of a cDNA microarray to analyze gene expression patterns in human cancer. Nat Genet 14:457–460 Duggan DJ et al (1999) Expression profiling using cDNA microarrays. Nat Genet 21(1 Suppl):10–14 Eisen MB, Spellman PT, Brown PO et al (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95:14863–14868 Kononen J, Bubendorf L, Kallioniemi A et al (1998) Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4:844–847 Mousses S, Bittner ML, Chen Y et al (2000) Gene expression analysis by cDNA microarrays. In: Livesey FJ, Hunt SP (eds) Differential gene expression: a practical approach. Oxford University Press, Oxford

Microcell-Mediated Chromosome Transfer

Microcell-Mediated Chromosome Transfer Maria Li Lung Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

Definition Microcell-mediated chromosome transfer (MMCT) is a method of transfer of intact or truncated chromosomes from a donor cell to a recipient cell through microcell fusion and hybrid cell selection. A microcell hybrid stably maintains the additional genetic material as functioning units within the cells. This technique is primarily used for transfer of a single donor chromosome (monochromosome transfer) of interest into a recipient cell.

Characteristics A method of mammalian gene transfer, termed microcell-mediated chromosome transfer (MMCT), allows the stable introduction of exogenous chromosomal material from a donor cell into a recipient cell. MMCT is generally used to transfer a portion of the genetic information (generally one chromosome) from one cell to another. This procedure is used for genetics, gene mapping, and gene expression/regulation studies in mammalian cells. One or more intact or truncated chromosomes from the donor cell can be transferred into the recipient cell. Transferred genes are under the control of their endogenous regulatory elements, ensuring their physiological expression in the recipient cell line. In ▶ cancer studies, the transferred donor chromosome is generally a normal human chromosome. Complete libraries of mouse microcell hybrid cell lines containing each individual normal human chromosome, tagged with a dominant

Microcell-Mediated Chromosome Transfer

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Microcell-Mediated Chromosome Transfer, Table 1 MMCT tumor suppression studies in human cancers Chromosomes studied 11 13 6 8

Cancers Bladder Breast

6, 11 11 11, 17 16 17 2 4

Cervix

6 11

7 5, 18 5, 17, 18 8 17

Choriocarcinoma Colon

3 9 14 1 11 1, 11, 17 10

Esophagus

19 8 3, 11 11

1, 6 6

Fibrosarcoma

Glioblastoma

Liver Lung

Melanoma

References Kugoh H et al. (2000) Cancer Genet Cytogenet 116:158 Banerjee A et al. (1992) Cancer Res 52:6297 Theile M et al. (1996) Oncogene 13:677 Wilson P et al. (2003) Cancer Genet Cytogenet 143:100; Seitz S et al. (2005) Oncogene 24:869; (2006) Genes Chromosomes Cancer 45:612 Negrini M et al. (1994) Cancer Res 54:1331 Phillips KK et al. (1996) Cancer Res 56:1222; Negrini M et al. (1992) Oncogene 7:2013 Yang X et al. (1999) Int J Oncol 15:629 Reddy DE et al. (1999) Oncogene 18:5100 Casey G et al. (1993) Hum Mol Genet 2:1921; Plummer SJ et al. (1997) Oncogene 14:2339 Uejima H et al. (1995) Genes Chromosomes Cancer 14:120 Backsch C et al. (2001) Genes Chromosomes Cancer 31:196; (2005) Genes Chromosomes Cancer 43:260; Forsythe NR et al. (2002) Oncogene 21:5135; Bryce SD et al. (2002) Neoplasia 4:544 Steenbergen RD et al. (2001) J Natl Cancer Inst 93:865 Saxon PJ et al. (1986) EMBO J 5:3461; Srivatsan ES et al. (1986) Cancer Res 46:6174; Koi M et al. (1989) Mol Carcinog 2:12; Misra BC, Srivatsan ES (1989) Am J Hum Genet 45:565 Miyamoto S et al. (1991) Hum Cell 4:38 Tanaka K et al. (1991) Nature 349:340 Goyette MF et al. (1992) Mol Cell Biol 12:1387 Gustafson CE et al. (1996) Cancer Res 56:5238 Flanagan JM et al. (2003) Int J Cancer 106:505; (2004) Genes Chromosomes Cancer 40:247 Lo PH et al. (2007) Oncogene 26:148–157 Yang L et al. (2005) Oncogene 24:697 Ko JMY et al. (2005) Genes Chromosomes Cancer 43:284 Klein KG, Bouck NP (1994) Cancer Genet Cytogenet 73:109 Benedict WF et al. (1984) Cancer Res 44:3471 Anderson MJ et al. (1994) Genes Chromosomes Cancer 9:266 Pershouse MA et al. (1993) Cancer Res 53:5043; Kon H et al. (1998) Oncogene 16:257 Drucker KL et al. (2009) Genes Chromosomes Cancer 48:854 Liu H et al. (2006) Zhonghua Yi Xue Yi Chuan Xue Za Zhi 23:540 Satoh H et al. (1993) Mol Carcinog 7:157 Murakami Y et al. (1998) Proc Natl Acad Sci U S A 95:8153; (2002) Oncogene 21:6930; Kuramochi M et al. (2001) Nat Genet 27:427; O’Briant K et al. (1997) Anticancer Res 17:3243 Miele ME et al. (1996) Mol Carcinog 15:284 Trent JM et al. (1990) Science 247:568; Ray ME et al. (1996) Oncogene 12:2527; Welch DR et al. (1994) Oncogene 9:255; (1997) Int J Cancer 71:1035; Miele ME et al. (1997) Clin Exp Metastasis 15:259; (2000) Int J Cancer 86:524 (continued)

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Microcell-Mediated Chromosome Transfer

Microcell-Mediated Chromosome Transfer, Table 1 (continued) Chromosomes studied 10 11 7, 11 3 6, 11, 17 11

Cancers

Myeloid leukemia Nasopharynx

13 14 1 3 11 17 11 3 3 6 11

6, 18 22 18 5 10

Neuroblastoma

Neuroepithelioma Oral cavity Ovary

Pancreas Prostate

11 12 13 17 18

19

Y 3 11 1, 6, 9, 18 11

Renal cell Rhabdomyosarcoma Uterine endometrium Wilms tumor

References Robertson GP et al. (1999) Cancer Res 56:3596 Robertson G et al. (1996) Cancer Res 56:4487; (1999) Oncogene 18:3173 Wilding J et al. (2002) Genes Chromosomes Cancer 34:390 Cheng Y et al. (1998) Proc Natl Acad Sci USA 95:3042 Cheng Y et al. (2000) Genes Chromosomes Cancer 28:82 Cheng Y et al. (2002) Genes Chromosomes Cancer 34:97; Lung HL et al. (2004) Int J Cancer 112:628; (2005) Oncogene 24:6525; (2006) Cancer Res 66:9385; Lung HL et al. (2008) Int J Cancer 122:1288 Cheng Y et al. (2004) Int J Cancer 109:357 Cheng Y et al. (2003) Genes Chromosomes Cancer 37:359; Cheung AKL et al. (2009) Proc Natl Acad Sci USA 106:14478 Martinsson T et al. (1989) Genes Chromosomes Cancer 1:67 Hoebeeck J et al. (2009) Cancer Invest 27:857 De Preter K et al. (2005) BMC Genomics 6:97 Bader SA et al. (1991) Cell Growth Differ 2:245 Chen P et al. (1995) Oncogene 10:577 Uzawa N et al. (1995) Oncogene 11:1997; (1998) Cancer Genet Cytogenet 107:125 Rimessi P et al. (1994) Oncogene 9:3467 Wan M et al. (1999) Oncogene 18:1545 Cao Q et al. (2001) Cancer Genet Cytogenet 129:131; Stronach EA et al. (2003) Cancer Res 63:8648; Abeysinghe HR et al. (2003) Cancer Genet Cytogenet 143:125 Dafou D et al. (2009) Int J Cancer 124:1037 Kruzelock RP et al. (2000) Oncogene 19:6277 Lefter LP et al. (2002) Genes Chromosomes Cancer 34:234; (2004) Asian J Surg 27:85 Ewing CM et al. (1995) Cancer Res 55:4813 Fukuhara H et al. (2001) Oncogene 20:314; Ichikawa T et al. (1992) Cancer Res 52:3486 Murakami Y et al. (1996) Cancer Res 55:3389; Sanchez Y et al. (1996) Proc Natl Acad Sci USA 93:2551; Kawana Y et al. (1997) Prostate 32:205 Berube NG et al. (1994) Cancer Res 53:3077 Banerjee A et al. (1992) Cancer Res 52:6297 Murakami YS et al. (1995) Cancer Res 55:3389; Chekmareva MA et al. (1997) Prostate 33:271 Lefter LP (2003) Clin Cancer Res 9:5044; Padalecki SS et al. (2001) Genes Chromosomes Cancer 30:221; (2003) Urol Oncol 21:366; Gagnon A et al. (2006) Genes Chromosomes Cancer 45:220 Gao AC et al. (1999) Prostate 38:46; Astbury C et al. (2001) Genes Chromosomes Cancer 31:143; Padalecki SS et al. (2001) Genes Chromosomes Cancer 30:221 Vijayakumar S et al. (2005) Genes Chromosomes Cancer 44:365 Shimizu M et al. (1990) Oncogene 5:185 Loh WE et al. (1992) Proc Natl Acad Sci U S A 89:1755 Yamada H et al. (1990) Oncogene 5:1141; (1995) Genes Chromosomes Cancer 13:18 Weissman BE et al. (1987) Science 236:175; Dowdy SF et al. (1991) Science 254, 293; Reid LH et al. (1996) Hum Mol Genet 5:239

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Microcell-Mediated Chromosome Transfer, Fig. 1 Schematic diagram of MMCT procedure

Microcell-mediated chromosome transfer (MMCT) ^ A A A A

Microenucleation U

ter

A

U

A

A

Fil

A

A-P

Microcells

U U

U

U U

U

U

U

U

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selectable antibiotic resistance gene, originating from a diploid fibroblast cell, have been generated. These cell line resources have been invaluable for gene function and mapping studies. Panels of selected donor chromosome cell lines, containing random truncations induced by irradiation to narrow down genomic regions containing tumor suppressive activity, have also been established for MMCT studies. The recipient cells are often human cancer cell lines or murine cell lines. Using MMCT approaches, functional complementation of genetic defects present in the original recipient cancer cell line by the introduction of a normal exogenous chromosome provides evidence for the existence of a ▶ tumor suppressor gene having a role in the cancer cell line studied. This approach has been used in specific tumor cell lines to detect candidate tumor suppressor genes on individual chromosomes and to aid in their identification and mapping to critical regions associated with tumorigenesis. This allowed both validation of the tumor suppressive (▶ tumor suppression) function of known tumor suppressor genes, such as RB (▶ retinoblastoma) and WT1 (▶ Wilms tumor), for example, and identification of yet other previously unidentified candidate

U U U

U

PEG for cell fusion

G418 HAT for hybird selection

U

U U U

PH

U

U

B sin ala e h toc rc Cy g fo

U U U

U

Chromosome donor cell

+

^

H

Colcemid

U

^ ^

U

U

Microcell hybird with recipient + donor chromosomes

tumor suppressor genes. A summary of some of these MMCT tumor suppressive studies of human cancers is shown in Table 1. Using MMCT approaches, researchers were able to localize tumor suppressor genes to specific chromosomes to provide direct functional evidence for their role in tumor suppression and to demonstrate that restoring a normal copy of a single tumor suppressor gene in a cancer cell line reversed its tumorigenic potential, despite multiple defects on several chromosomes contributing to a malignancy. MMCT approaches are also useful for identifying genes responsible for growth suppression, senescence, and ▶ metastasis. The MMCT procedure consists of the following key steps, as illustrated schematically in Fig. 1. Exponentially growing donor cells containing the stable exogenous donor human chromosome of interest, which is tagged with a selectable marker such as neo for G418 selection, are subjected to prolonged colcemid (mitotic inhibitor) treatment. Micronuclei (▶ micronucleus) are formed, when the nuclear envelope reforms around one or more of the chromosomes. Micronuclei are then enucleated by treatment with ▶ cytochalasin B and centrifugation. Microcells are then formed. They are filtered

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through the membranes to eliminate contaminating whole cells and karyoplasts in order to obtain the fraction containing only the smaller microcells harboring single chromosomes. These microcells are then added to recipient cells, agglutinated with phytohemagglutinin-P, and then induced to fuse in the presence of PEG. The fusion of microcells to the recipient cell results in hybrid heterokaryon cells. The heterokaryons, containing the exogenous chromosome of interest, are selected with HAT to eliminate mouse HPRT-negative donor cells from the culture and with G418 to eliminate human recipient cells that do not receive the donor chromosome of interest. Microcell hybrids contain the transferred chromosome in a new recipient cell environment. Generally the successful transfer of the donor chromosome is monitored by the FISH analysis.

Cross-References ▶ Cancer ▶ Cytochalasin B ▶ Fluorescence in Situ Hybridization ▶ Metastasis ▶ Retinoblastoma ▶ Tumor Suppression ▶ Tumor Suppressor Genes ▶ Wilms’ Tumor

References Dowdy SF, Scanlon DJ, Fasching CL et al (1990) Irradiation microcell-mediated chromosome transfer (XMMCT): the generation of specific chromosomal arm deletions. Genes Chromosomes Cancer 2:319–327 Fournier REK, Ruddle FH (1977) Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc Natl Acad Sci U S A 74:319–323 Satoh H, Barrett JC, Oshimura M (1991) Introduction of new genetic markers on human chromosomes. Exp Cell Res 193:5–11 Saxon PJ, Srivatsan ES, Leipzig GV et al (1985) Selective transfer of individual human chromosomes to recipient cells. Mol Cell Biol 5:140–146 Stanbridge EJ, Dowdy S, Bader S et al (1991) Monochromosome transfer provides functional evidence for tumor suppressor genes. In: Brugge J,

Microcephalin Curran T, Harlow E, McCormick F (eds) Origins of human cancer: a comprehensive review. Cold Spring Harbor Laboratory Press, New York, pp 393–401

See Also (2012) Enucleate. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1259. doi:10.1007/978-3-642-16483-5_1914 (2012) FISH. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1415–1416. doi:10.1007/978-3-642-16483-5_2197 (2012) HAT. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1632–1633. doi:10.1007/978-3-642-16483-5_2573 (2012) Heterokaryon. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1689. doi:10.1007/978-3-642-16483-5_2697 (2012) Karyoplast. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1941. doi:10.1007/978-3-642-16483-5_3199 (2012) Microcell. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2292. doi:10.1007/978-3-642-16483-5_3714 (2012) Microcell Hybrid. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2292. doi:10.1007/978-3-642-16483-5_3715 (2012) Micronucleus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2300. doi:10.1007/978-3-642-16483-5_3726 (2012) Senescence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3370. doi:10.1007/978-3-642-16483-5_5236

Microcephalin ▶ BRIT1 Gene

Microcystin-LR Hirota Fujiki Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Saga, Japan

Definition Cyanobacterial hepatotoxin, a potent tumor promoter; there are three forms of microcystins:

Microcystin-LR Microcystin-LR, Fig. 1 Structures of microcystins. (a) Microcystin-LR, microcystin-YR, and microcystin-RR. (b) Geometrical isomers (6Z)Adda microcystin-LR and microcystin-RR

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a

Glu

Mdha H CO2H

Adda

N

HN H3C H H

H OCH3 H

H CH3 CH3

H

NH O

H2C

O NH

H O

O

CH3

Y

O

X

Y

Leu

Arg

Microcystin-YR

Try

Arg

Microcystin-RR

Arg

Arg

b

X

H CO H O 2

Masp

Microcystin-LR

O

HN

H CH3 H N

H N

Ala

H H3C

CH3

H CO2H

O

N H OCH3 CH3 H

NH

HN O

H3C H H

H CH3

H

H2N

O

H

NH

O

NH

H2C

H N H

H H3C

H CH3 H N

H N O

H CO2H O

O

HN H

R

(6Z )-Adda microcystin-LR R=CH(CH3)2 (6Z )-Adda microcystin-RR R=CH2CH2NH(=NH)NH2

• Microcystin-LR is cyclo(-D-Ala-L-LeuD-erythro-beta methylisoaspartic acid(Masp)L-Arg-(2S,3S,8S,9S)-3-amino-9methoxy-2,6,8-trimethyl-10-phenyldeca-(4E,6E)dienoic acid (Adda)-D-isoGlu-N-methyldehydroalanine (Mdha)-). • Microcystin-YR (microcystin containing tyrosine instead of the leucine in microcystin-LR). • Microcystin-RR (microcystin containing arginine instead of the leucine in microcystin-LR) (Fig. 1a).

Characteristics Microcystin-LR is a hepatotoxin, one of the microcystins isolated from cyanobacteria (bluegreen algae), which include Microcystis aeruginosa, Microcystis viridis, Microcystis wesenbergii, Microcystis flos-aquae, and Oscillatoria agardhii. Microcystin-LR is found in waterblooms of toxic cyanobacteria and in eutrophic freshwater municipal and residential water supplies; it is associated with increasing

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Microcystin-LR

Microcystin-LR, Fig. 2 Induction of glutathione S-transferase placental form (GST-P) positive foci. (a) The liver treated with a single administration of DEN. (b)

The liver treated with DEN plus microcystin-LR 50 mg/kg body weight together 20 times for 10 weeks

environmental hazards in various areas of the world. Toxins of cyanobacteria are grouped into cytotoxin, neurotoxin, and hepatotoxin. The hepatotoxins are microcystin-LR, microcystin-YR, and microcystin-RR, and there are 50 microcystin derivatives. Microcystin-LR is a tumor promoter in rat liver, a potent inhibitor of serine/threonine protein phosphatase 1 and 2A (PP1 and PP2A) (IC50 values 0.1 nM and 0.1 nM, respectively). Protein dephosphorylation by PP1 and PP2A is an opposite biochemical reaction of protein phosphorylation by protein kinases. Thus, inhibition of protein phosphatase causes accumulation of phosphoproteins in the cells. The World Health Organization (WHO) International Agency for Research on Cancer (IARC), Lyon, in 2006 assessed the carcinogenicity of microcystin-LR for humans, based on three perspectives: carcinogenicity in rodents, epidemiological evidence, and unique mechanisms of action of the compound; WHO concluded that microcystin-LR is “possibly carcinogenic to humans” (group 2B).

uptake into the liver of mice – 17.0  4.1% of the total administered radioactivity after 5 min and 71.5  6.9% after 1 h – whereas oral administration results in 0.68% of the total administered amount in the liver from 6 to 19 h after treatment. After a single intraperitoneal injection of 1.5 mg microcystin-YR, i.e., 75 mg/kg body weight, the liver became dark red and the mouse died within 2 h. The histological examination revealed massive intrahepatic hemorrhages resulting in acute death of liver cells by necrosis. Two-stage carcinogenesis of microcystin-LR in the liver of male F344 rats showed tumorpromoting activity. Initiation was done with a single administration of diethylnitrosamine (DEN) 200 mg/kg. Tumor promotion was achieved by repeated intraperitoneal administrations of microcystin-LR 25 mg/kg, twice a week, a total of 20 times for 10 weeks (initiation and promotion). As an indicator for detecting tumorpromoting activity, autopsied liver sections were stained by the avidin-biotin-peroxidase complex method for immunohistochemistry of glutathione S-transferase placental form (GST-P) positive foci and neoplastic nodules, and only those foci larger than 50 mm in diameter were counted (Fig. 2). GST-P is a biochemical marker detecting neoplastic change, and the GST-P positive foci in the liver

Tumor-Promoting Activity in Rat Liver Liver organotropy of microcystins is unique because intraperitoneal administration of [3H] dihydromicrocystin-LR results in the highest

Microcystin-LR

were assessed by the number of foci/cm2 of the liver, area of foci/liver (mm2/cm2), and volume of foci/liver (v/v%). Group 4, treated with initiator, DEN plus microcystin-LR, had larger numbers, areas, and volumes of foci per liver than did the control groups. Group 3, treated with microcystinLR alone, did not induce any foci. Microcystins are not mutagenic in the Ames test with Salmonella typhimurium. Thus, microcystin-LR has a tumor-promoting activity in rat liver and does not have any initiating activity. Microcystin-YR and microcystin-RR also are assumed to be liver tumor promoters, because they have the same specific activity in various biochemical assays as does microcystin-LR. Inhibition of Specific [3H]okadaic Acid Binding The tertiary structure of microcystin-LR is similar to that of okadaic acid, since okadaic acid is thought to have a flexible cavity formed by an intramolecular hydrogen bond between C-1 carbonyl and C-24 hydroxyl groups. MicrocystinLR, microcystin-YR, and microcystin-RR inhibited the specific [3H]okadaic acid binding to cytosolic fraction with the same potencies. Thus, microcystins bind to the okadaic acid receptors PP1 and PP2A as strongly as okadaic acid does. Inhibition of Protein Phosphatases 1 and 2A Microcystin-LR inhibits the catalytic subunit of PP1 purified from rabbit skeletal muscle (IC50 value 0.1 nM) and that of PP2A purified from human erythrocytes (IC50 value 0.1 nM). Microcystin-LR inhibited both PP1 and PP2A with the same potencies, but it did not inhibit protein tyrosine phosphatase. Microcystin-LR, microcystin-YR, and microcystin-RR inhibited protein phosphatase 2A, with the similar potencies, and the results correlate well with inhibition of specific [3H]okadaic acid binding. Cellular Biochemical Response Microcystin-LR and okadaic acid have different effects on primary human fibroblasts. Microcystin-LR at concentrations up to 9.6 mM did not induce any biochemical or biological effects on primary human fibroblasts, but

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250 nM okadaic acid was enough to induce morphological changes in cells, from spindle-like to a round form, within 2 h of incubation. A solution of microcystin-YR at a very high concentration of 670 mM was injected into primary human fibroblasts resulted in morphological changes from spindle-like to a round form 45 min after microinjection. Obviously, there are differences in tissue specificity between microcystins and okadaic acid. Structure-Function Relationship of Microcystins The long hydrophobic portion of microcystins (Adda) is significant in their activity. Two geometrical isomers at C-7 in the Adda portion of microcystins, (6Z)-Adda microcystin-LR and microcystin-RR, were isolated from cyanobacteria (Fig. 1b). The specific [3H] dihydromicrocystin-LR binding to the cytosolic fraction was inhibited by microcystin-LR and microcystin-RR (IC50 values 0.38 and 0.42 nM, respectively) and by (6Z)-Adda microcystin-LR and microcystin-RR (IC50 values 32 and 52 nM, respectively). Thus, (6Z)-Adda microcystin-LR and microcystin-RR are 100 times weaker than their maternal (6E)-Adda microcystins, and the conjugated diene with (4E,6E) geometry in the Adda portion is essential in the interaction with protein phosphatases. Furthermore, microcystin-LR and microcystinLA have the same specific activity on the inhibitions of PP1 and PP2A and specific [3H]okadaic acid binding, suggesting that the arginine residue in microcystin-LR does not significantly contribute to its biochemical activity. Crystal Structure of PP1-Microcystin-LR Complex The crystal structure of PP1-microcystin-LR complex at 2.1 Å was initially reported in 1995. Microcystin-LR binds to catalytic subunit of PP1 through interaction with the metalbinding site, the hydrophobic groove, and the edge of the C-terminal groove near the active site (Fig. 3). The covalent linkage of microcystin-LR to PP1 was noted previously,

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Microcytoma

Microcystin-LR, Fig. 3 Electron density map of microcystin-LR binding to PP1 catalytic subunit (From: Goldberg et al. 1995)

but it is not essential for the inhibition of the enzyme by microcystin-LR.

Cross-References ▶ Okadaic Acid

References Carmichael WW (1992) Cyanobacteria secondary metabolites – the cyanotoxins. J Appl Bacteriol 72:445–459 Farber E, Solt D (1978) A new liver model for the study of promotion. Slaga TJ, Sivak A, Boutwell RK Eds, Carcinogenesis 2:443–451 Fujiki H, Suganuma M (2010) Tumor promoters – microcystin-LR, nodularin and TNF-a and human cancer development. Anticancer Agents Med Chem 11:4–18 Goldberg J, Huang H-b, Kwon Y-g, Greengard P, Nairn AC, Kuriyan J (1995) Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 376:745–753 Grosse Y, Baan R, Straif K, Secretan B, Ghissassi FEI, Cogliano V (2006) Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins. Lancet Oncol 7:628–629 Harada K-I, Ogawa K, Matsuura K, Murata H, Suzuki M, Watanabe MF, Itezono Y, Nakayama N (1990) Structural determination of geometrical isomers of microcystins LR and RR from cyanobacteria by two-dimensional NMR spectroscopic techniques. Chem Res Toxicol 3:473–481

Matsushima R, Yoshizawa S, Watanabe MF, Harada K-I, Furusawa M, Carmichael WW, Fujiki H (1990) In vivo and in vitro effects of protein phosphatese inhibitors, microcystins and nodularin, on mouse skin and fibroblasts. Biochem Biophys Res Commun 171:867–874 Nishiwaki-Matushima R, Ohta T, Nishiwaki S, Suganuma M, Kohyama K, Ishikawa T, Carmichael WW, Fujiki H (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J Cancer Res Clin Oncol 118:420–424 Sato K, Kitahara A, Satoh K, Ishikawa T, Tatematsu M, Ito N (1984) The placental form of glutathione S-transferease as a new marker protein for preneoplasia in rat chemicall heaptocarinogenesis Gann 75:199–202 Suganuma M, Suttajit M, Suguri H, Ojika M, Yamada K, Fujiki H (1989) Specific binding of okadaic acid, a new tumor promoter in mouse skin. FEBS Lett 250:615–618

Microcytoma ▶ Extrapulmonary Small Cell Cancer

Microglia ▶ Macrophages

Micrometastasis

Micrometastasis Catherine Alix-Panabieres1 and Klaus Pantel2 1 University Medical Center, Lapeyronie Hospital, Montpellier, France 2 Universitäts-Krankenhaus Eppendorf, Hamburg, Germany

Definition Micrometastases were originally defined by pathologists as small occult metastases ( tenfold increase in relapse after hematopoietic stem cell transplant (HSCT). For CML taking tyrosine kinase inhibitors (such as Gleevec), the BCR-ABL level predicts longterm outcome. In AML and ALL, detection of MRD by PCR or flow cytometry is highly associated with subsequent relapse. The same holds for ▶ chronic lymphocytic leukemia (CLL) and ▶ multiple myeloma, both B-cell malignancies where MRD can be detected by PCR assays directed at clonal immunoglobulin gene rearrangements. Not all patients with detectable MRD progress to relapse. The persistence of MRD without subsequent relapse has been referred to as ▶ dormancy (Fig. 1). Dormancy has been seen in t (8;21) AML following conventional chemotherapy, where most long-term survivors remain PCR-positive. Dormancy has also been seen in CML following HSCT. Furthermore, late relapses in pediatric ALL (who relapse >10 years after the establishment of remission) have the same clonal IgH V-D-J gene rearrangement as at diagnosis, suggesting a very long-term dormancy of highly malignant cells. The mechanism of dormancy is unknown. It is likely that the clinical definition of “cure” in cancer does not mean the elimination of all cells involved in the leukemia process. The further study of dormancy may clarify what it takes to “cure” leukemia.

Cross-References

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Minodronate Sho-ichi Yamagishi Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan

Synonyms Amino-bisphosphonate; [1-Hydroxy-2-(imidazo [1,2-a] pyridin-3-yl)ethylidene]-bisphosphonate; Minodronic acid; Nitrogen-containing bisphosphonate; ONO-5920; Third-generation bisphosphonate; Third-generation nitrogencontaining bisphosphonate; YM-529

Definition Bisphosphonates are potent inhibitors of bone resorption and are widely used drugs for the treatment of osteoporosis, osteolytic bone metastasis, and tumor-associated hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Minodronate, a nitrogen-containing bisphosphonate, acts intracellularly by inhibiting farnesyl pyrophosphate synthase, thus leading to the inhibition of posttranslational prenylation of small molecular weight G proteins, which could also contribute to its antiresorptive activity on osteoclasts and its antitumor effects in vivo.

▶ Circulating Tumor Cells

Characteristics References Campana D, Pui CH (1995) Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 85:1416–1434 Chung NG, Buxhofer-Ausch V, Radich JP (2006) The detection and significance of minimal residual disease in acute and chronic leukemia. Tissue Antigens 68:371–375

Minodronate is one of the most potent nitrogencontaining bisphosphonates. Farnesyl pyrophosphate synthase is a molecular target of nitrogencontaining bisphosphonates, and inhibition of posttranslational prenylation of small molecular weight G proteins is likely involved in their antitumor effects both in vitro and in vivo.

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Further, since nitrogen-containing bisphosphonates accumulate in human vessels as well, it is also probable that they might have pleiotropic effects on vascular cells by blocking the protein prenylation of small G proteins, which serve as lipid attachments for a variety of intracellular signaling molecules. Effects of Minodronate on Endothelial Cells (ECs) ▶ Angiogenesis, a process by which new vascular networks are formed from preexisting capillaries, is required for tumors to grow, invade, and metastasize. It is generally considered that a major event in tumor growth and expansion is the “angiogenic switch.” ▶ Vascular endothelial growth factor (VEGF), a specific mitogen to endothelial cells, is a crucial factor for tumor angiogenesis. VEGF also acts as a proinflammatory cytokine in tumorigenesis. Several lines of evidence implicate VEGF as the key factor involved in tumor growth, expansion, and metastasis. Indeed, VEGF expression levels are associated with ▶ angiogenesis and macrophage infiltration, the extent of which being correlated with the prognosis in various types of tumors. These observations suggest that inhibition of the VEGF signaling in tumor endothelium is a therapeutic target for preventing the development and progression of tumors. Minodronate inhibits the VEGF-induced increase in DNA synthesis and tube formation in ECs by suppressing NADPH oxidase-mediated reactive oxygen species generation and consequent RAS activation via suppression of Rac-1 geranylgeranylation. In addition, minodronate blocks the VEGF-induced upregulation of intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in ECs, subsequently suppressing the VEGFmediated T cell adhesion to ECs. Since small G proteins such as Rac-1 and Ras are involved in the VEGF signaling to ▶ inflammation as well, minodronate may exert antitumor effects in vivo by blocking the VEGF signaling in tumor endothelium. Reducing sugars, including glucose, fructose, and trioses, can react nonenzymatically with the amino groups of proteins to form reversible Schiff

Minodronate

bases and then Amadori products. These early glycation products undergo further complex reactions such as rearrangement, dehydration, and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives termed “advanced glycation end products (AGEs).” The formation and accumulation of AGEs in various tissues are known to progress during normal aging and at an extremely accelerated rate in diabetes mellitus. AGE-their receptor (RAGE) interaction elicits oxidative stress generation in vascular wall cells, thereby being involved in the pathogenesis of various types of disorders including melanoma growth, expansion, and metastasis. Minodronate inhibits the AGE-induced reactive oxygen species generation and ▶ nuclear factor-kB activation and consequently suppresses vascular cell adhesion molecule-1 (VCAM-1) expression in ECs. This also suggests that minodronate may have therapeutic potentials for the treatment of AGE-related tumor growth and expansion. Since prostaglandin F2a (PGF2a) is involved in tumor ▶ angiogenesis and minodronate suppresses PGF2a-induced VEGF overexpression in cultured osteoblasts, minodronate may exert antitumor effects by suppressing tumor VEGF expression. Effects of Minodronate on Tumor Cells In Vitro Minodronate induces S-phase cell cycle arrest and ▶ apoptosis in myeloma cells by inhibiting protein geranylgeranylation and subsequently decreasing the levels of phosphorylated extracellular signalregulated kinase 1/2 (ERK1/2), which could act as a survival signal in myeloma cells. Minodronate also induces apoptosis of other types of tumor cells; it decreases bcl-2 expression and induces bax expression as well as caspase-3 activation, promoting apoptotic cell death of prostate cancer cells. In addition, minodronate inhibits proliferation of cultured murine osteosarcoma cells with induction of apoptosis. Moreover, minodronate decreases DNA synthesis and increases apoptotic cell death in cultured human melanoma cells via suppression of Ras farnesylation. Effects of Minodronate on Tumor Cells In Vivo Minodronate inhibits ▶ melanoma growth and improves survival in nude mice by two

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Minodronate, Fig. 1 Possible mechanisms for antitumor effects of minodronate

independent mechanisms; one is by suppressing the tumor-associated ▶ angiogenesis and macrophage infiltration via inhibition of the VEGF signaling in ECs, and the other is by inducing directly apoptotic cell death of ▶ melanoma. These antitumor effects of minodronate observed in vivo could partly be ascribed to its suppressive properties on protein prenylation in melanoma cells themselves and tumor-associated endothelium (Fig. 1). Minodronate is reported to exert antitumor effects in other animal models. It inhibits prostate cancer cell invasion into the bone matrix of an intratibial tumor injection murine model by suppressing CXC-R-4 expression in the lesions. Minodronate also suppresses osteoclast-mediated bone invasion by oral squamous cell carcinoma in mice by inhibiting cytokine expressions (tumor necrosis factor, interleukin-6, etc.). In addition, minodronate is reported to augment the anticancer effects of interferon on renal cell carcinoma in mice by further reducing serum levels of VEGF. Furthermore, in vivo combination therapy with docetaxel and minodronate is found to significantly reduce the tumor incidence compared with the control and also growth of intraossal transitional cell carcinoma of urinary tract in athymic nude mice compared with the control. Therapy with minodronate significantly enhances the inhibition of proliferation by docetaxel in

osteoclasts of bone tumors compared with single therapy with docetaxel. These observations suggest that combination therapy with minodronate and docetaxel or minodronate plus interferon may also be beneficial for the treatment of tumors. In conclusion, in vitro and in vivo data with minodronate support the concept that minodronate is a novel potential therapeutic agent for preventing the development and progression of various types of tumors. Further clinical investigation is needed to evaluate the efficacy of this new pharmacological approach as an antitumor therapy.

Cross-References ▶ Angiogenesis ▶ Inflammation ▶ Nuclear Factor-kB ▶ Vascular Endothelial Growth Factor

References Green JR, Clezardin P (2002) Mechanisms of bisphosphonate effects on osteoclasts, tumor cell growth, and metastasis. Am J Clin Oncol 25:S3–S9 Yamagishi S, Abe R, Inagaki Y et al (2004) Minodronate, a newly developed nitrogen-containing bisphosphonate, suppresses melanoma growth and improves survival in nude mice by blocking vascular endothelial growth factor signaling. Am J Pathol 165:1865–1874 Yamagishi S, Matsui T, Nakamura K et al (2005) Minodronate, a nitrogen-containing bisphosphonate, inhibits advanced glycation end product-induced vascular cell adhesion molecule-1 expression in endothelial cells by suppressing reactive oxygen species generation. Int J Tissue React 27:189–195 Yamagishi S, Nakamura K, Matsui T et al (2006) Minodronate, a nitrogen-containing bisphosphonate, is a promising remedy for treating patients with diabetic retinopathy. Med Hypotheses 66:273–275 Ylitalo R, Kalliovalkama J, Wu X et al (1998) Accumulation of bisphosphonates in human artery and their effects on human and rat arterial function in vitro. Pharmacol Toxicol 83:125–131

See Also (2012) AGEs. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 101. doi:10.1007/978-3-642-16483-5_140 (2012) Angiogenic switch. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 186. doi:10.1007/978-3-642-16483-5_277

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Minodronic acid ▶ Minodronate

Mismatch Repair in Genetic Instability Margherita Bignami Istituto Superiore di Sanita’, Rome, Italy

Definition Mismatch repair acts after replication to correct mismatches which have escaped proofreading by the replication apparatus. Thus mismatch repair corrects DNA bases in non-Watson–Crick pairings or small distortions generated by incorrect strand alignment in repetitive DNA sequences. Structural abnormalities such as these can also be generated during recombination between DNA molecules which are not perfectly homologous (▶ Repair of DNA).

Characteristics The first step in human mismatch repair is the binding of DNA mispairs by either the MutSa or the MutSb heterodimer. These comprise of the MSH2/MSH6 and hMSH2/MSH3 proteins,

Minodronic acid

respectively. Biochemical evidence obtained with purified complexes indicates that MutSa selectively recognizes single base mispairs and one base loop, whereas MutSb prefers loops of 2–10 unpaired bases. Both bind to the two base loops that are the most common abnormalities in repetitive DNA sequences (Fig. 1). MutSa most likely forms a sliding clamp, which encircles DNA in the vicinity of the mismatch. The next step usually involves formation of a ternary complex with the MutLa heterodimer which is composed of the MLH1 and PMS2 proteins. Two minor heteroduplexes of undefined function with PMS1 (MutLb or MLH3) and MutLg together account for 50 mode. EXO1 is an ATP- and mismatch-dependent 50 ! 30 exonuclease that paradoxically appears to be sufficient to direct correction in both directions. In the presence of MutSa, EXO1-mediated excision is highly processive and results in removal of >1,000 nucleotides. MutSa, the replication processivity factors PCNA, and RFC activate a latent endonuclease of MutLa which yields new 50 termini for EXO1 entry to permit 30 ! 50 correction. PCNA, in its role as a processivity factor for DNA polymerase d, is also involved together with the single stranded binding protein, replication protein A (RPA) in the subsequent repair synthesis step. It is not known which of the several DNA ligases catalyzes the ligation of the newly synthesized DNA stretch to complete repair. Biological Consequences Loss of mismatch repair leads to the accumulation of replication errors particularly in repetitive sequences such as microsatellites (long stretches of mono- and dinucleotide repeats scattered throughout the genome). During replication, template and daughter DNA strands in microsatellites are particularly prone to transient dissociation followed by realignment with the repeat units out of phase. This generates extrahelical loops that if uncorrected produce frameshift mutations.

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Mismatch 5′ 3′

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Mismatch recognition 5′ 3′

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Mismatch Repair in Genetic Instability, Fig. 1 The mismatch bound by MutSa recruits MutLa. The ternary complex forms a sliding clamp which can move in both 50 and 30 direction. MutSa, PCNA, and RFC activate a latent MutLa endonuclease, in a mismatch- and ATP-dependent manner. This activity provides incisions that give a 50 terminus that serves as entry sites for the MutSa-activated

EXOI, which removes the mismatch via its 50 !30 exonucleolytic activity. These strand interruptions can be on either side of the mispair and EXOI is able to direct corrections in both directions. The single strand gap is stabilized by RPA. Once the mismatch is removed and the EXOI activity is inhibited by MutLa and RPA, the gap is filled by polymerase d

Thus loss of mismatch repair results in profound ▶ microsatellite instability (MSI) and accumulation of frameshifts in mono- or dinucleotide repeats. Thus inactivation of this pathway in human cell lines and in knockout mice is associated with large increases in spontaneous mutation rates at functional genes (even by two or three order of magnitude) (▶ Mutator phenotype). Molecular analysis of spontaneous mutations occurring in mismatch repair-defective cells shows that both frameshifts and base substitutions are strongly increased in the absence of a functional mismatch repair. It is acknowledged that mismatch repair proteins process some altered or damaged DNA bases and loss of mismatch repair can modify sensitivity to some therapeutic DNA-damaging agents.

Mismatch repair deficiency is invariably associated with high level of resistance (tolerant phenotype) to methylating agents (▶ Alkylating agents), to the base analog 6-thioguanine, and, to a minor extent, to ▶ cisplatin. Although the route of killing is not completely understood, it is clear that it involves mismatch repair processing of lesioncontaining DNA mismatches. Thus hMutSa can indeed bind duplex oligonucleotides containing single O6-methylguanine, 6-methylthioguanine, or 1,2 GpG cisplatin adducts. Following recognition, aberrant processing by the MutLa complex (“futile cycles” of repair attempts) might transform these lesions into strand interruptions, which at the next round of replication leads to the formation of lethal lesions, probably double strand breaks.

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Mismatch recognition MutSa, MutLa

6

O -methylguanine:T mispairs

Processing by MMR (EXOI, pold, RPA) Direct signaling to the apoptotic machinery

DSBs

ATR

Activation of the G2 checkpoint Cell death

Mismatch Repair in Genetic Instability, Fig. 2 Treatment with methylating drugs introduce O6methylguanine into DNA. Error-prone bypass of the methylated base will generate O6-methylguanine:T mispairs, which are recognized by MutSa. Processing by MutSa/ MutLa complexes might remain incomplete and result in “futile cycles” of attempted repair leading to strand interruptions, probably DSBs, which become lethal. Lethal lesions trigger a G2 arrest mediated by the ATR/CHK1dependent pathway. Alternatively, mismatch repair might signal DNA damage directly to the apoptotic machinery

Mismatch repair is also required for triggering a G2 arrest, occurring in the second cycle after treatment, via the ATR/CHK1-dependent pathway. The late timing in checkpoint activation suggests that the signal might be abnormal DNA structures produced by mismatch repair processing of lesion-containing mismatches. As an alternative to the “futile cycles” hypothesis, it has been proposed that mismatch repair proteins might signal directly to the apoptotic machinery (Fig. 2). Finally, mismatch repair also contributes to the control of endogenous levels of DNA oxidation – probably by removing incorporated oxidized bases from the nascent DNA strand. Thus, embryo stem cells, mouse embryo fibroblasts, and several organs of mice defective in the msh2 mismatch repair gene show higher steady-state levels of DNA 8-hydroxyguanine,

than wild-type controls. The attenuation of the mutator phenotype of mismatch repair-defective mammalian cells by efficient exclusion of 8hydroxyguanine at replication implicates oxidized bases in the genetic instability, including MSI, associated with loss of mismatch repair. Clinical Relevance Mutations in genes encoding mismatch repair proteins underlie the predisposition to colorectal tumors in the familial syndrome hereditary nonpolyposis colon cancer (HNPCC) (▶ Lynch syndrome). The inherited germline mutation in at least one of four mismatch repair genes (hMSH2, hMLH1, hMSH6, hPMS2) and the subsequent functional inactivation of the second allele predisposes to colon cancer at an early age. It is proposed that the mutator phenotype, following loss of mismatch repair, facilitates the occurrence of mutations in genes controlling proliferation and/or apoptosis thus leading to an increased cancer risk. The majority of the mutations in the HNPCC families occur in the hMSH2 and hMLH1 genes. A peculiar characteristic of the hMLH1 gene is that this gene is often silenced through methylation of its promoter. Some HNPCC families with unusual tumor spectra have mutations in the hMSH6 gene. At variance with the classical “tumor suppressor” pathway that usually displays gross genome instability (large chromosomal changes and aneuploidy), mismatch repair defective tumors are usually pseudodiploid. It has been proposed that the multiple genetic changes needed for malignancy can be obtained in two alternative and mutually exclusive ways: in a minority of cases instability at gene level because of mismatch repair deficiency and gross chromosomal changes in the majority of cases. Indeed, colorectal cancer occurring in HNPCC patients show a specific pattern of genetic changes compared with sporadic tumors with chromosomal instability. Runs of mononucleotide repeats in coding regions of genes become a preferential target of mutagenic events as a consequence of mismatch repair loss. For example, tumors with MSI preferentially accumulate frameshift mutations in genes involved in the control of proliferation and

Mitochondrial DNA

death. Examples of frameshifts that are likely to confer direct or indirect proliferative advantages and which are particularly prevalent in mismatch repair defective tumors include transforming growth factor b receptor (TGFbRII), insulin-like growth factor type II (IGF-II), b2-microglobulin, Bax, and caspase 5 as well as the mismatch repair genes hMSH6 and hMSH3. MSI is also quite common in some apparently sporadic tumors suggesting that loss of mismatch repair can be a relatively common event in human cancer. In particular, 15% of sporadic colorectal tumors and, to a varying degree, tumors of several other organs (gastrointestinal tract, bladder, endometrium, and ovary) display the mutator phenotype characteristic of mismatch repair defects.

References Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7:335–346 Karran P, Bignami M (1996) Drug-related killings: a case of mistaken identity. Chem Biol 3:875–879 Kunkel TA, Erie DA (2005) DNA mismatch repair. Annu Rev Biochem 74:681–710 Lagerstedt Robinson K et al (2007) Lynch syndrome (hereditary nonpolyposis colorectal cancer) diagnostics. J Natl Cancer Inst 99:291–299 Modrich P (2006) Mechanisms in eukaryotic mismatch repair. J Biol Chem 281:30305–30309

Mitochondrial DNA Mukesh Verma1 and Deepak Kumar2 1 Division of Cancer Control and Population Sciences, National Cancer Institute (NCI), National Institutes of Health (NIH), Rockville, MD, USA 2 Department of Biological and Environmental Sciences, University of the District of Columbia, Washington, DC, USA

Definition Mitochondria are double membrane bound organelles, 1–2 mm in length and 0.5–1 mm in diameter,

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and provide energy to the cell. Human cells contain two types of DNA: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Before the term “mitochondria” was used, these oval subcellular granules, similar in size and shape to bacteria, were called “bioblasts” and were considered the basic unit of cellular activity.

Characteristics Mitochondria now are considered as centers of energy metabolism and produce up to 80% of the energy needs of a cell. The inner membrane forms a series of folds called cristae and harbors enzymes involved in oxidative phosphorylation. Oxidative phosphorylation enzymes are located in the inner membrane, and depending on the metabolic activity of a cell, the size of the inner membrane increases or decreases. Human cells contain two types of DNA: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Before the term “mitochondria” was used, these oval subcellular granules, similar in size and shape to bacteria, were called “bioblasts” and were considered the basic unit of cellular activity. Mitochondria now are considered as centers of energy metabolism and produce up to 80% of the energy needs of a cell. The inner membrane forms a series of folds called cristae and harbors enzymes involved in oxidative phosphorylation. Oxidative phosphorylation enzymes are located in the inner membrane and depending on the metabolic activity of a cell, the size of the inner membrane increases or decreases. A typical mammalian cell contains about 1,000–10,000 copies of mitochondrial DNA. The mitochondrial genome is 16.5 kb which is closed circular double-helical molecule encoding 2 rRNAs, 22 tRNAs, and 13 polypeptides and does not contain introns and histones. mtDNA represents about 1% of the cellular DNA. mtDNA is double stranded except for the D-loop which is triple stranded (contains extra 7S DNA). Promoter of mtDNA is located in the D-loop, which is the only noncoding region of mtDNA. The D-loop contains cis elements involved in mtDNA replication and transcription. All 13 peptides of

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mitochondria are located in a mitochondrial respiratory chain complex. Mitochondria are an important biological source and target of ▶ reactive oxygen species (ROS) and free radicals and sensitive to environmental mutagens. Normal assembly and operation of the respiratory chain requires an intact and functional mitochondrial genome. Depending on the energy demand of a cell, the number of mitochondria per cell varies. Under physiologic conditions, as many as 2% of electrons leak from the mitochondrial electron transport chain and reduce oxygen into superoxide anion, triggering the formation of free radicals that indescribably damage biological molecules. Damage due to ROS and radiation is severe for mtDNA, and unlike nDNA, mtDNA have limited repair capability. The accumulation of mutations in mtDNA is about tenfold greater than in the nDNA. A continuing cycle of worsening mitochondrial dysfunction with increasing ROS production might be expected from such damage to mtDNA. Mutations in mtDNA can vary widely among tissues in an individual, and mutation load may change over time. Tissues are differentially sensitive to levels of mtDNA mutations. Genetic and/or metabolic alterations in mitochondria may lead to different diseases. The mitochondrial genome has drawn increasing attention in ▶ cancer research due to its unique genetics, such as maternal inheritance, and lack of recombination as well as functional importance in oxidative phosphorylation. mtDNA is a critical target for cellular reactive oxygen species, and the level of oxidative damage is more severe and persistent in mtDNA than in nDNA. MITOMAP (http:// www.MITOMAP.org) is a comprehensive database of human mtDNA variation and its relation with human diseases. In humans, 99.99% of mtDNA is inherited from the mother because sperm mtDNA is actively degraded. Sperm carries its mitochondria around a portion of its tail and has only about 100 mitochondria compared to 100,000 in the oocyte. As the cells develop, more and more of the mtDNA from males is diluted out and less than 0.01% of the mtDNA is paternal. Thus, mutations of mtDNA can be passed from the mother to child.

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Its implication is in cloning of mammals using somatic cells. For example, when the sheep “Dolly” was cloned, the nDNA was from the donor cells, but the mtDNA was from the host cells. Abnormality in mtDNA may lead to different diseases such as optic neuropathy (degeneration of the optic nerve accompanied by increasing blindness), myopathy, exercise intolerance, diabetes, deafness, MELAS, Pearson’s syndrome, ataxia, and cancer. As an example, cancer and mtDNA has been described in details. mtDNA has been used in epidemiology. Admixture populations have been identified based on mtDNA characteristics. To understand the utility of mtDNA in cancer epidemiology, one approach is to evaluate in patients with cancer and matching controls for somatic mutations in mitochondrial DNA. Another approach is to look for disease-associated haplotypes. The inheritance pattern of mitochondria in patients with cancer has been studied by haplotype analysis. Polymerase chain reaction (PCR) of key polymorphic sites in the mitochondrial genome was performed in samples from cancer patients and controls to determine presence of an association between mitochondrial genotype and cancer. Such analysis has been accomplished in a few studies of ▶ prostate and renal cancer. For example, haplogroup U, one of the nine major haplotype groups, was associated with an odds ratio of 1.95 in a prostate cancer case control study and with an odds ratio of 2.65 in a renal cancer study. There are very large differences across racial and ethnic groups in the distributions of the major haplotypes. Haplotyping is performed by restriction analysis, whereas for mutation analysis whole mitochondrial genome is sequenced. Microdissected samples have been successfully used to identify mtDNA mutations from clinical samples. Thus, a pure population of cells could be isolated from heterogeneous cells of the surgically isolated samples. To understand the etiology of the disease and to do mitochondrial genotyping, a pure population of cells is needed. A variety of clinical samples have been utilized for mtDNA mutation detection. For example, nipple aspirate and paraffin-embedded specimens for

Mitochondrial DNA

breast cancer, urine for bladder cancer, buccal cells for head and neck cancer, cerebrospinal fluid (CSF) for medulloblastoma, and sputum for ▶ lung cancer have been used. High-throughput technology has been developed for somatic mutation detection. Since each copy may be independently replicated, mutations can accumulate in various proportions of the genomes. The presence of a mutation in 100% of the genomes is termed homoplasmy, while heteroplasmy is a mixture of mutated and wild-type sequences for a given locus. About 50 years ago, Nobel-Laureate Otto ▶ Warburg reported the fundamental difference between normal and cancerous cells to be the ratio of glycolysis to respiration. Now we see a linking impaired mitochondrial function as well as impaired respiration to growth, division, and expansion of cancer cells. Recent work has confirmed that this is, indeed, a promising approach in the treatment of cancer. Cancer cells accumulate defects in the mitochondrial genome, leading to deficient mitochondrial respiration and ATP generation. In some cases, mitochondrial germ line mutations have been shown to provide predisposition to cancer development. Mostly, such mutations are acquired during or after oncogenesis. Tumors rely heavily on glycolysis to meet their metabolic demands. Compared to normal cells, cancer cells exhibit metabolic imbalances and enhanced resistance to mitochondrial ▶ apoptosis. Mitochondrial mutations have been reported in preneoplastic lesions, suggesting that mutations occur early in multistep tumor progression and hence may be used as a tool for ▶ early detection of cancer in clinical samples. Alterations in mitochondrial structure and function might provide clinical information either for early detection of cancer or as unique molecular sites against which chemotherapeutic agents might be targeted. The field of mitochondria has seen a surge of interest among cancer researchers as alterations in mtDNA have been reported in various neoplasms. Population-based studies involving environmental and occupational exposure, infectious agents, personal susceptibility factors, and acquired genetic factors may identify high-risk populations

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that are likely to develop cancer; additionally, such studies are very informative and significant in designing future community-based health initiatives. mtDNA biomarkers can be used to follow disease prevalence by determining their level in cohort studies with the potential for identifying high-risk populations.

Cross-References ▶ Apoptosis ▶ Cancer ▶ Lung Cancer ▶ Prostate Cancer ▶ Prostate Cancer Clinical Oncology ▶ Reactive Oxygen Species ▶ Warburg Effect

References Baysal BE (2006) Role of mitochondrial mutations in cancer. Endocr Pathol 17:203–212 Chatterjee A, Mambo E, Sidransky D (2006) Mitochondrial DNA mutations in human cancer. Oncogene 25:4663–4674 Jakupciak JP, Dakubo GD, Maragh S et al (2006) Analysis of potential cancer biomarkers in mitochondrial DNA. Curr Opin Mol Ther 8:500–506 Maitra A, Cohen Y, Gillespie SE et al (2004) The human mitochip: a high-throughput sequencing microarray for mitochondrial mutation detection. Genome Res 14:812–819 Modica-Napolitano JS, Kulawiec M, Singh KK (2007) Mitochondria and human cancer. Curr Mol Med 7:121–131

See Also (2012) Admixture Population. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 84. doi:10.1007/978-3-642-16483-5_115 (2012) Epidemiologic Studies. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1269. doi:10.1007/978-3-642-16483-5_1929 (2012) Heteroplasmy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1689. doi:10.1007/978-3-642-16483-5_2700 (2012) Histones. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1706. doi:10.1007/978-3-642-16483-5_2762 (2012) Homoplasmy. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1729. doi:10.1007/978-3-642-16483-5_2805

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(2012) Mitochondrium. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2336. doi:10.1007/978-3-642-16483-5_3768 (2012) Oxidative Phosphorylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2730. doi:10.1007/978-3-642-164835_4308 (2012) Recombination. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3208. doi:10.1007/978-3-642-16483-5_4996 (2012) ROS. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3321. doi:10.1007/978-3-642-16483-5_5125

and caspase-independent proteins such as apoptosis-inducing factor, AIF, and EndoG. The major consequence of these protein movements is the coordination of the final cellular degradation phase of apoptosis. Concomitantly, MMP provokes a mitochondrial failure, which manifests by an arrest of oxidative phosphorylation and ATP synthesis, an increased ROS level, and dissipation of the inner membrane potential (DCm). Therefore, MMP constitutes a point of no return of the activation cascade of apoptosis.

Characteristics

Mitochondrial Membrane Permeabilization in Apoptosis Catherine Brenner INSERM UMR-S 769, Labex LERMIT, Ch^atenay-Malabry, University of Paris Sud, Paris, France

Synonym MMP

Definition During ▶ apoptosis, many signals can converge to the mitochondrion to trigger the so-called mitochondrial membrane permeabilization (MMP), a rate-limiting step in the execution of the death process. These signals are mainly endogenous proteins, which translocate from an intracellular compartment (e.g., nucleus, cytosol, lysosomes, etc.) to the mitochondrial outer membrane (OM). Accumulation of modified lipids (e.g., oxidized cardiolipin, ceramide, etc.) and ions (e.g., Ca2+) by the mitochondrion can also influence MMP. Moreover, the intracellular milieu, such as pH, ▶ reactive oxygen species (ROS), and ATP levels, can contribute to define a permissive environment for MMP execution. Once initiated, MMP leads to the release of intermembrane space factors into the cytosol, caspase-dependent proteins, such as cytochrome c, Smac/DIABLO, and pro-caspases,

Mitochondria are intracellular organelles involved in the cell energy metabolism and also in the control of the intrinsic pathway of apoptosis. They are surrounded by two different membranes that differ in terms of composition, surface, permeability, and function. The OM is permeable to solutes of MM  6 kDa due to the presence of channels, such as the voltage-dependent anion channel (VDAC), which belongs to the porin subfamily. However, with an estimated pore diameter about 2.6–3 nm, VDAC would not allow the passage of a folded protein like cytochrome c. In contrast, the inner membrane (IM) is almost totally impermeable. Specialized membrane proteins, namely, the mitochondrial carriers, carry out the transport of ions and solutes across this membrane. Most mitochondrial proteins, which have been implicated in apoptosis, exhibit dual functions, a vital metabolic function and a lethal pro-apoptotic function. This applies to various channels (VDAC, adenine nucleotide translocase (ANT), Bax, tBid, Bak), receptors (e.g., TOM22), chaperones (cyclophilin D, CypD), as well as oxidoreductases (AIF). Mechanisms of MMP Mechanisms mediating MMP may be multiple depending on the cell type and the death stimuli. They can affect (only) the OM, or both mitochondrial membranes (OM+IM). Among numerous hypothetic models, experimental evidence support models involving members of the conserved ▶ BCL-2 family, which can be divided into

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OM+IM model

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Mitochondrial Membrane Permeabilization in Apoptosis, Fig. 1 Two models of mitochondrial membrane permeabilisation are depicted. A model, which

involves outer membrane (OM) permeabilisation and a model, which involves OM permeabilisation and also inner membrane permeabilisation (IM+OM)

pro-apoptotic members (Bax, Bak, Bid, Bik, Bnip3, etc.) and anti-apoptotic members (Bcl-2, Bcl-XL, Mcl-1, etc.), and members of the permeability transition pore complex (VDAC, ANT, CypD) and lipids. At the onset of MMP, cytosolic and nuclear proteins such as Bax, truncated Bid (tBid), p53, and Nurr77 can undergo conformational changes and/or posttranslational modifications favoring their translocation to the mitochondrial membranes. Subsequently, proteinprotein interaction changes operate and novel homo- or hetero-oligomers form to promote the release of pro-apoptotic factors into the cytosol. In OM models, Bax would oligomerize with Bak and/or VDAC to form megachannels. In OM +IM models, the permeability transition pore would undergo long-lasting openings of high conductance (1.5nS), allowing a massive entry of water and solutes (MM1.0, consistent with a polygenic model of inherited predisposition to cancer. Such a model predicts that genetic susceptibility to tumorigenesis may be conferred by multiple genes with small effects and a powerful approach to identify the genetic loci involved may lie in linkage/association analyses of the entire genome in large samples. Rodent Models In mice, genome-wide analyses require only a few hundred genetic markers due to the genetic homogeneity of inbred strains, the limited number of genetic recombinations in crosses, and the consequent tight linkage of genetic markers over wide chromosomal regions. The availability of a large number of genetic markers easy to genotype (Microsatellites) allowed the first genome-wide linkage studies in mouse models and leads to the mapping of hepatocellular cancer susceptibility loci (Hcs); the pulmonary adenoma susceptibility 1 locus (Pas1); the Mom1 locus as a modifier of the germ-line Apc gene mutation (▶ APC Gene in Familial Adenomatous Polyposis), which induces intestinal tumorigenesis; and loci affecting plasmacytomagenesis (▶ Plasmacytomas). The hepatocellular cancer model provided the first formal demonstration of the polygenic nature of inherited cancer predisposition in mice. Since then, cancer modifier loci affecting almost all types of tumors have been mapped in the mouse. Loci affecting different types of rat carcinogenesis have also been mapped; among them, loci affecting susceptibility to hepatocarcinogenesis have been characterized in more detail and showed to control the multiplicity and size of neoplastic lesions.

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Therefore, in liver, as well as in other models, cancer modifier loci may affect either single or multiple stages of carcinogenesis. Humans In humans, some authors proposed a polygenic model of inherited predisposition to cancer, whereas others propose models based on rare dominant alleles or additive gene effects or on common alleles of low penetrance. Although all these models agree in part with epidemiological evidence, no combination of genetic polymorphisms that convincingly defines an additive or polygenic inheritance of predisposition to sporadic cancer has yet been described in humans. Despite many reports indicating that a higher or lower risk of cancer is associated with metabolic polymorphisms, several meta-analyses showed that the relative risks associated with most of the metabolic polymorphisms are low. The exceptions include arylamine N-acetyltransferase 2 (NAT2) polymorphism [▶ Arylamine N-Acetyltransferases (NAT)] and bladder cancer risk, where mechanistic and epidemiological evidence is strong. The discovery of millions of single-nucleotide polymorphisms (SNPs) in humans and in the genome of other mammalian species now makes feasible large-scale genome-wide association studies, also thanks to the introduction of SNP array systems that enable screening of thousands of SNPs. Because of the genetic heterogeneity of the human population, complete analysis of the human genome by association studies might require a huge number of genetic markers and large sample collections. However, the presence of haplotype blocks in the human genome suggests that even a less dense marker coverage might be sufficient for a whole-genome analysis. Although the characterization of these haplotype blocks awaits completion, population-based association studies are complicated by complex ▶ linkage disequilibrium (LD) patterns, possible population admixture in the selection of cases or controls, differences in environmental factors, and other factors. However, as these studies test hundreds of thousands of polymorphisms in cases and controls, the need for robust replication in one or, preferably, several independent studies is

Modifier Loci

paramount to distinguish the true-positive results from the large number of expected false positives. Genome-wide association studies and familybased linkage studies mapped several modifier loci affecting different types of tumors, including breast, colon, lung, or prostate cancer. Mechanisms Few studies to date have addressed the site of action of cancer modifier genes, i.e., whether cancer modifier genes act directly within the tumor cells or instead in a cell-autonomous way, to determine the host response to the tumor. This issue likely bears on the mechanism of action of cancer modifier genes and on the targeting of these gene products. Experiments using chimeric mice generated by aggregation of embryos derived from genetically susceptible and resistant strains indicate that at least for hepatocarcinogenesis, intestinal tumorigenesis, and plasmacytomagenesis, most of the tumors in these mice derive from the susceptible strain. Thus, the cancer modifier loci in these models appear to act in the target cells, with no apparent systemic effects. In humans, most inherited major germ-line defects appear to confer predisposition to one major tumor type, with several others exhibiting a minor increased incidence in families. For example, carriers of the BRCA1 mutations (▶ BRCA1/BRCA2 germ-line mutations and breast cancer risk) are at high risk of breast and ovarian cancer and may have an increased risk of colon, pancreas, and stomach cancers. BRCA2 mutation carriers show a high risk of breast cancer and an increased risk of pancreatic cancer. Compared with the general population, carriers of the mutations causing hereditary nonpolyposis colorectal cancer (HNPCC) syndrome (▶ Lynch Syndrome) show, in addition to colorectal cancer, an increased incidence of endometrial, ovarian, gastric, biliary tract, and kidney cancers. On the other hand, ▶ Li-Fraumeni syndrome is characterized by a wide spectrum of neoplasms in children and young adults. Thus, both specificity and pleiotropy, i.e., single-gene allelism that influences two or more types of tumorigenesis, in genetic susceptibility to cancer in humans have been described.

Modular Transporters

Perspectives Genetic loci affecting risk of several types of tumors have been identified in mouse models, but the relationships with the risk of corresponding human cancer have not yet been established. The major locus affecting inherited susceptibility to lung cancer in mouse (Pas1) seems either lacking its human counterpart or playing a much weaker effect on lung cancer phenotype, whereas a human homology has been found for the rat Mcs5a locus affecting breast cancer risk. The difficulties in the identification of human homologs of mouse cancer modifier genes are obvious considering that some mouse alleles may not be carried in humans and that the human population is much more genetically complex than the relatively few mouse inbred strains used so far for mapping cancer modifier loci. Overall, it seems highly unlikely that the enormous genetic variability underlying susceptibility and resistance to tumorigenesis in mice evolved only in rodents and not in all mammals, including humans. In fact, quantitative trait loci affecting several noncancer phenotypes have been mapped in cattle, pigs, and humans, and it seems reasonable to expect that cancer modifier QTLs are present in humans. Even in cases where the human homologous gene does not display allelism and cancer modifier effects, identification of cancer modifier genes in any given species would provide a step toward understanding the biochemical mechanisms of inherited resistance/susceptibility to cancer. Eventually, it may become possible to devise new chemoprevention and therapeutic strategies for cancer based on the biochemical effects of such genes. Also, new diagnostic markers might be devised to identify groups of individuals at higher risk of sporadic cancer as compared with the general population. This is now possible for members of families with monogenic cancer syndromes, which represent a minority of cancer cases. Identification of individuals at high risk of cancer raises the possibility of chemoprevention. For example, FAP carriers (APC gene in familial adenomatous polyposis) benefit from long-term use of nonsteroidal ▶ anti-inflammatory drugs.

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In conclusion, the identification of cancer modifier genes may allow understanding of the genetic mechanisms responsible for the variability in the individual risk to develop a cancer, and it may also provide new opportunities for diagnosis and cure.

References Botstein D, Risch N (2003) Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. Nat Genet 33(Suppl):228–237 Dragani TA (2003) 10 Years of mouse cancer modifier loci: human relevance. Cancer Res 63:3011–3018 Dragani TA, Canzian F, Pierotti MA (1996) A polygenic model of inherited predisposition to cancer. FASEB J 10:865–870 Feo F, De Miglio MR, Simile MM et al (2006) Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition. Biochim Biophys Acta 1765:126–147 Ponder BAJ (1990) Inherited predisposition to cancer. Trends Genet 6:213–218

Modular Nanotransporters ▶ Modular Transporters

Modular Recombinant Transporters ▶ Modular Transporters

Modular Transporters Alexander S. Sobolev Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

Synonyms Modular recombinant transporters; Modular nanotransporters

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Definition Modular transporters are engineered polypeptides consisting of several interchangeable parts or modules designed for delivery of anticancer drugs to the target cancer cell and its specific subcellular compartment. Modular transporters can also be considered as nanomedical drug vehicles (▶ nanotechnology), which recognize the cancer cells of choice and, once in those cells, are transported to the most sensitive compartment of the cell (e.g., nucleus). In order to reach the desired compartment of the cancer cell, the modular transporters are first passively delivered to the surface of the cell in the bloodstream. Once within the cell, depending upon the nature of the polypeptide modules, they are transported to a particular subcellular compartment utilizing the cell’s intrinsic transport machinery.

Characteristics Objectives To minimize side effects, many antitumor agents need to be delivered (▶ drug delivery) not only to the target cancer cell but also into a specific subcellular compartment, usually into the most sensitive/vulnerable site of the cancer cell. Examples of such antitumor agents are (i) foreign DNA for cancer gene therapy, (ii) photosensitizers for ▶ photodynamic therapy, or (iii) radionuclides emitting alpha-particles for endoradiotherapy (▶ radioimmunotherapy). All of the above should be delivered into the nuclei where they can perform their specific function. On the other hand, (iv) toxins, most of which are active in the cytosol, require a different modular transport strategy to retain in the cytoplasm. Principles This goal can be achieved with the use of modular transporters with preset properties, which would ensure recognition of the desired target cell and subsequent directed transport to the subcellular compartment of choice (Slastnikova et al. 2012, 2015). The necessity of different modules is

Modular Transporters

determined by the following considerations. First, cell-type specificity together with internalization into the target cell can be achieved if the engineered transporter possesses a ligand module, which has high binding affinity to the ▶ receptor overexpressed on the target cancer cell but not on noncancer cells. This highly specific ligandreceptor binding will ensure recognition of the target cell as well as a subsequent receptormediated ▶ endocytosis. The internalized transporter will then be delivered to endocytotic vesicles, or endosomes, localized in the cytoplasm (▶ endosomal compartments). Second, because the internalized transporter moves along the endocytotic pathway, it is necessary to provide the transporter with an endosomolytic module enabling the transporter’s escape from the endosome. Third, a specific subcellular delivery can be achieved if the transporter has a specific localization amino-acid sequence, e.g., a nuclear localization sequence to target the cell nucleus. Finally, the modules as well as the antitumor agent should be integrated into one moiety; this goal can be achieved by inclusion of the fourth module, a carrier module. Therefore, modular transporters for nuclear drug delivery should include the following parts: (i) an internalizable ligand module providing for target cell recognition and subsequent receptor-mediated endocytosis, (ii) an endosomolytic module ensuring escape of the transporter from endosomes, (iii) a module containing a nuclear localization sequence (a sequence of amino acids that is recognized by importins needed for the active translocation into the nucleus), and (iv) a carrier module for attachment of an antitumor agent (Fig. 1) (Slastnikova et al. 2012, 2015). Features Fundamental to the success of this strategy is that the modules are functional within the transporter, i.e., they retain their activities within the chimeric molecule. Depending on the type of target cancer cells, the ligand module can be replaced; the module with subcellular localization signal can be replaced or omitted (e.g., omission of the nuclear localizing signal will leave the transporter in the cytoplasm of the target cell).

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Modules:

receptor (▶ cytokine receptor as target for immunotherapy and immunotoxin therapy), respectively. Antitumor agents carried by these modular transporters acquired a significantly higher efficacy aside from cell specificity. In cases when they are delivered into the most sensitive sites of the target cancer cells, the agents become 10–3,000 times more effective (Slastnikova et al. 2012, 2015; Urieto et al. 2004).

Endosomolytic carrier NLS ligand

Stages: Receptor Binding Out In Endocytosis

α / β importins

H+ Endosomal exit Endosome

Nuclear pore

Nuclear import

Nucleus

Modular Transporters, Fig. 1 Scheme of a modular transporter and stages of its transportation within the target cell. NLS, nuclear localization sequence. Arrows indicate successive steps of the transporter binding to overexpressed internalizable receptors on the target cancer cell, internalization, endosomal localization within an acidifying milieu, escape from endosomes, binding to importins, and transport through the nuclear pores into the cell nucleus (From Rosenkranz et al. (2003), with permission)

Several types of modular transporters have been created that can deliver photosensitizers into the nuclei of ▶ melanoma cells, photosensitizers and radionuclides into the nuclei of glioma and epidermoid carcinoma cells, and toxins into the cytoplasm of ▶ acute myeloid leukemia cells. In all these cases, cell specificity was achieved by inclusion of a specific ligand module into the transporter that bound to a corresponding internalizable receptor overexpressed on the surface of the target cancer cell: melanocortin-1 receptor, epidermal growth factor receptor (▶ epidermal growth factor receptor inhibitors) (▶ receptors), and interleukin-3

Cross-References ▶ Acute Myeloid Leukemia ▶ Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶ Drug Delivery Systems for Cancer Treatment ▶ Endocytosis ▶ Endosomal Compartments ▶ Epidermal Growth Factor Inhibitors ▶ Melanoma ▶ Nanotechnology ▶ Photodynamic Therapy ▶ Radioimmunotherapy ▶ Receptor Tyrosine Kinases ▶ Receptors

References Gilyazova DG, Rosenkranz AA, Gulak PV et al (2006) Targeting cancer cells by novel engineered modular transporters. Cancer Res 66:10534–10540 Slastnikova TA, Rosenkranz AA, Gulak PV et al (2012) Modular nanotransporters: a multipurpose in vivo working platform for targeted drug delivery. Int J Nanomedicine 7:467–482 Slastnikova TA, Rosenkranz AA, Zalutsky MR, Sobolev AS (2015) Modular nanotransporters for targeted intracellular delivery of drugs: folate receptors as potential targets. Curr Pharm Design 21:1227–1238 Urieto JO, Liu TF, Black JH et al (2004) Expression and purification of the recombinant diphtheria fusion toxin DTIL3 for phase I clinical trials. Protein Expr Purif 33:123–133

See Also (2012) Alpha-Particles. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 147. doi:10.1007/978-3-642-16483-5_208 (2012) Importins. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1836. doi:10.1007/978-3-642-16483-5_3017

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2894 (2012) Ligands. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2040. doi:10.1007/978-3-642-16483-5_3352 (2012) Module. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2354. doi:10.1007/978-3-642-16483-5_3806 (2012) Photosensitizer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 28812882. doi:10.1007/978-3-642-16483-5_4559 (2012) Polypeptide. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2955. doi:10.1007/978-3-642-16483-5_4676 (2012) Radionuclide. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3154. doi:10.1007/978-3-642-16483-5_4921 (2012) Subcellular Compartments. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3552. doi:10.1007/978-3-64216483-5_5548 (2012) Tyrosine Kinase Receptors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3824. doi:10.1007/978-3-642-16483-5_6081

Modulation of Colon Carcinogenesis ▶ Colorectal Cancer Nutritional Carcinogenesis

Mole ▶ Melanocytic Tumors

Molecular Chaperones Marissa V. Powers1 and Paul Workman2 1 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton, London, UK 2 Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK

Definition Proteins that transiently interact with nascent polypeptide substrates to protect them from

Modulation of Colon Carcinogenesis

misfolding and aggregation. They also have an important role in helping to achieve the native conformation of the newly synthesized protein without forming part of the final folded product. The molecular chaperone ▶ HSP90 is responsible for the stability and activity of a range of oncogenic client proteins and is a target for cancer drugs.

Characteristics Proteins are mediators of a vast array of biological processes, and their activity is dependent on obtaining their correct three-dimensional conformation. It has been widely accepted from in vitro folding experiments that the formation of the native state is a spontaneous process that is dictated by the amino acid sequence of the protein. However, protein folding in vivo is complicated by the crowded cellular environment which naturally favors protein misfolding and aggregation. Under certain conditions, aggregation may lead to the production of amyloid fibrillar aggregates that are associated with pathological conditions including Alzheimer or Huntington disease. To prevent this, nascent or partially folded proteins interact with various chaperone proteins that encourage the formation of the correct threedimensional structure. Generally, chaperones interact with nonnative proteins by recognizing hydrophobic residues and regions of the polypeptide backbone that are not normally exposed to solvent when the correct conformation is achieved. Chaperones may promote de novo protein folding by binding directly to the non-nascent protein to shield regions which may be involved in intermolecular aggregation and intramolecular misfolding. In addition to promoting productive de novo protein folding and preventing nonspecific aggregation under normal conditions, molecular chaperones also have an important role in the cellular response to stress. This is because conditions such as elevated temperatures, ionizing radiation, heavy metals, and oxidative stress may cause some proteins in their native state to unfold. Many molecular chaperones demonstrate

Molecular Chaperones

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NAC

NAC HSP40

HSP70

GimC

TRiC

HSP90 Proteasome

HOP

Ub +ATP +ATP +co-chaperones

Protein degradation

+ATP

Native polypeptide

Molecular Chaperones, Fig. 1 Chaperone-mediated protein quality control in the eukaryotic cytosol. Nascent polypeptides leave the ribosome via the NAC. The majority of small polypeptides require no further assistance to achieve the correct conformation following release from the ribosome. For longer polypeptides, the native conformation may be achieved via an interaction with HSP70 and its co-chaperone HSP40. The chaperone function of HSP70 may be extended via its interaction with

the HSP90 molecular chaperone via the adaptor protein p60/HOP. According to the compliment of co-chaperones associated with HSP90, its function can be altered from assisting protein folding to promoting protein degradation via the ubiquitin (Ub)-proteasome pathway. A limited number of proteins have been shown to be reliant on transfer to the prefoldin/TRiC pathway; these include the abundant proteins actin and tubulin

increased expression under such conditions and are referred to as heat shock proteins (HSPs). In the mammalian cytosol, a number of chaperones are involved in native protein folding and the ▶ stress response (Fig. 1). Chaperone involvement begins as newly synthesized proteins exit the ribosome. This is particularly important because the tendency to aggregate at this stage is increased due to exposure of nonnative features and the close proximity of nascent polypeptides of the same type at the polyribosome complex. Nascent polypeptide chains emerge from the peptide exit tunnel of the ribosome and interact with the nascent-chain-associated complex (NAC). It has been hypothesized that the NAC exerts a passive chaperone function by simply binding to and protecting the exposed hydrophobic regions of the newly synthesized polypeptides. This is because it lacks an ATPase domain which is characteristic of several molecular chaperones (see below). After release from the ribosome, small,

single-domain proteins may not require additional chaperones. Longer polypeptide chains may require additional help by association with the HSP70 chaperones which can act in higher eukaryotes both co- and posttranslationally. The HSP70 family consists of both constitutively expressed (e.g., HSC70) and stress-inducible forms (e.g., HSP72) which promote the correct folding of nascent proteins through ATP-dependent cycles of binding and release. HSP70 stably interacts with substrates in its ADP-bound form which is encouraged by its co-chaperone HSP40, a J-domain protein that significantly stimulates the ATPase activity of HSP70 to prolong its association with substrate. The substrate binding sites for HSP70 are typically extended hydrophobic stretches of seven residues which statistically occur every 40 residues. In eukaryotes, cytosolic HSP70 can further promote the folding of a particular group of

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cellular proteins by interacting with the ATP-dependent HSP90 chaperone family. Proteins that are reliant on HSP90 for their correct folding, stability, and functional regulation include many kinases and other ▶ signal transduction proteins such as steroid hormone receptors. Substrate transfer from HSP70 to HSP90 is mediated by the adapter protein p60/HOP which binds to the carboxy-terminus of the two chaperones via its tetratricopeptide repeat (TPR) domains. Similar to HSP70, HSP90 chaperone function is reliant on the orchestrated interaction of a number of co-chaperones that collectively control substrate binding, ATPase activity, and substrate release. In addition, co-chaperone interaction may alter HSP90 function from polypeptide folding to protein sorting either into intracellular organelles or to the ubiquitin-proteasome system for degradation. For example, in mammals the HSP90 chaperone system can interact with the E3 ubiquitin ligases Cullin-RING ligase Cullin-5 (CUL5) and CHIP (carboxyl-terminus of HSP70-interacting protein) to direct proteins for proteasomal degradation. CHIP interacts with HSP90 via its TPR domain, promoting the ▶ ubiquitination of nonnative or misfolded proteins and their degradation by the proteasome. In addition, HSP70 and CHIP can associate with BAG-1 which contains an ubiquitin-like domain that can interact with the proteasome. Therefore, the chaperone function of the HSP70-HSP90 system can be modified by co-chaperone interaction to control protein quality in the cell by mediating a balance between protein folding versus proteasomal degradation. HSP70 can recruit the assistance of chaperonins (HSP60) for the co-translational folding of multidomain proteins that are slow folding or sensitive to aggregation. Chaperonins are a conserved class of large double-ring complexes of 800–1000 kDa with a central chamber that provides shelter to protect the nascent polypeptide from the crowded, aggregate promoting cytosol. Chaperonins are exemplified in the eukaryotic cytosol by the group II chaperonin, TRiC (tailless complex polypeptide-1 ring complex). As with HSP70 and HSP90, TRiC mediates folding by undergoing a conformational cycle which is driven by ATP hydrolysis. TRiC interacts with a

Molecular Chaperones

wide range of substrates including several proteins rich in b-propeller and WD40-repeat domains, but the most abundant are the cytoskeletal proteins actin and tubulin which are entirely dependent on TRiC for folding. Substrate recognition and transfer to the TRiC complex can also be mediated by the cytosolic ATP-independent chaperone prefoldin (GimC). HSP90 as an Anticancer Drug Target At first glance molecular chaperones may not appear as obvious candidates for selection as anticancer drug targets because, unlike many oncogenic targets, they are not subject to mutation or amplification. However, the expression of HSP70 and HSP90 is frequently increased in a number of different human ▶ cancers. In addition, the client proteins which are reliant on HSP90 function for their stability and function include a wide range of oncoproteins including ERRB2, BCR-ABL, C-RAF (▶ Raf Kinase), ALK, mutant EGFR, mutant B-RAF, AKT/PKB, CDK4, mutant p53, PLK-1, HIF-1a, ▶ estrogen receptor, and ▶ androgen receptor (for an up-to-date compilation, see www.picard.ch/downloads/Hsp90 interactors.pdf). Inhibition of HSP90 function has been shown to cause the depletion of client proteins via the ubiquitin-proteasome pathway, suggesting that modulation of HSP90 function may offer a novel mechanism to simultaneously inhibit multiple oncogenic pathways, all of which may contribute to the hallmark traits of malignancy. These combinatorial effects will be valuable not only in treating cancers which are driven by multiple molecular abnormalities but should also reduce the opportunity for resistance developing. The increased expression of HSP90 in malignant cells may be a reflection of the stress imposed on cancer cells by the effects of deregulated ▶ oncogenes and ▶ tumor suppressor genes. These molecular stresses may be coupled with the microenvironmental stress of solid tumors caused by hypoxia, acidosis, and nutrient deprivation. Collectively these factors may lead to greater dependence on HSP90 and other chaperones in cancer versus normal cells, offering the prospect of therapeutic selectivity.

Molecular Chaperones

As described above, HSP90 (along with many of the other molecular chaperones) are ATPase enzymes. The natural products radicicol and geldanamycin bind specifically to the intrinsic N-terminal ATPase domain of HSP90 to inhibit its function and cause client protein degradation and growth arrest. Since the original discovery of these two natural products, there has been an explosion of interest in the development of novel HSP90 inhibitors. The proof of concept for the clinical use of HSP90 inhibitors was provided by the completion of over 30 clinical trials (phase I/II) on the first-in-class HSP90 inhibitor 17-AAG (tanespimycin), an analogue of the natural product geldanamycin. Biomarker evidence demonstrated client protein depletion and increased HSP72 expression in the tumor tissue and peripheral blood lymphocytes of treated patients at doses that were well tolerated. Pharmacological limitations of 17-AAG have halted its clinical progression but have provided the foundation for the subsequent development of rationally designed synthetic HSP90 inhibitors. Among the most advanced examples is the isoxazole resorcinol AUY922 (luminespib) that has shown promising activity during phase II clinical trials. The exciting observations made in vitro and in vivo with HSP90 inhibitors highlight the possibility of modulating chaperone function, and research efforts are expanding to identify novel pharmacological modulators of other chaperone families in particular HSP70. This may have benefits not only in the treatment of cancer but also in the management of other protein folding diseases.

Glossary ATPase Enzymes that use ATP hydrolysis to yield energy to drive an energetically unfavorable reaction. Proteasome A protein degradation complex which can digest a variety of proteins into short polypeptides and amino acids. Ubiquitination A posttranslational modification involving the attachment of ubiquitin molecules to specific lysine residues in target proteins. Often this modification acts as a tag for

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the rapid cellular degradation of the protein by the proteasome. WD40 repeat A poorly conserved motif consisting of a repeat sequence containing 40–60 amino acids, which usually contains a Trp-Asp (WD). Several consecutive repeats fold into a domain structure known as a b-propeller in which each section of the structure is a four-stranded b-sheet.

Cross-References ▶ Androgen Receptor ▶ Cancer ▶ Estrogen Receptor ▶ Hsp90 ▶ Hypoxia-Inducible Factor-1 ▶ Oncogene ▶ Proteasome ▶ Raf Kinase ▶ Signal Transduction ▶ Stress Response ▶ Tumor Suppressor Genes ▶ Ubiquitination

References Butler LM, Ferraldeschi R, Armstrong H et al (2015) Maximizing the therapeutic potential of HSP90 inhibitors. Mol Can Res; 13(11):1445–51 Kim YE, Hipp MS, Bracher A et al (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82:323–355 Lopez T, Dalton K, Frydman J (2015) The mechanism and function of group II chaperonins. J Mol Biol.11; 427(18): 2919–30 Neckers L, Workman P (2012) HSP90 molecular chaperone inhibitors: are we there yet? Clin Cancer Res 18(1):64–76 Powers MV, Jones K, Barillari C et al (2010) Targeting HSP70: the second potentially druggable heat shock protein and molecular chaperone? Cell Cycle 9(8):1542–1550

See Also (2012) ATPase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 302. doi:10.1007/978-3-642-16483-5_442 (2012) Biomarkers. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 408–409. doi:10.1007/978-3-642-16483-5_6601

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Molecular Imaging Tobias Bäuerle1 and Wolfhard Semmler2 1 Institute of Radiology, University Medical Center Erlangen, Erlangen, Germany 2 Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany

Definition Molecular imaging is an interdisciplinary approach to noninvasively assess disease-specific structures in a living organism. When using the term molecular imaging in the narrow sense, imaging of molecular structures is referred to, but in the broader sense also, the assessment of morphology in high resolution; functional and metabolic parameters are included. When applying the broader definition, the following imaging techniques are most relevant: magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), computed tomography (CT), ultrasound (US), optical imaging (OI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Each imaging modality produces data with advantages and disadvantages containing complementary information from a region of interest. For probe design, data acquisition, and image postprocessing, knowledge from biology, chemistry, physics, pharmacology, informatics, and medicine is required. MR spectroscopy and functional and

Molecular Imaging

metabolic techniques from MRI, CT, US, PET, and SPECT are frequently used in patients, but the assessment of molecular targets particularly with MRI, US, and OI is primarily restricted to experimental research. Thakur and Lentle define the term molecular imaging as follows: Molecular imaging techniques directly and indirectly monitor and record the spatiotemporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic, or therapeutic applications (Thakur and Lentle 2005). In the following, characteristics of molecular imaging probes and modalities are briefly described (adapted from Semmler 2008).

Characteristics Molecular Imaging Probes Molecular imaging probes can be divided into three classes: nonspecific, targeted, and activatable probes. Nonspecific probes are rather conventional contrast agents and radiotracers assessing functional or metabolic parameters such as blood flow or perfusion as surrogate markers. In contrast, targeted probes show high affinity toward molecular structures such as receptors, enzymes, transporters, or reporter genes. Activatable or “smart” probes are activated exclusively at the site of the target structure resulting in low background signals. Schematically, targeted and activatable probes consist of a specific ligand with high affinity to a certain target structure and a signal generator (Fig. 1). Besides the high affinity of the ligand to the target structure, molecular probes require certain properties to be applied in vivo. In this regard, probes have to maintain specificity and affinity for the target structure after the signal generator is coupled to the specific ligand. Furthermore, the accumulation at the extra- or intracellular target within the region of interest must be high enough to reach concentrations of the probe that can be detected by the respective modality. Upon application in a living organism, in vivo stability, favorable pharmacokinetics (particularly concerning plasma half-life,

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excretion and elimination of the probe, etc.), and low toxicity have to be guaranteed. Imaging Modalities Modalities used for molecular imaging differ significantly concerning the acquired data (Fig. 2). Information assessed from currently available imaging modalities includes aspects from morphology, function, metabolism, and molecular structures. Below, the main characteristics of molecular imaging modalities are given, whereas technical and methodological basics cannot be described in detail. Signal generator Specific ligand

Epitope

Linker

Molecular Imaging, Fig. 1 Schematic design of a specific molecular probe. Probes for imaging a molecular target consist of a signal generator, linker, and specific ligand. The epitope of the specific ligand is required for specific binding of the molecular probe to the target structure (e.g., cell-surface receptor or intracellular structure) (Adapted from Semmler 2008)

Molecular Imaging, Fig. 2 Overview of molecular imaging modalities. The given modalities cover different aspects on the morphological, functional, and molecular levels. MRI magnetic resonance imaging, MRS magnetic resonance spectroscopy, CT computed tomography, US ultrasound, OI optical imaging, PET positron emission tomography, SPECT single-photon emission computed tomography (Adapted from Semmler 2008)

MRI MRS CT US OI PET SPECT PET/MRI PET/CT

Magnetic Resonance Imaging and Spectroscopy

The strength of MRI is its excellent soft-tissue contrast for morphological imaging, which is even increased when using an intravenous contrast agent. A major drawback of this technique is the low physical sensitivity impeding imaging on the molecular level. Increasing the magnetic field, however, improves the signal-to-noise ratio significantly. Experimental scanners with field strengths of up to approx. 20 tesla are currently available reaching a spatial resolution in the low micrometer range. Contrast or non-contrastenhanced functional imaging including techniques to quantify surrogate parameters for blood volume, perfusion, vessel permeability, etc., is another feature of this imaging modality producing quantifiable biomarkers for noninvasive tissue characterization. Imaging on the molecular level is feasible with MRS, enabling the direct measurement of cellular metabolites. For many years, MRS has been regularly used in patients, e.g., to discriminate tumor from regular tissue in the brain or the prostate. Signal enhancement for MRI is achieved by increasing the relaxivity upon application of paramagnetic or superparamagnetic substances. Commonly used contrast agents in clinical use are paramagnetic substances (gadopentetate dimeglumine, Gd-DTPA; gadoterate meglumine,

Morphology

Function

Anatomic and pathologic structures

Blood flow, blood volume, perfusion, cell metabolism etc.

Molecules Receptors, cell signalling, gene expression etc.

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Gd-DOTA; etc.), which might be coupled to a specific ligand although the achieved relaxivities are rather low. More promising in this regard are superparamagnetic nanoparticles that contain iron oxides altering the magnetic field locally depending on their diameter (e.g., ultrasmall paramagnetic iron oxide, approx. 10–50 nm), which can be coupled to target-specific molecules. Ultrasound

Ultrasound captures the complete imaging spectrum ranging from morphological to functional and molecular imaging in high spatial and temporal resolution. For small animal use, US systems are commercially available including dedicated hardware and software, particularly for mice and rats. Besides assessing morphology with excellent soft-tissue contrast, non-contrast- and contrastenhanced techniques are used for the quantification of functional vascular parameters. Thereby, the Doppler effect requires no contrast media injection, which can be employed to measure blood flow. On the contrary, intravenously injected microbubbles might be applied using dynamic contrast-enhanced US to produce a pixel-by-pixel analysis of a certain region of interest. Contrast-enhanced US is a method of high sensitivity allowing the detection of even single microbubbles, which can be applied for imaging of molecular structures. The gas-filled microbubbles possess low acoustic impedance, which results in a stronger reflection of sound waves as compared to surrounding tissue. Most frequently, either bubbles encapsulated with a soft shell (lipids or proteins) or hard shell (polylactide or cyanoacrylate) are suitable for coupling targetspecific ligands to their surface. However, due to the bubble size between 1 and 5 mm, US contrast agents are restricted to the intravascular space. Therefore, targeted microbubbles might be used to detect endothelial structures, e.g., integrins and VEGF receptors. For quantitative assessment of molecular structures upon binding of targeted microbubbles, stimulated acoustic emission (SAE) and sensitive-particle acoustic quantification (SPAQ) were developed.

Molecular Imaging

Optical Imaging

Optical imaging is frequently used in vitro and in vivo due to its simple application and high sensitivity. For in vitro use, penetration depth of emitted light is not a major issue, in contrast to in vivo imaging where scattering of optical photons plays an increasing role when the lightemitting target is located deeper within the animal, thus preventing “sharp” imaging. Here, the following optical imaging methods are described briefly: bioluminescence imaging, fluorescence imaging, and optical tomography. The photon source in bioluminescence imaging is within the cell, e.g., green fluorescent protein (GFP) or luciferase. Commercially available imaging systems contain a detector (CCD camera), a light-tight chamber, and dedicated software for display and evaluation of the acquired data. The strength of this method is the high contrast (lack of background signal) enabling tracking of single cells depending on the depth of the light source in the tissue. However, quantitation of results is highly difficult using planar imaging. Comparing bioluminescence imaging to fluorescence imaging, the latter technique uses a light source that excites endogenous or exogenous fluorochromes. For optical tomography, the light is directed toward the object to excite the fluorochrome, and the emitted light is assessed in multiple projections. Subsequently, this information is reconstructed tomographically to derive the three-dimensional distribution of fluorochromes in the organism enabling absolute quantification of data. Optical signal generators include dyes and luminescence peptides (GFP, luciferin/luciferase). For optical signal detection, the photophysical properties of the compound are pivotal and include high absorption yields, appropriate absorption wavelengths, favorable fluorescence emission characteristics, appropriate lifetimes of the intermediate state, and low photobleaching. Positron Emission Tomography and Single-Photon Emission Computed Tomography

Nuclear medicine techniques PET and SPECT allow for the assessment of metabolism and

Molecular Imaging

molecular structures and are therefore molecular imaging techniques in the narrow sense because of their high physical sensitivity. For both techniques, concentrations of radioactively labeled molecules in the picomolar range can be detected as compared to MRI. However, PET and SPECT do not provide sufficient anatomical information and are therefore often combined with CT or MRI as hybrid techniques as described below. For PET, positron emitters are used, and for SPECT, longlived radioisotopes emitting gamma rays are injected. The spatial resolution of a clinical PET scanner is around 4 mm or below, whereas small animal scanners achieve a resolution down to 1 mm. The construction of PET and SPECT scanners and detection and reconstruction algorithms are similar for both techniques. Besides their application in experimental molecular imaging, these nuclear medicine techniques are routinely in clinical use for patients in oncology, cardiology, and neurology. Positron emitters for PET comprise, among others, 11C, 13N, 15O, 18F, and 68Ga which are produced in accelerators or generators, the latter resulting in easier availability. For the synthesis of molecular imaging PET probes, a major obstacle is the relatively short half-lives of these positron emitters ranging between approx. 2 and 110 min. For SPECT, 99mTc, 11In, 123I, and 125I or others are used as signal generators with half-lives in the range of hours or days. Imaging molecular structures is feasible by labeling these radioactive isotopes to target-specific biomolecules.

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long imaging times or the use of high X-ray power have to be taken into account. To increase the soft-tissue contrast, iodine-containing agents of various formulations (intravascular, liverspecific contrast agents, etc.) might be applied. Flat-panel volumetric CT scanners contain a whole-body gantry equipped with flat-panel detectors, which can be used for small animals but also larger animals such as sheep and primates. The advantages of these systems are functional (dynamic contrast-enhanced CT) and morphological imaging of relatively large volumes in high resolution. Micro-CT requires either the rotation of the object or the gantry. The rotation of the object, however, may result in unphysiologic conditions, while scanning may result in an alteration of functional parameters such as blood flow or blood volume. Hybrid Techniques

Due to the lack of morphological and anatomical information of PET and SPECT, these techniques are often combined with CT or MRI. Also hybrid devices combining techniques from nuclear medicine and optical imaging are being developed at the moment. Hybrid techniques therefore compensate for the low spatial resolution on the one side (PET, SPECT, OI) and the low sensitivity on the other (CT, MRI). Currently, PET/CT is most frequently used, but also integrating PET and MRI by simultaneous or subsequent image acquisition is available for human and small animal use.

Computed Tomography

In the narrow definition as indicated above, CT cannot be considered as molecular imaging modality. However, morphological imaging in high resolution and functional imaging is possible with this technique. Resolutions of approximately 250–500 mm are regularly achieved with current clinical CT scanners, whereas flat-panel CT systems and dedicated micro-CT systems reach 100–200 mm and below 100 mm, respectively. The disadvantages of CT images are the poor soft-tissue contrast, high image noise levels, and, for high resolution CT, high scan doses due to

Cross-References ▶ Ultrasound Microimaging

References Semmler W (2008) Molecular imaging. In: Reiser MF, Semmler W, Hricak H (eds) Magnetic resonance tomography, 3rd edn. Springer, Heidelberg, pp 1381–1410 Thakur M, Lentle BC (2005) Report of a summit on molecular imaging. Radiology 236:753–755

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Molecular Morphology ▶ Immunohistochemistry ▶ Molecular Pathology

Molecular Pathology Mark F. Evans1 and Kumarasen Cooper2 1 Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT, USA 2 Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

Synonyms Molecular morphology

Definition Molecular pathology is the study of the molecular genetic causes of abnormal cell and tissue functioning with the goal of improved disease diagnosis and treatment. As a medical discipline, molecular pathology is a specialty training that incorporates the subject matter of genetics, inherited cancers, solid tumors, neoplastic hematopathology, infectious diseases, identity testing, HLA typing, and laboratory management and impacts both anatomic and clinical pathology practice.

Characteristics Historical and Clinical Background ▶ Pathology, the study of the origin, nature, and courses of diseases, has its foundation in the studies of Giovanni Battista Morgagni of Padua (1682–1771) (the “father of anatomic pathology”) and Rudolf Virchow of Berlin (1821–1902) (the “father of cellular pathology”). Dr. Morgagni

Molecular Morphology

documented the relationship between diseases and the gross changes observed in autopsy specimens, and Dr. Virchow established the correlation of cellular changes with disease having formulated the cellular theory of life, omnis cellula e cellula (all cells come from cells). Modern anatomic pathology retains gross and microscopic tissue examination as the basis of clinical diagnostics via standardized classifications; tumor specimens are described in terms of organ of origin (e.g., breast, colon, etc.), cell type (e.g., carcinoma, describing cells of epithelial type; adenocarcinoma, referring to a cancer of glandular origin), and ▶ tumor staging and ▶ tumor grade. The “TNM staging” characterizes solid tumors by the extent to which a tumor (T) has spread locally, the amount of regional lymph node (N) involvement, and whether or not there are metastases (M) to distant organs or lymph nodes. Tumor grade refers to the degree of cellular divergence from the normal condition. These criteria are increasingly supplemented by tests for additional ▶ biomarkers to improve disease diagnosis. The discoveries of molecular cell biology and the human genome project together with technological advancements resulting in methodologies that are reliable, rapid, and cost-effective have set the stage for molecular pathology as an essential and routine discipline for improved patient care. Potentially, molecular pathology will yield a comprehensive molecular classification of cancer improving early tumor diagnosis, resolution of equivocal diagnoses including tumors of uncertain grade or unknown organ of origin, tumor chemoresistance/drug therapy investigation, and individualized medicine. Numerous putative markers are reported in the scientific literature; the challenge is to identify those individual or sets of biomarkers that have high sensitivity, specificity, and predictive value (negative predictive value, positive predictive value) for vital clinical parameters. The following illustrates by way of current common techniques how molecular pathology can support cancer diagnosis. Immunohistochemistry Immunohistochemistry (IHC) allows by microscopic inspection the correlation of protein

Molecular Pathology

staining patterns with cytology or histology and is the most routinely used adjunct technique of anatomic pathology. Aberrant molecular genetic changes frequently result in an altered immunophenotype (nuclear, cytoplasmic, or membrane protein expression staining patterns). Typically, IHC involves application of a primary antibody to the antigen of investigation to tissue sections, followed by secondary antibodies labeled to allow chromogenic or fluorescent detection. IHC has been a routine part of pathology practice since the mid-1970s, often to identify cell or tissue origin. For example, the lineage of an undifferentiated tumor or the organ of origin of a metastasis can be identified after IHC with panel of antibodies for cytokeratins as the expression fingerprint of these cytoskeletal markers is not compromised by malignant transformation. IHC is also used to differentiate lymphomas and sarcomas. Panels of markers (e.g., CD5, CD23, CD43, cyclin D1, and bcl-6) are used to help distinguish different categories of lymphomas such as follicular, mantle cell, marginal zone, and chronic lymphocytic leukemias. Similarly, IHC markers including desmin, myogenin, CD99, CD43, and FLI-1 may be used to distinguish ▶ rhabdomyosarcomas, ▶ Ewing’s sarcoma, ▶ desmoplastic small round cell tumor (DSRCT), ▶ neuroblastoma, and lymphoblastic lymphoma. “Genogenic” IHC supports the demonstration of genomic translocations that result in novel chimeric proteins; for example, 90% of Ewing sarcoma cases harbor a t(11;22)(q24;q12) translocation resulting in the EWS-FLI-1 chimeric product; overexpression of the FLI-1 protein can be used to distinguish Ewing’s sarcoma from histologically similar tumors. IHC can also be used as a marker of gene mutations that result in protein underor overexpression; hereditary nonpolyposis colon cancer (HNPCC) is characterized by ▶ microsatellite instability (MSI) due to mutations in genes such as hMLH1 and hMSH2. These mutations translate as under-expression of MLH1 and MSH2 proteins detected by IHC. Routinely, IHC markers are employed to assess tumor aggressiveness and the appropriate course of treatment; breast tumor diagnostics requires

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IHC for ▶ estrogen receptor (ER), progesterone receptor (PR), and ▶ HER-2 status. ER and PR positive tumors are generally less aggressive and are more responsive to hormone suppression treatments such as tamoxifen; HER-2 positive tumors tend to be aggressive but are candidates for treatment with ▶ herceptin. Defective cell-cycle checkpoint control may be critical for tumor development, and there is a variety of potential IHC markers. For example, p57Kip2 expression may be associated with poor prognosis, whereas p21WAF1 and p27Kip1 overexpression may signify a favorable prognosis; p16INK4a is widely used as a surrogate marker of human papillomavirus (HPV) infections and preinvasive cervical lesion grade. High-risk HPV type E7s openreading frame product inactivates pRb (▶ retinoblastoma protein), thereby disrupting pRb negative feedback of p16INK4a with consequent overexpression of the latter and increased staining the more severe the lesion (Fig. 1). IHC can also be used in the direct demonstration of oncogenic viruses. Detection of ▶ EpsteinBarr virus (EBV) latent membrane proteins (LMPs) and/or nuclear antigens can be used in the diagnosis of ▶ nasopharyngeal carcinoma, Burkitt lymphoma, and ▶ Hodgkin lymphoma. Detection of human herpesvirus 8 (HHV-8) may assist in the diagnosis of ▶ Kaposi sarcoma, and detection of the HPV L1 capsid protein of infective virions may be a marker of low-grade cervical lesions. In Situ Hybridization Fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) allows the demonstration of DNA or RNA in tissue samples by hybridization with labeled nucleic acid probes or synthetic analogs; like IHC, in situ hybridization (ISH) supports the direct correlation of test data with specimen morphology. FISH techniques represent an efficient and easier option than classical metaphase cytogenetic techniques for the detection of chromosomal rearrangements; additionally, ISH supports interphase cytogenetic applications. FISH is utilized in the diagnosis of hematological malignancies. For example, chronic

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Molecular Pathology

Molecular Pathology, Fig. 1 Differential nuclear and cytoplasmic p16INK4a staining patterns in low-grade (a) and high-grade (b) cervical lesions

myelogenous leukemia (CML) is characterized by the ▶ BCR-ABL reciprocal translocation involving fusion of the BCR region of chromosome 22 with the ABL region of chromosome 9. The fusion results in a shortened chromosome 22 (the Philadelphia chromosome). Predictable FISH signal patterns can be observed in interphase cells depending on whether the probes span or flank the translocation breakpoint. Other hematological diagnoses by FISH include ▶ acute promyelocytic leukemia (APL) (t(15;17)(q22; q12)) and follicular lymphoma (t(14;18)(q32; q21)). Soft tissue tumors such as synovial sarcomas can also be diagnosed by FISH; the t(X;18) (p11;q11) translocation is present in 90% of these tumors and results in the juxtaposition of the SYT gene (18q11) and the SSX gene (Xp11). Among solid tumors, FISH is routinely used to confirm HER-2 amplification indicated after IHC assay. The PathVysion™ (Vysis Inc., IL, USA) FISH assay involves dual hybridization with a probe for the centromeric region of chromosome 17 together with a probe to the HER-2 locus at 17q11.2-12; comparison of the ratio of signals is used to score amplification (Fig. 2). The UroVysion™ (Vysis Inc., IL, USA) FISH assay is a noninvasive test for ▶ bladder cancer. The test is applied to urinary cytology specimens and uses

a panel of four probes to targets frequently altered in bladder cancer (centromeres 3, centromeres 7, and centromeres 17, and 9q21 (site of the p16 gene)); if 4/25 cells show multiple chromosome gains, or there is loss of both copies 9q21 in 12/ 25 cells, this is taken as predictive of cancer. ISH is the preferred choice for the demonstration of EBV by the detection of EBV-encoded RNAs (EBER) in circulating B lymphocytes of patients with suspected lymphoma. ISH supports the detection of low- or high-risk HPV types, and HPV signal types may be useful in determining (cervical) lesion grade. “Diffuse” signals are associated with episomal HPV, whereas “punctate” signals may be demonstrative of HPV integrated into the cell genome (Fig. 3). In addition to cervical tissues, HPV ISH is applicable to head and neck, esophageal, and other tumors with a suspected HPV etiology. Other ISH applications include investigation of chromosome instability/aneusomy in tumors or cell lines using panels of centromeric probes. Locus-specific probes can be used to investigate the association of defined abnormalities with tissue morphology. Centromeric and locus-specific probes can also be used to examine intra-tumoral heterogeneity and to investigate the relationship between tumors and putative precursor lesions.

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Molecular Pathology, Fig. 2 (a) Non-amplification of HER-2 indicated by a 1:1 ratio of chromosome 17 centromeric signals (green) to HER-2 FISH signals (red). (b) Amplification indicated by abundant HER-2 signals relative to centromere 17 signals

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Molecular Pathology, Fig. 3 HPV detection by CISH in cervical tissues. Diffuse signals (blue arrows) detected mainly in upper epithelial layers characterize low-grade lesions (a). In high-grade lesions (b) diffuse and punctate

signals (red arrows) may be detected throughout the epithelial thickness. Punctate signals alone in an invasive cervical carcinoma (c)

Combining ISH with IHC allows observation of the correlation between nucleic acid detection and protein expression patterns. Specialized FISH techniques such as spectral karyotyping (SKY) or multiplex (M) FISH utilize chromosome paints specific for each of the 24 human chromosomes and detectable by fluorescent microscopy and computer imaging. Application of the paints to metaphase spreads from tumors allows the identification of chromosomal rearrangements with a resolution down to 2–3 Mb.

Polymerase Chain Reaction Pathology specimens are frequently highly limiting with respect to the amounts of nucleic acids that can be recovered for diagnostic or research applications. The polymerase chain reaction (PCR) utilizes a thermostable DNA polymerase, DNA primers, and deoxyribonucleic acid building blocks to amplify a DNA template; theoretically, after 30 cycles of denaturation, annealing, and extension, the starting template DNA sequences are amplified one billion-fold.

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Consequently, PCR techniques have been at the vanguard of molecular pathology investigations. Using fluorescent-labeled primers, PCR can be adapted for real-time quantitative (Q) PCR to allow an estimation of relative DNA load or mRNA expression. By combining PCR with microdissection of specific tissues or cells off a slide, it is possible to correlate PCR data directly with tissue morphology. PCR is used diagnostically to screen for gene mutations (by PCR product sequencing or by techniques such as single-strand conformation polymorphism (SSCP)). Mutation screening of all 19 hMLH1 exons, all 16 hMSH2 exons, or all 10 exons of the hMSH6 is possible in the diagnosis of HNPCC; MSI can be investigated directly by PCR for a panel of 5–12 standardized microsatellite loci that includes mono- and dinucleotide repeat sites (▶ colon cancer). PCR mutation screening is also applicable in the diagnosis of other familial tumors such as the ▶ APC gene in familial adenomatous polyposis, which can lead to an autosomal dominant condition resulting in the development of myriad colon polyps, some of which may have the potential to progress to colon carcinoma. Mutation screening is also performed for ▶ BRCA1/BRCA2 germ line mutations and breast cancer risk. Numerous candidate genes associated with sporadic tumors have been described; however, uncertainty about the relationship of mutations in these genes to tumor diagnosis and prognosis has stalled translation to routine screening tests. For example, mutations leading to aberrant expression of the tumor suppressor TP53 protein (“guardian of the genome”) are estimated to be present in up to 50% of sporadic tumors. Despite the undoubted contribution of p53 mutations to tumor pathology, p53 mutation screening is of uncertain clinical utility. PCR is used in the diagnosis of oncogenic viruses; in particular, several PCR strategies are available for detecting HPV and determining which of more than 40 HPV types associated with cervical lesions are present in a patient (cervical smear) sample. In the reverse line blot assay, labeled PCR product is hybridized to a blot spotted with HPV type-specific probes. Patients

Molecular Pathology

positive for high-risk HPV types may be at an increased risk for high-grade lesions/cancer. An elevated HPV DNA load as determined by Q-PCR may also correlate with increased tumor risk. Reverse-transcriptase (RT) PCR converts RNA to DNA for PCR amplification. RT-PCR allows the detection of RNA fusion transcripts and is widely used in soft tissue tumor diagnostics. For example, alveolar rhabdomyosarcoma diagnosis is aided by the detection of mRNA transcripts created after the t(2:13)(q35;q14) reciprocal translocation that juxtaposes part of the PAX3 gene on chromosome 2 with part of the FKHR gene on chromosome 13. The API2/ MALT1 RT-PCR test can be applied to stomach or other gastrointestinal tissues for the diagnosis of ▶ B-cell tumors of mucosa-associated lymphoid tissue (MALT lymphoma). This condition commonly develops against a background of chronic inflammation caused by ▶ Helicobacter pylori infection. Diagnosis of DSRCT can be aided by RT-PCR for transcripts from the t(11:22)(p13;q12) translocation associated with DSRCT and involving the EWS and WT1 genes. RT-PCR is also used in the diagnosis of Ewing’s sarcoma, synovial sarcomas, and BCL-ABL/CML. Q-RT-PCR is used in the diagnosis of APL to detect transcripts of the fusion of the retinoic acid receptor alpha gene (RARA) with the promyelocytic leukemia (PML) gene; monitoring the tumor burden may be helpful in predicting disease relapse. The PCR-based methods have greater sensitivity than their FISH counterparts for detecting tumor-specific rearrangements; nevertheless, “false-negative” diagnoses may occur because of variant translocation points not detected by PCR primer sets. RT-PCR also supports the assessment of HPV integration. The amplified papillomavirus oncogenic transcript (APOT) assay confirms integration by the detection of HPV transcripts that are contiguous with human sequence transcripts. Future Developments Improved techniques and new findings are a constant in molecular pathology. For example, application of a sophisticated high-throughput PCR

Molecular Therapy

mutation screening strategy of more than 13,000 genes in 11 breast and 11 colorectal cancers has shown that individual tumors typically accumulate 90 mutant genes and that only a subset of these contribute to the neoplastic process; an average of 11 genes per tumor were mutated at significant frequency. Follow-up of such studies in course of time may reveal (sets of) mutations that have high utility as sporadic tumor diagnostic markers. Similarly, ▶ microarray cDNA technology and the findings of ▶ proteomics studies are expected to greatly increase and refine knowledge of the molecular basis of cancer, translating into effective clinical tests including improved IHC, ISH, or PCR, or the development of cost-effective microarray or proteomic tests for limited sets of biomarkers. Additionally, there remains much to be learned about genome regulation; for example, the significance of ▶ microRNAs in the control of gene expression and contribution to tumor biology has been recognized, and there is increasing awareness of the importance of ▶ epigenetics in tumor etiology. Advances in the conceptual frameworks within which cancer is understood will also direct the interpretation of biomarkers. While ▶ oncogenes and ▶ tumor suppressor genes remain key concepts, there is a growing appreciation of cancer as a process involving a multi-interaction of cellular systems.

Cross-References ▶ Pathology

References Calin GA, Croce C (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 68:57–866 Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of cancer. Nat Rev Cancer 7:21–33 Leonard DG (ed) (2007) Molecular pathology in clinical practice, 1st edn. Springer, New York Simpson PT, Reis-Filho JSR, Gale T et al (2005) Molecular evolution of breast cancer. J Pathol 205:248–254 Sjoblom T, Jones S, Wood LD et al (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314:268–274

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Molecular Therapy Ingo Kausch von Schmeling1 and Christian Doehn2 1 Klinik für Urologie und Kinderurologie, Ammerland Klinik GmbH, Westerstede, Germany 2 Urologikum Lübeck, Lübeck, Germany

Definition Molecular therapy is a therapy with compounds which are rationally targeted against certain specific molecular structures.

Characteristics There is yet no generally accepted definition of the term molecular therapy. A frequent opinion is that molecular therapeutics target diseases on an early molecular level and thus modulate the disease at its roots. This would comprise the classic ▶ gene therapy as well as other DNA- or RNA-based strategies. However, also other nonmolecular therapeutics such as ▶ chemotherapy interact with nucleic acids. Additionally, nucleic acidbased drugs may also interact with proteins. Therefore, it seems more useful to define molecular therapy as a strategy which specifically targets certain molecular structures, although all therapies that work have a target with some kind of molecular structure. In some cases, the target is discovered first, while in others the drug is discovered before the target. In contrast to other therapeutic concepts such as chemotherapy or immunotherapy, molecular therapy is usually directed against a biologically important process with a measurable target in the clinic. Based on this definition, certain compounds which are frequently designated as “targeted therapy” such as monoclonal antibodies or ▶ small-molecule drugs should in addition to DNA- or RNA-based strategies also belong to the class of molecular therapeutics.

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Gene Therapy Classic gene therapy is the direct use of genetic material in the treatment of diseases. The various strategies for inhibiting cancer growth by gene therapy approaches comprise the reversion of the malignant phenotype by correcting aberrant gene expression, induction of genes that inhibit tumor growth, immune gene concepts, and cytoreductive or oncolytic approaches. Additive or corrective gene transfer involves on the one hand the introduction of foreign genes such as suicide genes or antiangiogenesis genes. On the other hand, physiological but mutated and malfunctioning genes which are associated with tumor development and progression such as tumor suppressor genes may be restored. Since the cellular uptake of naked longer nucleic acid molecules is inefficient, gene therapy is dependent on effective delivery. The clinically relevant gene transfer techniques can be classified into viral (vector) delivery systems and nonviral delivery systems. DNA molecules may be packed in viral vectors. Viruses can transfer these molecules into cells, and the transgene of interest enters the nucleus and is integrated into the host gene pool and eventually expressed. Common clinically used viruses are adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, the herpes simplex virus, and parvoviruses. The gene transfer by viral vectors in general is characterized by a high effectivity but considerable toxicities such as, for instance, immune responses. Common nonviral delivery systems are polymeric systems and liposomal systems. These systems are characterized by lack of immune response as well as ease of industrial production but also by significantly lower transfection efficiency compared to viral vectors. Clinically, several gene transfer phase I–III studies in different tumors have been performed or are in progress. Earlier trials suffered especially from inefficient expression of the transgene. Currently, there are no gene therapy products approved for clinical use in the Western world. Oncolytic gene therapy viruses are designed to infect and destroy cancer cells by expression of cytotoxic proteins while remaining innocuous to the rest of the body. Several different viruses such

Molecular Therapy

as adenoviruses and herpes virus have been used clinically. However, only few trials with oncolytic therapy have been performed. The most advanced (phase III) is the approach with an adenovirus that replicates and mediates cell lysis only in cancerous cells with p53 mutation (ONYX-015). Since cancer cells tend to develop mechanisms to evade immune detection, traditional immunotherapy is often characterized by limited effectivity. In order to overcome these problems, immune gene therapy concepts have been advocated. One popular strategy is recombinant cancer vaccines. Autologous or allogeneic cancer cells are engineered to be more recognizable to the immune system by the addition of one or more genes. These genes are mostly cytokine genes or highly antigenic genes. Another approach is the direct delivery of immunostimulatory genes such as cytokines to the tumor. The goal is to unmask the cells from immune evasion and enhance an antitumor response. A third strategy is the direct alteration of the patient’s immune system by adding tumor antigens or stimulatory genes to the patient’s immune cells. Although early clinical trials were quite successful, long-lasting immune responses were the exception rather than the rule. Several large phase III studies are currently underway. Nucleic Acid Constructs The various nucleic acid therapeutics include antisense oligonucleotides, ▶ small interfering RNAs (siRNA), aptamers, ribozymes, and DNAzymes. Nucleic acid-based drugs have emerged to yield extremely positive candidates for therapy of various diseases including cancer. In opposite to gene therapy, the major goal of nucleic acid-based drugs is the inhibition and silencing of specific cancer-associated genes such as ▶ oncogenes. The most extensive applications have been performed with antisense oligonucleotides. These are short chemically modified DNA sequences (10–25 nucleotides), designed to modulate the information transfer from gene to protein. Sequence-related hybridization with the mRNA of a specific protein results via different mechanisms such as RNAse H-dependent mRNA cleavage in selective inhibition of gene expression

Molecular Therapy

and downregulation of protein expression. Antisense oligonucleotide inhibitors can be designed directly from genomic sequence information by simply making the reversed complement of the desired sequence. However, active compounds are more effectively generated by computerbased approaches. Two antiviral antisense drugs have already been approved by the FDA. Phase III studies with several antisense constructs in oncological patients are underway or have been finished. Some results have been particularly encouraging although antitumoral effects have to a certain extent been attributed to unspecific immunostimulatory mechanisms. In clinical studies, antisense oligonucleotides have mostly been applied without any delivery system. In the majority of the current trials, oligonucleotides are combined with one or more traditional chemotherapeutic agents or another targeted agent. Ribozymes are RNA molecules which may selectively bind to a target mRNA and form a duplex which results in cleaving of the target mRNA. Two types of ribozymes, the hammerhead and hairpin ribozymes, have been extensively studied for therapeutical purpose. In DNAzymes, the RNA backbone chemistry is replaced by DNA motifs which confers greater biological stability. DNAzymes have high catalytic activity. Aptamers are small single-stranded or double-stranded nucleic acid segments that directly interact with proteins. In comparison to protein inhibition by antibodies, aptamers are highly specific, nonimmunogenic, and stable. In some cases and as opposed to antisense oligonucleotides, effects can be mediated against extracellular targets, thereby preventing a need for intracellular transportation. The three latter classes of nucleic acidbased drugs have been evaluated in preclinical oncological studies or have entered early clinical trials. Small interfering RNAs are short doublestranded RNA segments with a length of typically 21–23 nucleotides. siRNA are also complementary to a target mRNA sequence. They are upon application incorporated into RNA-induced silencing complexes (RISCs) which bind to the mRNA of interest and stimulate mRNA degradation via different mechanisms such as nuclease

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activation. These compounds are, compared to other gene-silencing instruments, substantially more effective and nonimmunogenic. Initial nononcological phase I studies have been initiated. A new class of noncoding small RNA molecules are the microRNAs (miRNAs). Many miRNAs are known to be up- or downregulated in a variety of cancers, suggesting a role for miRNAs in tumorigenesis. These compounds are beginning to be used therapeutically. Nucleic acid-based drugs offer in comparison to viral vectors or plasmids greater safety since they do not integrate into the genome. Furthermore, delivery is mostly easier since most of these compounds do not have to enter the nucleus for their activity and because of the small size (especially antisense oligonucleotides and siRNA). However, for most medical applications, several obstacles related to toxicity, stability, affinity, cellular delivery, and specificity remain to be clarified and may be overcome by new chemical modifications. Small Molecule Drugs The small molecules of highest interest in cancer therapy are protein tyrosine kinases (PTK). They comprise over 100 ▶ oncogenes and more than 40 ▶ tumor suppressor genes. Overexpression of PTK receptors is correlated with poor prognosis in some malignancies. Tyrosine kinase inhibitors (TKIs) are orally available, synthetic chemicals with a small molecular weight. They reversibly bind to the ATP-binding site on the receptor and prevent its activation as well as signal transduction. Small-molecule agents can translocate through plasma membranes and interact with the cytoplasmic domain of cell surface receptors and intracellular signaling molecules. TKIs have shown objective tumor responses in chronic myeloid leukemia (CML), gastrointestinal stromal tumors, and non-small cell lung cancer. The TKIs gefitinib and erlotinib are specific for the ▶ epidermal growth factor receptor (EGFR). In contrast, imatinib inactivates the kinase activity of the BCR–ABL fusion protein in CML. Sorafenib is a dual kinase inhibitor inhibiting different isoforms of Raf serine kinase as well as various receptor tyrosine kinases such as VEGFR,

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EGFR, and PDGFR. This results in an inhibition of tumor proliferation and angiogenesis. Sunitinib is another multi-targeted tyrosine kinase inhibitor of VEGFR, PDGFR, KIT, and fms-like tyrosine kinase 3 (FLT3). Both drugs are approved for the treatment of metastatic kidney cancer. Bortezomib, which was first developed as a selective, reversible inhibitor of the chymotryptic protease in the 26S proteasome, has been reported to be effective against hematological malignancies. Antibody Therapy In 1975, Köhler and Milstein first described the generation of murine monoclonal antibodies (mAbs). The typical antibody consists of two antigen-binding fragments (Fabs) which are linked via a flexible region to a constant (Fc) region. This structure comprises two pairs of polypeptide chains, each pair containing a heavy and a light chain of different sizes. Antibodies bind to antigens on the surface of cells or pathogens. The first therapeutic antibody for cancer therapy (rituximab) was approved in 1987 for the treatment of non-Hodgkin lymphoma. mAbs are relatively large proteins with a high molecular weight. They are generally administered via an intravenous infusion and often have a half-life of several days. The disadvantages of mouse antibodies are a short half-life in serum, the inability to trigger human effector function, and the induction of human antimouse antibodies (HAMA) which in turn inactivate murine mAbs. Chimeric antibodies such as rituximab combine human constant regions and mouse variable antigen-binding regions to reduce their immunogenicity. However, they can also induce human anti-chimeric antibody response (HACA). Thus, human antibodies seem ideal to overcome the latter problems. Today, about 200 antibodies are in clinical trials for cancer therapy, chronic inflammation, transplantation, and infectious and cardiovascular diseases. For cancer therapy, more than ten mAbs are approved by the FDA, predominantly for the treatment of non-Hodgkin lymphoma, breast cancer, and colorectal cancer.

Molecular Therapy

Antibody cancer therapy aims to target tumorassociated antigens and/or tumor-specific antigens (e.g., CEA, EGFR, HER2, MUC1, CD20) to alter their signaling. They can function by three principal modes of action: blocking the action of specific molecules such as growth factors or cytokines, targeting specific cells, or functioning as signaling molecules. Also, mAb therapy may result in an elimination of tumor cells by immune effector cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Limitations beside immunogenicity of murine mAbs and chimeric mAbs are a potential lack of selectivity and poor penetration into solid tumors. The latter problem is due to the size of mAbs and a poor permeability of the tumor surface. Monoclonal antibodies are relative specific therapies associated with low toxicity. Clinically, antibodies are often safe well tolerated with some fever and chills at first infusion. A possible side effect of antibody therapy is the cytokine release syndrome that is probably mediated through recruitment of the immune effector cells. However, treatment-associated deaths have been described in few patients. Another limitation is the restriction of targets to those on the surface of host cells. This problem could be solved by the development of functional antibodies within cells, so-called intrabodies. Besides the development of unconjugated or naked mAbs, a conjugation with cytotoxic drugs, cytokines, toxins, radionuclides, and DNA molecules has been tested in order to optimize delivery and enhance specificity and effectivity.

Cross-References ▶ Chemotherapy ▶ Epidermal Growth Factor Receptor ▶ Gene Therapy ▶ Oncogene ▶ Small Molecule Drugs ▶ Tumor Suppressor Genes

Monoclonal Antibodies for Cancer Therapy

References Carter PJ (2006) Potent antibody therapeutics by design. Nat Rev Immunol 6:343–357 Cross D, Burmester JK (2006) Gene therapy for cancer treatment: past, present and future. Clin Med Res 4(3):218–227 Imai K, Takaoka A (2006) Comparing antibody and smallmolecule therapies for cancer. Nat Rev Cancer 6:714–727 Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR (2014) Site-specific antibody drug conjugates for cancer therapy. AMbs 6(1):34–45 Patil SD, Rhodes DG, Burgess DJ (2005) DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J 7(1):61–77

See Also (2012) Small Interfering RNA. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3448. doi:10.1007/978-3-642-16483-5_5372

Monoclonal Antibodies for Cancer Therapy Ingegerd Hellstrom and Karl Erik Hellstrom Department of Pathology, University of Washington, Seattle, WA, USA

Definition A monoclonal antibody (Mab) is made by a population of identical immune cells that are the progeny of a unique parent cell. Mab therapy of cancer is a form of ▶ immunotherapy in which a patient is injected, most often intravenously, with a Mab with the purpose to destroy the tumor or at least delay its growth. Most Mabs used for cancer therapy are of the IgG subtype 1.

Characteristics Background More than a century ago, Paul Ehrlich proposed that antibodies can be used as “magic bullets” to

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specifically destroy cancer cells, and it has been known since the 1960s that one can make antibodies which have greater specificity for neoplastic cells than their normal counterparts, for example, by immunizing rabbits and absorbing the sera with normal tissues. However, injecting cancer patients with such sera gave no therapeutic benefit and often caused toxicity because of allergic reactions. Antibodies regained interest for cancer therapy in 1975 when Köhler and Milstein discovered that spleen cells from an immunized mouse can be fused with cells from a mouse myeloma (a hematologic cancer) to establish rapidly growing clones of hybridomas where each clone makes large amounts of the same, monospecific antibody, i.e., the antibody-producing ability of the spleen cells is combined with the unrestricted life span of the myeloma. Many antigens were soon identified which are overexpressed in neoplastic cells by using Mabs made by hybridomas from immunized mice. Mouse Mabs (suffix -omab) to some of these antigens were tested for clinical efficacy in patients with various tumors, including lymphoma, melanoma, and colon carcinoma. There was only modest success, since the half-lives of the Mabs were short and their biological function was hampered as a result of species differences between mice and humans. Retreatment was rarely possible since the patients rapidly formed antibodies to the foreign antigens of the mouse Mabs. Nevertheless, early studies using mouse Mabs gave the first evidence that an injected antitumor Mab can have therapeutic efficacy. More effective Mabs were obtained as a result of advancements in genetic engineering during the 1980s and 1990s. Chimeric Mabs (suffix -ximab), which contain the murine immunoglobulin variable regions fused to human constant domains, and humanized Mab (suffix -zumab), where only the actual antigen-binding regions are of mouse origin, soon replaced the mouse Mabs. Subsequent development of phage display libraries and transgenic mice expressing human immunoglobulin genes has now led to the creation of fully

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human antibodies (suffix -umab). Hundreds of therapeutic anticancer Mabs have been evaluated in the clinic, and many are marketed for clinical application. Applications of Mabs for Cancer Therapy Mabs can be used “naked,” i.e., without any further modification as well as to target, to tumors, various cytotoxic drugs, toxins, radionuclides, or other molecules that can cause tumor destruction after the Mab part binds to the tumor cells. Therapeutic anticancer Mabs now provide one of the largest classes of new anticancer agents approved by FDA. Eleven Mabs were approved as of July 1, 2011, and this figure almost doubled by May 2015 (Table 1). However, the cost of Mab-based therapy has been high, and there is a need to more effectively produce and modify the Mabs. “Naked” (Unmodified) Mabs Which Interfere with Growth-Controlling Mechanisms Many tumors have an increased expression of growth factor receptors whose signaling function plays a key role to maintain the malignant phenotype. Mabs can induce ▶ apoptosis, decrease proliferation, and increase sensitivity to ▶ chemotherapy drugs by blocking ligand binding to the receptor or otherwise interfere with the receptor function. For example, the anti-CD20 Mab ▶ Rituximab, which was the first Mab approved for cancer therapy, can induce apoptosis as well as kill tumor cells via immunological mechanisms (see below). Members of the ▶ epidermal growth factor receptor (EGFR) family are frequently overexpressed in solid tumors, for example, ▶ colorectal cancer. They can serve as target for Mabs, such as Erbitux, which interfere with the interaction between the receptor and its ligand. Another growth factor receptor, HER2, is overexpressed in about 30% of ▶ breast cancers and in a small population of some other tumors, for example, ovarian carcinomas. Mabs to HER2 are therapeutically effective by inhibiting dimerization and internalization of the receptor and also by immunological mechanisms. ▶ Herceptin is a humanized such antibody of the IgG isotype, which is

Monoclonal Antibodies for Cancer Therapy

used for treatment of breast cancers that overexpress HER2. Tumor Killing via Complement-Dependent Cytotoxicity (CDC) and Antibody-Dependent Cellular Cytotoxicity (ADCC) Antibodies contain both an antigen-binding variable fragment (Fab) and a constant fragment (Fc). Following the binding of an antibody to the tumor cell surface, its Fc part can activate complement to cause ▶ complement-dependent cytotoxicity (CDC). It can also cause the attachment of ▶ natural killer cells (NK cells) via their Fc receptors and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC). Antibodies whose oligosaccharides are defucosylated have enhanced ability to mediate ADCC in vitro and a greater antitumor activity in vivo. Mabs can be made more effective also by increasing their affinity for the tumor antigens and by manipulating antibody Fcg domains. The antiCD20 Mab Rituximab is more effective in lymphoma patients who have high responder Fcg receptor gene polymorphism. Interference with Tumor Vasculature Many solid tumors express ▶ vascular endothelial growth factor (VEGF) which binds to their receptors on the vascular endothelium to stimulate ▶ angiogenesis. Avastin is a VEGF-specific humanized Mab which blocks binding of VEGF to its receptor. It is approved for the treatment of ▶ colorectal cancer in combination with ▶ chemotherapy. Antibodies to Lymphocyte Antigens Applied to Modify Antitumor Immunity via CheckPoint Inhibition The immune system of cancer patients (as well as that of mice) can form a tumor cell destructive immune response, also when the tumor is advanced when the tumorbearer’s lymphoid cells, tumor cells, and certain Mabs (e.g., to CD3 and CD28) are co-cultured in vitro, and many studies have demonstrated that Mabs to checkpoint inhibitors can have dramatic antitumor activity in vivo.

Monoclonal Antibodies for Cancer Therapy

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Monoclonal Antibodies for Cancer Therapy, Table 1 Therapeutic monoclonal antibodies approved or in review European Union or United States International non-proprietary name Necitumumab

Trade name (Pending)

Target; Format EGFR; Human IgG1

Dinutuximab

Unituxin

GD2; Chimeric IgG1

Nivolumab

Opdivo

PD1; Human IgG4

Melanoma, non-small cell lung cancer

Blinatumomab

Blincyto

Pembrolizumab

Keytruda

CD19, CD3; Murine bispecific tandem scFv PD1; Humanized IgG4

Acute lymphoblastic leukemia Melanoma

Ramucirumab Obinutuzumab

Cyramza Gazyva

Adotrastuzumab emtansine Pertuzumab Brentuximab vedotin

Kadcyla

VEGFR2; Human IgG1 CD20; Humanized IgG1; Glycoengineered HER2; humanized IgG1; immunoconjugate

Gastric cancer Chronic lymphocytic leukemia Breast cancer

Perjeta Adcetris

HER2; humanized IgG1 CD30; Chimeric IgG1; immunoconjugate

Ipilimumab Ofatumumab

Yervoy Arzerra

CTLA-4; Human IgG1 CD20; Human IgG1

Catumaxomab

Removab

Panitumumab Bevacizumab Cetuximab TositumomabI131 Ibritumomab tiuxetan Alemtuzumab

Vectibix Avastin Erbitux Bexxar

EPCAM/CD3; Rat/mouse bispecific mAb EGFR; Human IgG2 VEGF; Humanized IgG1 EGFR; Chimeric IgG1 CD20; Murine IgG2a

Breast Cancer Hodgkin lymphoma, systemic anaplastic large cell lymphoma Metastatic melanoma Chronic lymphocytic leukemia Malignant ascites

Zevalin

CD20; Murine IgG1

MabCampath, Campath-1H; Lemtrada Mylotarg

CD52; Humanized IgG1

Herceptin MabThera, Rituxan

HER2; Humanized IgG1 CD20; Chimeric IgG1

Gemtuzumab ozogamicin Trastuzumab Rituximab

CD33; Humanized IgG4

Indication first approved or reviewed Non-small cell lung cancer Neuroblastoma

Colorectal cancer Colorectal cancer Colorectal cancer Non-Hodgkin lymphoma Non-Hodgkin lymphoma Chronic myeloid leukemiab; multiple sclerosis Acute myeloid leukemia Breast cancer Non-Hodgkin lymphoma

First EU approval year In review

First US approval year In review

EC decision pending EC decision pending In review

2015

EC decision pending 2014 2014

2014

2013

2013

2013 2012

2012 2011

2011 2010

2011 2009

2009

NA

2007 2005 2004 NA

2006 2004 2004 2003b

2004

2002

2001b; 2013

2001b; 2014

NA

2000b

2000 1998

1998 1997

2014

2014

2014 2013

M

Source: Janice M. Reichert, PhD, Reichert Biotechnology Consulting LLC; table updated May 26, 2015 NA not approved or in review a Country-specific approval b Withdrawn or marketing discontinued for the first approved indication

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Mabs to cytotoxic T-lymphocyte antigen 4 (CTLA4) were the first shown to have in vivo antitumor activity. CTLA4 is a homologue of CD28 and binds to CD80 and CD86 with much higher affinity than CD28. It is expressed on ▶ regulatory T cells (Treg) and involved in their ability to downregulate an immune response. Treatment with a CTLA4-specific Mab can reverse antigen-specific T-cell tolerance by inhibiting Treg cells and improve a tumordestructive immune response. Ipilimumab, an anti-CTLA4 Mab, has been approved by the FDA for treating patients with ▶ melanoma and is being evaluated clinically also for the treatment of certain other tumors. Mabs to PD1 have also attracted much attention for tumor therapy. PD1 has homology with CD28 and is expressed on CD4+ and CD8+ T lymphocytes, B cells, and monocytes. It has two ligands, PD-L1, also known as B7-H1, and PDL-2, a.k.a. as B7-DC. Many human tumors express PDL-1 which correlates with poor prognosis. Interaction of PD-1 with its ligands inhibits T-cell activation and proliferation. Mabs to PD-1 and PD-L1 have antitumor efficacy and have been approved by FDA for clinical use. Efficacy, as seen primarily but not exclusively in patients with melanoma, has been best when Mabs to CTLA4 (ipilimumab) and PD-1 (nivolumab) have been combined. Several other Mabs are also attracting attention for modification of antitumor responses. CD137 (4-1BB) is a member of the ▶ tumor necrosis factor (TNF) receptor family. It is expressed on activated T cells, including Treg cells and NKT cells, cytokine-activated NK cells, and activated DCs, and its ligand is expressed on activated DCs, B cells, and macrophages. Agonist Mabs to CD137 have induced regression and cures of many established syngeneic mouse tumors by a mechanism thought to involve the activation of T cells that are specific for tumor antigens which are cross-presented by DCs. Tumor regression generally requires CD8+ T lymphocytes and NK cells, and CD4+ T cells are needed in most mouse models. The clinical use of anti-CD137 Mabs has so far been hampered by significant liver toxicity, but attempts are being made to overcome that problem.

Monoclonal Antibodies for Cancer Therapy

CD40 is another member of the TNF receptor family and is expressed by B cells, DCs, monocytes, ▶ macrophages, and certain tumors. Engagement of CD40 on antigen-presenting cells (APC) upregulates their expression of CD80 and CD86, causes the production of proinflammatory ▶ cytokines, and facilitates cross-presentation of antigens. Antitumor effects have been observed in some preclinical models, and several anti-CD40 Mabs are tested in cancer patients. OX40 is still another member of the TNF receptor family. It is expressed on activated CD4+ and CD8+ T lymphocytes and acts as a late costimulatory receptor for these cells. An agonist Mab to OX40 can increase tumor immunity against several transplanted mouse tumor lines. CD28, a member of the immunoglobulin superfamily, is constitutively expressed on most resting CD4+ and on about 50% of resting CD8+ T lymphocytes. After CD28 is engaged with CD80 and CD86 on DC, there is a dramatic increase of lymphocyte proliferation and cytokine secretion. Mabs to CD28 can potentiate tumor immunity when combined with antibodies to CD3. Immunomodulatory Mabs offer much promise for cancer therapy. However, these Mabs can also cause toxicity, including death, by interfering with mechanisms that normally protect against autoimmunity. In a clinical trial in March 2006, volunteers were injected with 0.1 mg per kg of TGN1412, a humanized anti-CD28 Mab, which was 500 times lower than the dose found safe in animals. Six antibody recipients were hospitalized, at least four of whom suffered from multiple organ dysfunction. There are also Mabs which can counteract the immunosuppressive tumor microenvironment by interfering with immunostimulatory molecules produced by the tumor cells and/or their stroma. ▶ Transforming growth factor-beta (TGFb) is one of the best characterized of these molecules and has been shown to promote tumor escape from the immune system. The production of TGFb can result in the accumulation of suppressive CD4+CD25+ FoxP3+ Treg cells which is

Monoclonal Antibodies for Cancer Therapy

associated with a poor clinical outcome. GC1008 is a human antibody that binds to all three isoforms of TGFb. Another approach to deplete Treg cells is treatment with CD25-specific Mab and has shown efficacy in preclinical models. A humanized CD25-specific Mab, Zenapax, has been tested in the clinic and may synergize with peptide tumor vaccines. Drug Immunoconjugates One way to improve the therapeutic efficacy of an anticancer drug is to conjugate it with a Mab to tumor antigen, i.e., an antigen that is expressed more in malignant than in normal cells. The antibody and drug are joined chemically so that the conjugate combines the tumor-binding activity of the Mab with the biological activity of the drug. Antibodies that internalize following binding to the surface of the tumor cells are preferable, and one cannot use Mabs which bind to critical tissues such as the brain. Furthermore, it is important that the immunoconjugates remain stable in the circulation and release active drug first after the conjugate has been internalized by the tumor cells via endocytosis. An immunoconjugate comprising a humanized anti-CD33 IgG with the highly potent bacterial toxin calicheamicin was approved by the FDA in 2000 for the treatment of relapsed ▶ acute myeloid leukemia. However, the approval was withdrawn 10 years later because the immunoconjugate was not more therapeutically efficient than other available therapies. Another immunoconjugate, brentuximab vedotin, combines a humanized anti-CD30 Mab with the antitubulin agent monomethyl auristatin E. It has displayed dramatic responses in patients with relapsed Hodgkin lymphoma or ▶ anaplastic large cell lymphoma, two hematologic tumors that express CD30. The conjugate was approved by the FDA in 2011 and is marketed under the trade name Adcetris for Hodgkins patients who failed bone marrow transplantation and patients with anaplastic large cell lymphoma. A different approach has been developed which is commonly referred to as ADEPT (▶ antibody-directed enzyme prodrug therapy). In this case, one first targets an antibody-enzyme conjugate to the tumor which is followed by a

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prodrug after the conjugate has attached to the tumor and been cleared from the blood. The prodrug, which is not biologically active, is converted into an active drug by the enzyme conjugate in the tumor. In spite of original promise, there is no clinically approved product based on the ADEPT concept. Immunoconjugates delivering modulators of antitumor immunity for systemic delivery and tumor localization should also be considered. Toxin Mab Constructs In the early studies, the toxic ricin A chain was combined with the cell-binding ability of an antitumor Mab. Today, most immunotoxins are recombinant molecules in which the cell-binding part of a ribosome-inactivating Pseudomonas exotoxin PE40 has been replaced with the antigen-binding (scFv) part of an antitumor Mab. The small size of the recombinant immunotoxins facilitates their penetration into tumors; only a few molecules are needed to kill a tumor cell, and the immunotoxins can be made relatively cheaply. However, since the toxins are foreign proteins, neutralizing antibodies are often formed and interfere with retreatment. Several immunotoxins have been tested in the clinic. Therapy of B-cell lymphomas with ricin A chain conjugates and a Mab to CD19 or CD22 was hampered by the development of vascular leak syndrome, while a recombinant PE40 antiCD22 immunotoxin has been effective in patients with ▶ hairy cell leukemia and caused few side effects. An analogous immunotoxin which combines PE40 with scFv from an anti-Lewis Y Mab was tested in 46 patients with Lewis Y-positive carcinomas. Therapeutic responses were observed but were too few to justify development for clinical use. Work is ongoing to overcome problems of immunogenicity and toxicity, including the vascular leak syndrome. Radioimmunotherapy ▶ Radioimmunotherapy is the targeted delivery of radiation to tumors by antitumor Mabs. Radionuclide conjugates have the advantage over most drug conjugates and ▶ immunotoxins in that they can exert a ▶ bystander effect destroying adjacent

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tumor cells that lack antigen expression. Most radioimmunotherapy uses beta particles. In clinical trials, less than 0.01% of an intravenously injected dose has been found per gram of tumor tissue, while most of the conjugates bind to normal tissues, including cells in the bone marrow to cause dose-limiting myelotoxicity. This probably explains why the therapeutic efficacy against solid tumors has been low. Localization of radioimmunoconjugates to cancer cells might be enhanced through pretargeting where the Mab is first targeted to the tumor. After clearance of circulating antibody, a radioactive agent is administered for selective capture by antibody bound to the tumor cells. An approach to decrease myelotoxicity, which has yielded promising preclinical results in rat tumor models, is to remove circulating radiolabeled Mab via plasmapheresis performed 24–48 h after injection of the radiolabeled Mab. Another approach to increase therapeutic efficacy, which is supported by preclinical findings, is to combine radioimmunotherapy with immunoconjugates, check point inhibiting Mabs or a tumor vaccine. There are two FDA-approved radiolabeled anti-CD20 IgG Mabs for the treatment of patients with ▶ follicular lymphoma and transformed non-Hodgkin lymphoma who failed or relapsed from prior therapies,90Y-ibritumomabtiuxetan (Zevalin) and131I tositumomab (Bexxar). Patients given these radiolabeled Mabs showed a significant increase in overall response compared with patients given an unlabeled version of the same Mab (▶ Rituximab). Bispecific Antibodies ▶ Bispecific antibodies simultaneously target tumor epitopes and surface molecules expressed on lymphocytes to engage a T-cell-mediated immune response to tumor antigens. Their clinical efficacy has been limited, but more effective approaches are being developed. Bispecific T-cell engager (BiTE) molecules are formed by linking two Fv fragments using flexible linkers to target both CD3 and a tumor marker. BiTE molecules have shown to be stable, give enhanced tumor cell lysis, and display efficacy at low T-cell/target cell ratios.

Monoclonal Antibodies for Cancer Therapy

Induction of an Immune Response by Mabs to Tumor Antigens Tumor peptides released from cells that have been killed via CDC or ADCC or some other mechanism can combine with antitumor Mabs (e.g., Rituximab) and taken up, processed, and presented by ▶ dendritic cells (DCs) to cause the generation of an adaptive T cell response, and they can be crosspresented by major histocompatibility complex (MHC) class I molecules to activate CD8+ cytolytic T cells. For example, MUC-1 (▶ mucins)-specific T-cell responses have been described in patients treated with an anti-MUC-1 antibody. Uptake of tumor antigen combined with antibody can induce either immunity or tolerance, depending on several factors including the nature of the Fc receptors on the DC to which the antibody part binds. It may be possible to engineer antibody molecules to selectively engage activating as opposed to inhibitory Fcg receptors. It is likely that induction of an immune antitumor response plays a role in the therapeutic activity of “naked” Mabs.

Anti-idiotypic Antibodies The variable regions of an antibody express antigenic determinants, called idiotype, which are unique to each antibody. These can trigger immune responses, including the formation of anti-idiotypic antibodies, some of which can act as surrogate antigen. Cancer patients can develop anti-idiotypic antibodies following the administration of antitumor Mabs. Although these antibodies shorten the half-life of the injected antitumor Mab, the presence of anti-idiotypic antibodies has been reported to be associated with a favorable clinical response in some patients who develop cellular and humoral immunity to the tumor. Anti-idiotypic Mabs with well-defined characteristics can be produced in large amounts for clinical use. Although therapeutic responses have been described in a few patients, these are few and there is no FDA-approved antiidiotypic Mab for therapeutic use at this time.

Monoclonal Antibodies for Cancer Therapy

Therapeutic Vaccination with Tumor Cells Expressing Mab-Derived scFv Fragments at Their Surface ▶ Cancer vaccines can be constructed to combine the immune stimulation of a tumor antigen together with that of a Mab by transfecting tumor cells to express, at their surface, singlechain Fv fragments (scFv) from an immunostimulatory Mab. Mouse melanoma cells expressing scFv from an anti-CD137 Mab induced a strong immune response involving CD4+ T lymphocytes and NK cells which caused the rejection of small established wild-type melanomas growing subcutaneously in the lung. Combination Therapy Many Mabs to tumor antigens work additively or even synergistically with anticancer drugs, and most naked Mabs to tumor antigens are used in combination with ▶ chemotherapy, since exposure to the Mabs often increases the response to the anticancer drug. For example, a combination of the anti-CD20 Mab Rituxan or of the antiHER2 Mab ▶ Herceptin with chemotherapy is more effective than either therapy used alone. Mabs to tumor antigens can also be combined with agents that promote antigen presentation, for example, toll receptor agonists, costimulation, or T-cell activation and expansion, with Mabs to checkpoint inhibitors, and with tumor vaccination. Combinations of Mabs to several antigens on the same tumor should also be considered. Mabs can also be combined with therapeutic cancer vaccines and with adoptive transfer of tumor-reactive T lymphocytes. Using GM-CSF-producing melanoma cells as a vaccine and adding a Mab to CTLA4 were more effective in eradicating established B16 tumors than vaccination alone. Complications of Mab-Based Therapy The most common side effects of Mab therapy have been fever, chills, weakness, headache, nausea, vomiting, diarrhea, low blood pressure, and rashes. In addition, side effects have been observed which are caused by binding of the Mab to normal tissues, for example, the binding of both the “naked” and doxorubicin-conjugated

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anti-Lewis-Y Mab BR96 to epithelial cells in the stomach and intestine to cause dose-limiting gastrointestinal toxicity. The disastrous effect of injecting volunteers with the anti-CD28 Mab TGN1412 is referred to above. Antitumor Mabs are often ineffective for the treatment of bulky lesions. This may be due to the low level of injected Mab that actually reaches its target within a large solid tumor mass. Studies with radiolabeled Mabs suggest that only 0.01–0.1% per gram of tumor tissue will reach the target antigen within a solid tumor. This low level of binding is due to a series of barriers confronting an i.v. administered Mab in route to tumor, including the endothelial barrier, interstitial pressure, and stromal impediments.

Cross-References ▶ Activated Natural Killer Cells ▶ Acute Myeloid Leukemia ▶ Anaplastic Large Cell Lymphoma ▶ Angiogenesis ▶ Antibody-Directed Enzyme Prodrug Therapy ▶ Apoptosis ▶ Bispecific Antibodies ▶ Breast Cancer ▶ Bystander Effect ▶ Cancer Vaccines ▶ Cetuximab ▶ Chemotherapy ▶ Colorectal Cancer Clinical Oncology ▶ Complement-Dependent Cytotoxicity ▶ Cytokine ▶ Dendritic Cells ▶ Epidermal Growth Factor Receptor ▶ Follicular Lymphoma ▶ Hairy Cell Leukemia ▶ Herceptin ▶ Hodgkin Disease ▶ Hodgkin Lymphoma, Clinical Oncology ▶ Immunotherapy ▶ Immunotoxins ▶ Lewis Antigens ▶ Macrophages ▶ Mucins ▶ Radioimmunotherapy

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▶ Regulatory T Cells ▶ Rituximab ▶ Targeted Drug Delivery ▶ Transforming Growth Factor-Beta ▶ Transgenic Mouse ▶ Tumor Necrosis Factor ▶ Vascular Endothelial Growth Factor

References Alewine C, Hassan R, Pastan R (2015) Advances in anticancer immunotoxin therapy. Oncologist 20:1–10 Curran M, Montalvo W, Yagita A, Allison J (2010) PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci 107:4275–4280 Dai M, Yip Y, Hellstrom I, Hellstrom K (2014) Curing mice with large tumors by locally delivering combinations of immunomodulatory antibodies. Clin Cancer Res 21:1127–1138 Hellstrom I, Ledbetter JA, Scholler N, Yang Y, Ye Z, Goodman G, Pullman J, Hayden-Ledbetter M, Hellstrom KE (2001) CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer. Proc Natl Acad Sci 98:6783–6788 Kyi C, Postow M (2014) Checkpoint blocking antibodies in cancer immunotherapy. FEBS Lett 588:368–376 Lameris R, de Bruin R, Schneiders F, van Bergen en Henegouwen P, Verheul H, de Gruijl T, van der Vliet H (2014) Bispecific antibody platforms for cancer immunotherapy. Crit Rev Oncol Hematol 92:153–165 Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen LP (2007) Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer 7:95–106 Mendelsohn J, Prewett M, Rockwell P, Goldstein N (2015) CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth. Clin Cancer Res 21:227–229. CCR 20th anniversary commentary Smaglo B, Aldeghaither D, Weiner L (2014) The development of immunoconjugates for targeted cancer therapy. Nat Rev Clin Oncol 11:637–648 Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10:317–327 Wolchok JD et al (2013) Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 369:122–133 Ye Z, Hellstrom I, Hayden-Ledbetter M, Dahlin A, Ledbetter JA, Hellstrom KE (2002) Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat Med 8:343–348 Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, Forero-Torres A (2010) Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 363:1812–1821

Monoclonal Antibodies for Cancer Therapy

See Also (2012) Alemtuzumab. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 127. doi:10.1007/978-3-642-16483-5_177 (2012) Avastin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 323. doi:10.1007/978-3-642-16483-5_6577 (2012) CD4. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 698. doi:10.1007/978-3-642-16483-5_914 (2012) CD20. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 693. doi:10.1007/978-3-642-16483-5_922 (2012) CD30. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 697. doi:10.1007/978-3-642-16483-5_926 (2012) CD33. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 698. doi:10.1007/978-3-642-16483-5_927 (2012) CD40. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 699. doi:10.1007/978-3-642-16483-5_6852 (2012) CD52. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 702. doi:10.1007/978-3-642-16483-5_932 (2012) FDA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1386. doi:10.1007/978-3-642-16483-5_2136 (2012) Gemtuzumab ozogamicin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1522. doi:10.1007/978-3-642-164835_2361 (2012) Growth factor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1607–1608. doi:10.1007/978-3-642-164835_2520 (2012) Growth factor receptors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1608. doi:10.1007/978-3-642-164835_2521 (2012) HER2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1678. doi:10.1007/978-3-642-16483-5_2676 (2012) Hybridomas. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1775. doi:10.1007/978-3-642-16483-5_2884 (2012) Idiotype. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1804. doi:10.1007/978-3-642-16483-5_2944 (2012) Immunoglobulin genes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1819. doi:10.1007/978-3-642-164835_2992 (2012) Isoform. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1920–1921. doi:10.1007/978-3-642-16483-5_3158 (2012) Lymphocytes. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2123. doi:10.1007/978-3-642-16483-5_3455

Monoclonal Gammopathy of Undetermined Significance (MGUS) (2012) Monoclonal antibody. In: Schwab M(ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Monocyte. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2371. doi:10.1007/978-3-642-16483-5_3825 (2012) Non-Hodgkin lymphoma. In: Schwab M(ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4110 (2012) Polymorphism. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2954–2955. doi:10.1007/978-3-642-16483-5_4673 (2012) Prodrug. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2989. doi:10.1007/978-3-642-16483-5_4751 (2012) Proliferation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3004. doi:10.1007/978-3-642-16483-5_4766 (2012) Radionuclide. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3154. doi:10.1007/978-3-642-16483-5_4921 (2012) Stroma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3541. doi:10.1007/978-3-642-16483-5_5532 (2012) Transgenic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3763. doi:10.1007/978-3-642-16483-5_5919

Monoclonal Antibody c-erb-2 ▶ Trastuzumab

Monoclonal Antibody HER2 ▶ Trastuzumab

Monoclonal Gammopathy of Undetermined Significance (MGUS) Siegfried Janz Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

Synonyms Monoclonal gammopathy of unknown significance and monoclonal gammopathy of uncertain

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significance are synonyms. MGUS was formerly known as benign monoclonal gammopathy (BMG), but usage of this term has been discouraged because it implies that the condition is not harmful in effect. To highlight findings that MGUS may cause kidney and skeletal damage in a subset of patients, the term has been modified in the biomedical literature to monoclonal gammopathy of renal significance and monoclonal gammopathy of skeletal significance, respectively.

Definition Monoclonal gammopathy of undetermined significance (MGUS) (Agarwal and Ghobrial 2013; Landgren 2013) is defined by a serum paraprotein (monoclonal immunoglobulin or mIg, M-spike, M-protein) that amounts to 30 g/L or less in individuals that (a) contain 10% clonal, immunoglobulin-producing cells in the hematopoietic bone marrow and (b) do not exhibit the sort of end-organ damage (e.g., anemia, lytic bone lesions, hypercalcemia, renal insufficiency) commonly seen in patients with multiple myeloma (MM), Waldenström macroglobulinemia (WM), and related B-cell and plasma-cell neoplasms. MGUS is usually detected as an incidental or random finding in the course of standard laboratory tests including serum protein electrophoresis as diagnostic tool. MGUS is a common condition, particularly in elderly individuals in which the prevalence may reach 5%. MGUS does not require treatment, but the need for monitoring is lifelong due to the propensity of MGUS cell clones to continue on the pathway of malignant cell transformation and progress to frank neoplasia. Thus, plasmacytic MGUS (~80% of cases), which is characterized by IgG or IgA M-spikes or IgD or IgE M-spikes in rare cases, may progress to MM. The rate of progression is slow but remarkably steady: 1% per year on average. Lymphoplasmacytoid MGUS clones expressing IgM (~20% of cases) may progress to MW at an annual rate of 1.5% on average. In both cases, MM and MW, frank neoplasia may be preceded by “smoldering” and largely asymptomatic precursor conditions, in which the underlying

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Monoclonal Gammopathy of Undetermined Significance (MGUS)

mIg-producing cell clone and concomitant serum paraprotein exhibit a significant increase, yet end-organ damage is lacking. These conditions have been designated smoldering MM (SMM) or smoldering WM (SWM). They are targets of ongoing clinical tumor prevention trials aimed at blocking the transition from smoldering disease to full-blown blood cancer. Our current understanding of MGUS is owed in large measure to the pioneering work of Dr. Robert A. Kyle at the Mayo Clinic in Rochester, Minnesota, who coined the terms MGUS and SMM in 1978 and 1980, respectively, and has educated hematologists and driven the field ever since (Fig. 1).

Characteristics Epidemiology A comprehensive population-based study on the prevalence of MGUS in persons 50 years of age or older used serum samples from more than 21,000 residents of Olmsted County, Minnesota, to find MGUS in 694 (3.2%) individuals. The age-adjusted rates were greater in men (4.0%) than in women (2.7%). The prevalence of MGUS was 5.3% among persons 70 years or older and 7.5% among persons 85 years or older. The concentration of M-protein was less than 10 g/L in the majority (64%) of cases. Of 79 patients tested, 21% had monoclonal urinary light chain (Bence-Jones protein). The prevalence of MGUS was 8.6% in black individuals compared with 3.6% in white individuals. This race-related difference was confirmed in independent studies of patients from US Veteran Affairs hospitals, demonstrating that the age-adjusted prevalence rate for MGUS was 3.0fold higher in African Americans than in Americans of Caucasian ancestry. Etiology The etiology of MGUS is not known. Epidemiological evidence points to age, gender, and race as genetic risk factors. Pathogenesis MGUS is a highly consistent precursor condition of MM and WM. Remarkably, studies on the

molecular genetic and genomic makeup of MGUS cell clones have revealed a large amount of similarities to their malignant counterparts, but very little in the way of differences. This has raised thorny questions why MGUS is a stable, nonmalignant disorder in the great majority of affected individuals but undergoes progression to frank neoplasia in a small subset of cases. The underlying mechanism of tumor progression may be complex, possibly involving the breakdown of immune surveillance pathways that normally limit MGUS to the premalignant disease stage. The acquisition of yet-to-be-discovered genetic and epigenetic changes in MGUS cells and changes in the microenvironment of resident MGUS clones may also play important roles. For example, with respect to the latter, a shift in the balance of pro- and anti-angiogenic factors that results in increased angiogenesis in the bone marrow may contribute to the MGUS-to-MM/WM transition. The reader is referred to the MM and WM sections of the encyclopedia for in-depth information on the natural history of these neoplasms. Diagnosis Serum protein electrophoresis in agarose gels provides a good screening method to detect the presence of a monoclonal immunoglobulin aka mIg, M-protein, M-spike, and paraprotein. Electrophoretic analysis is usually followed by immunofixation, which confirms the monoclonal immunoglobulin and determines the underlying isotype of the Ig heavy chain (g1-4, m, a, e, d) and Ig light chain (k, l). The concentration of the M-protein is then measured with a rate nephelometer, which permits an estimate of the size of the MGUS cell clone, which is, in turn, useful for patient monitoring. Serum M-protein higher than15 g/L should lead to a test for Bence-Jones proteinuria, i.e., the urinary secretion of a monoclonal Ig k or l light chain. This can be accomplished by electrophoresis and immunofixation of a concentrated aliquot from a 24-h urine specimen. Measurement of serum levels of free light chains (FLCs) is also useful for diagnostic purposes, as deviations from the normal FLC k-to-l ratio (0.26–1.65) are a strong indication of MGUS and related monoclonal plasma-cell disorders.

Monoclonal Gammopathy of Undetermined Significance (MGUS)

b IgG,

β globulins

Albumin

α globulins

β1

Normal

γ globulins

MGUS

β2

M spike M protein Extragradient Paraprotein mIg

IgA

IgM

SMM

MM

MGUS SWM

WM

Monoclonal Gammopathy of Undetermined Significance (MGUS), Fig. 1 Detection of MGUS using serum protein electrophoresis and evolution of MGUS into frank cancer. Panel (a) shows representative schemes of serum protein electropherograms from a healthy individual (bottom) or person with MGUS (top). Serum protein electrophoresis is a laboratory technique in which the blood serum (i.e., the liquid portion of the blood after it has clotted) is placed into a gel or capillary tube, followed by the exposure of the serum sample to an electric current that separates the serum proteins according to size and electrical charge into five major fractions: serum albumin, a1 globulins, a2 globulins, b globulins, and g globulins. In case of MGUS, a narrow spike in the g-globulin fraction is readily detected. This spike is caused by the abnormal abundance of a monoclonal immunoglobulin (M-component, paraprotein, M-spike) secreted by MGUS cells. The M-spike is narrow because the underlying protein is homogeneous (one molecular species with identical physicochemical features) and, therefore, exhibits the same electrophoretic mobility. Panel (b) depicts pathways of tumor progression by which MGUS evolves into fullblown neoplasia. MGUS is widely believed to derive from a mature post-germinal center B-lymphocyte that expresses immunoglobulin on the cell surface (not shown). This cell acquires tumor-initiating genetic

MGUS Variants • Biclonal gammopathies are characterized by the presence of two different M-proteins, which occurs in ~4% of patients with MGUS and related monoclonal gammopathies. Rare cases of triclonal gammopathies have also been reported. • Idiopathic Bence-Jones proteinuria is defined as urinary excretion of sometimes large amounts of monoclonal Ig light chain (i.e., Bence-Jones protein) in the absence of end-organ damage, particularly kidney damage. The condition is rare because BenceJones proteins are usually associated with B-cell and plasma-cell neoplasms. • IgD MGUS almost always indicates MM, amyloidosis, or plasma-cell leukemia. Nonetheless, 6–8-year follow-ups in rare patients without evidence of progression to malignancy have been reported. • IgE MGUS is extremely rare. • IgM MGUS is not always a precursor of WM; instead, in rare cases the condition can also evolve into IgM+ myeloma. Association of MGUS with Other Diseases • Lymphoproliferative disorders such as chronic lymphocytic leukemia/small lymphocytic lymphoma and marginal zone lymphoma. Rarely in cases of leukemia, e.g., acute leukemia, hairy cell leukemia, T cell leukemias, and chronic myelocytic leukemias. • Other hematological diseases including acquired von Willebrand’s disease, pernicious ä

a

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, Fig. 1 (continued) and epigenetic changes that lead to clonal expansion and establishment of an abnormal cell clone. MGUS producing IgG or IgA may undergo progression to multiple myeloma (MM), sometimes via an intermediate disease stage referred to as smoldering MM (SMM). Similarly, MGUS clones producing IgM exhibit proclivity to progress to Waldenström macroglobulinemia (WM), often via an asymptomatic intermediate called smoldering WM (SWM). MM may further progress to plasmacell leukemia (not shown), a fatal blood cancer. MGUS, SMM/SWM, and frank MM/WM comprise a continuum of tumor development characterized by steadily increasing neoplastic potential

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• •

• • •



Monoclonal Gammopathy of Undetermined Significance (MGUS)

anemia, refractory anemia, polycythemia vera, idiopathic myelofibrosis, and Gaucher’s disease. Connective tissue disorders, such as rheumatoid arthritis, lupus erythematosus, scleroderma, polymyositis, and ankylosing spondylitis. Motor neuron and other neurological diseases including amyotrophic lateral sclerosis and spinal muscular atrophy. Rare dermatological diseases, such as lichen myxedematosus, pyoderma gangrenosum, necrobiotic xanthogranuloma, and Schnitzler syndrome (chronic urticaria plus IgM monoclonal gammopathy). Liver disease associated with hepatitis C virus (HCV). Immunosuppression, frequently after transplantation. Miscellaneous conditions, e.g., acquired angioedema, systemic capillary leak syndrome, and idiopathic focal and segmental glomerulosclerosis. IgM MGUS deserves special attention because of IgM-related autoimmune disorders. For example, in IgM MGUS neuropathy, the M-protein exhibits binding activity to myelinassociated glycoprotein (MAG).

Antibody Activity of mIg and Associated Diseases M-proteins, particularly IgM, can exhibit immunologic specificity (antibody activity) to certain self-antigens and/or foreign antigens, possibly leading to full-blown autoimmune disorders, such as chronic cold agglutinin disease, type II mixed cryoglobulinemia, and peripheral neuropathy. The self/foreign antigens implicated in these disorders are I/i red cell antigens/bacterial lipopolysaccharides, IgG Fc/hepatitis C virus, and neural carbohydrates/bacterial lipopolysaccharides, respectively. M-proteins with specificity for actin, von Willebrand’s factor, thyroglobulin, insulin, riboflavin, dextran, antistreptolysin-O, double-stranded DNA, apolipoprotein, thyroxin, lactate dehydrogenase, and

several antibiotics have also been detected in rare cases. Aggregation and Deposition of mIg and Associated Diseases Independent of their immunologic specificity but dependent upon their concentration and physicochemical properties, M-proteins can become insoluble in the aqueous phase (serum, urine). This may result in homotypic aggregation (precipitation) and formation of mIg deposits in various tissue sites. Diseases associated with protein depositions of this sort include AL amyloidosis, light-chain deposition disease, light-chain cast nephropathy, adult Fanconi syndrome due to crystal-storing histiocytosis, and cryoglobulinemia type I. Predictors of Tumor Progression At the time of recognition of MGUS, one cannot distinguish with certainty an individual whose condition will remain stable from one in whom progression to MM of WM will occur. Parameters that can predict progression include the serum level of the M-protein and its heavy-chain isotype. For example, IgA and IgM carry a higher risk of progression compared to IgG. The abundance of bone marrow plasma cells and degree of deviation from a normal serum free light-chain (FLC) ratio are additional parameters. One should keep in mind that even in individuals at high risk of progression, death from unrelated diseases is greater than death from MM or WM. Based on advances in immunophenotyping of plasma cells (PCs) and methods of serum Ig and FLC determination, two independent US Mayo Clinic and Spanish PETHEMA study groups developed risk stratification schemes for the MGUS-to-MM progression. The Mayo Clinic scheme, which relies on serum Ig analysis, includes M-protein levels (>15 g/L), type of M-protein (non-IgG MGUS), and shift from a normal FLC ratio (1.65) as independent risk factors for tumor progression. Evidence shows that this scheme is useful for identifying high-risk SMM patients that may benefit from early treatment. The PETHEMA scheme, which relies on flow cytometric analysis

Mother Against Decapentaplegic, Drosophila, Homolog of 4

of surface markers and ploidy status of PCs, uses multiparameter flow cytometry of bone marrow cells to quantify the ratio of abnormal (neoplastic) and normal PCs. At 5 years of follow-up, three groups of MGUS exhibiting a small (2%), intermediate (10%), or high (46%) risk of tumor progression could be distinguished. A similar study on the MGUS-to-WM transition has identified two independent risk factors for this pathway of tumor progression: serum levels of mIgM and albumin (Kyle et al. 2013).

Management of MGUS and Development of Early Treatment Strategies Although patients with MGUS do not require treatment, they do need indefinite follow-up. Serum electrophoresis should be repeated 6 months after initial presentation of an M-protein and, if stable, annually thereafter. Low-risk MGUS – characterized by serum M-protein 12 mitosis figures per 10 highpower fields. Less than 5% of serous carcinomas are classified as “low grade.” They show a predominantly micropapillary architecture, less cytologic atypia, and a low mitotic rate (mostly 10 cm diameter) at the time of diagnosis. In contrast, metastatic mucinous tumors from the intestinal tract (including colorectum, stomach, and pancreas) tend to be bilateral and smaller (7 cm, two or more mitoses per 10 high-power fields, marked nuclear pleomorphism, hemorrhage, and necrosis. Sex Cord Tumor with Annular Tubules SCTAT: this extremely rare tumor is associated with ▶ Peutz-Jeghers syndrome in 30%. However, only cases not associated with this syndrome have been reported as malignant. Germ Cell Tumors

Germ cell tumors account for approximately 30% of ovarian tumors, 95% of which are benign mature cystic teratomas, usually dermoid cysts. Malignant germ cell tumors include immature teratomas, dysgerminomas, yolk sac tumors, embryonal carcinomas, polyembryomas, and choriocarcinomas. Choriocarcinoma is a type of gestational trophoblastic tumor: any of a group of tumors that develops from trophoblastic cells (cells that help an embryo attach to the uterus and help form the placenta) after fertilization of an egg by a sperm. The two main types of gestational trophoblastic tumors are hydatidiform mole and choriocarcinoma. They occur in children, adolescents, and young adults, a characteristic they have in common. They are highly aggressive neoplasms, but with the advent of modern oncotherapy, the prognosis has become much better, and many can be cured. Teratoma Immature teratoma accounts for 1% of ovarian cancers. They usually show an admixture of mature and immature tissues. The most commonly found immature component is embryonic neural tissue that typically forms the pathognomonic “neural tubules” (Fig. 7). Immature cartilage and muscle can also be seen. While immature teratomas are seen in young patients, malignization of a mature teratoma can occur in elderly people, most frequently as squamous cell carcinoma arising from a dermoid cyst. Many other types of malignancies have been encountered in ovarian teratomas, including carcinoid tumors and melanomas.

Ovarian Cancer Pathology, Fig. 7 Immature teratoma. Several immature neural tubules (solid arrows) adjacent to mature mucinous epithelial structures (bottom left) (dotted arrow). Hematoxylin/eosin; original magnification 40

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Ovarian Cancer Pathology, Fig. 8 Dysgerminoma. Tumor cells (arrows) have big nuclei, prominent nucleoli, and pale cytoplasm. Note lymphocytic infiltrate. Hematoxylin/eosin; original magnification 400

Dysgerminoma Dysgerminomas make up 1% of ovarian cancers. Microscopically, they are identical to seminomas of the testes, characterized by large aggregates of mostly uniform cells with clear or pale cytoplasm and high mitotic rate, and surrounded by connective tissue septa with a prominent lymphocytic infiltrate (Fig. 8). Yolk Sac Tumor These tumors can form a large variety of architectural patterns, including solid, microcystic, myxomatous, papillary, polyvesicular vitelline, and glandular areas. Schiller-Duval bodies (papillary structures containing a blood vessel

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Ovarian Cancer Pathology, Fig. 9 Yolk sac tumor. Schiller-Duval body in the center. Hematoxylin/eosin; original magnification 100

surrounded by malignant cells) are present in up to three fourths of these tumors and considered pathognomonic (Fig. 9). Most yolk sac tumors also show hyaline bodies (eosinophilic PAS-positive globules) and are positive for ▶ alpha-fetoprotein on ▶ immunohistochemistry. Embryonal Carcinoma This very rare tumor is identical to embryonal carcinoma of the testes. It is composed of large immature cells with numerous mitotic figures, forming sheets, nests, glandular, or papillary structures. Choriocarcinoma While most choriocarcinomas are related to a (molar) pregnancy, an identical tumor may very rarely occur in the ovaries without associated pregnancy. These tumors are very hemorrhagic. Microscopically, they are characterized by the admixture of two cell populations: uninucleated trophoblast cells with clear cytoplasm and distinct cell borders and large multinucleated syncytiotrophoblasts with vacuolated cytoplasm and irregular borders. Syncytiotrophoblasts are immunoreactive for human chorionic gonadotropin (b-hCG), which is also elevated in the patients’ serum. Polyembryoma This exceedingly rare tumor contains a variable number of embryoid bodies, which are structures resembling an amniotic cavity with embryonic disk and a yolk sac.

Ovarian Cancer Pathology

Ovarian Cancer Pathology, Fig. 10 Krukenberg tumor: metastatic gastric carcinoma to the ovaries, forming signet ring cells (arrows). Hematoxylin/eosin; original magnification 400

Metastatic Tumors to the Ovary

Many types of cancers can metastasize to the ovary. They can come from within the female genital tract (cervix, endometrium, fallopian tube) or from more distant origins. Generally, tumors metastatic to the ovaries tend to present bilaterally. The most frequent sites of distant primary tumors that metastasize to the ovaries include the breast (10% of breast carcinoma cases), stomach (the classic Krukenberg tumor is composed of signet ring cells, Fig. 10), large intestine, appendix (often associated with pseudomyxoma peritonei), intestinal carcinoid tumors, pancreas, and melanoma (may be darkly pigmented). Selected Diagnostic Markers Diagnostic markers are used to confirm a diagnosis if the tumor morphology is not entirely conclusive. The most useful markers include the following: • Epithelia of Müllerian origin are generally positive for cytokeratin (CK) 7, but negative for CK 20. The reverse is true for epithelium of the colorectum and appendix. In addition, intestinal epithelial express CDX2. These markers are used to differentiate primary ovarian carcinomas from metastatic intestinal carcinomas. • WT-1 (▶ Wilms tumor gene protein) is positive in serous ovarian carcinomas and helps distinguish them from poorly differentiated

Ovarian Cancer Stem Cells Ovarian Cancer Pathology, Table 1 Cancer staging systems AJCC T1

FIGO I

T1a

IA

T1b

IB

T1c

IC

T2

II

T2a

IIA

T2b

IIB

T2c

IIC

T3

III

T3a

IIIA

T3b

IIIB

T3c

IIIC

Any T, N1 Any T, M1

IIIC IV

Definition Tumor confined to one or both ovaries Tumor limited to one ovary; no surface involvement; pelvic washings negative for malignant cells Tumor involves both ovaries; no surface involvement; pelvic washings negative for malignant cells Tumor involves one or both ovaries; tumor is present on the serosal surface, or pelvic washings are positive for malignant cells Tumor involves one or both ovaries with pelvic extension Extension to uterus and/or fallopian tubes; pelvic washings negative for malignant cells Extension to other pelvic tissues; pelvic washings negative for malignant cells Extension to pelvic tissues; pelvic washings positive for malignant cells Tumor involves one or both ovaries with extension beyond the pelvis, but confined to the peritoneal cavity Microscopic (but not macroscopic) peritoneal metastases Macroscopic peritoneal metastases 2 cm in greatest dimension

endometrioid carcinomas and metastatic carcinomas from distant sites, e.g., breast. • a-Inhibin is expressed in sex cord stromal tumors. • a-Fetoprotein (alpha-fetoprotein) is expressed in yolk sac tumors. • b-hCG (human chorionic gonadotropin) is expressed in the syncytiotrophoblast cells of choriocarcinoma?.

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Staging of Ovarian Cancer The stage of ovarian cancer at the time of diagnosis is the single most important prognostic factor. While pathologists are required to provide a cancer stage in the tumor/lymph node/metastasis (TNM) format developed by the American Joint Committee on Cancer (AJCC), gynecologic oncologists tend to prefer the Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) stage classification. Table 1 summarizes both staging systems.

References Kurman RJ (2002) Blaustein’s pathology of the female genital tract, 5th edn. Springer, New York McCluggage WG (2008) My approach to and thoughts on the typing of ovarian carcinomas. J Clin Pathol 61: 152–163 Mills SE et al (2009) Sternberg’s diagnostic surgical pathology, 5th edn. Lippincott Williams & Wilkins, Baltimore Scully R, Young R, Clement P (1998) Tumors of the ovary, maldeveloped gonads, fallopian tube and broad ligament. Armed Forces Institute of Pathology Atlas of Tumor Pathology, Washington, DC

Ovarian Cancer Stem Cells Roman Mezencev Georgia Institute of Technology, School of Biology, Atlanta, GA, USA

Synonyms Ovarian cancer-initiating cells; Ovarian cancer stem-like cells

Definition Consistent with the cancer stem cell (CSC) hypothesis, ovarian cancer stem cells represent a subset of cancer cells lying at the top of hierarchy of all malignant cells that form the bulk of ovarian cancers. Cancer stem cells display ability to

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Ovarian Cancer Stem Cells

continually sustain tumor growth through their indefinite self-renewal and ability to generate all cancer cell lineages (transit-amplifying cells, differentiated cells) found in particular tumors through their (aberrant) differentiation.

Characteristics Classification of Ovarian Cancers Ovarian cancers represent a highly heterogeneous group of diseases with distinct cellular origin, epidemiology, pathogenesis, morphological and molecular characteristics, and clinical course. Considering cell types from which ovarian cancers presumably originate, most ovarian cancers can be classified into three major categories: epithelial (~90%), sex cord-stromal (~8%), and germ cell (~2%) malignancies, each of which includes a number of subtypes. Serous ovarian carcinomas, which represent approximately 70% of epithelial ovarian cancers, can be subclassified based on their cytological features (degree of nuclear atypia and mitotic rate) into high-grade serous ovarian carcinoma (HGSOC) and low-grade serous ovarian carcinoma (LGSOC). HGSOC and LGSOC do not represent two grades of the same disease but rather two distinct tumor types with different molecular pathogenesis, clinical behavior, and prognosis. HGSOC accounts for about 90% of serous ovarian cancers and majority of ovarian cancer stem cell research employed cells and tissues related to this specific tumor type. As a result, the body of knowledge on ovarian cancer stem cell available at the present time and discussed in this text largely reflects characteristics of cancer stem cells relevant to the high-grade serous ovarian carcinoma. Isolation, Identification, and Characterization of Ovarian CSCs Originally, cancer stem cells were identified as a small subset (20% CD44-positive cells tend to display poorer response to carboplatin-paclitaxel treatment and significantly shorter progression-free survival compared to patients with 10 cm diameter) at the time of diagnosis. In contrast, metastatic mucinous tumors from the intestinal tract (including colorectum, stomach, pancreas) tend to be bilateral and smaller (12 mitosis figures per ten high-power fields. Less than 5% of serous carcinomas are classified as “low grade.” They show a predominantly micropapillary architecture, less cytologic atypia, and a low mitotic rate (mostly < five mitotic figures per ten high-power fields), tend to occur in a slightly younger population, and have a somewhat better prognosis. Low-grade and high-grade serous carcinomas appear to evolve through different pathogenetic mechanisms. This theory is supported by divergent molecular genetic profiles, including the frequent occurrence of ▶ KRAS/BRAF1 (▶ BRaf-Signaling) mutations (but wild-type ▶ TP53) in low-grade

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Ovarian Small Cell Carcinoma

Ovarian Serous Carcinoma, Fig. 1 Ovarian serous carcinoma, high grade. Note papillary structures and slit-like spaces between them. Hematoxylin and eosin staining, original magnification  40, ▶ ovarian cancer pathology

carcinomas, whereas the majority of high-grade serous carcinomas have TP53 mutations but wild-type KRAS/BRAF (Fig. 1).

Ovarian Small Cell Carcinoma ▶ Ovarian Tumors During Childhood and Adolescence

Cross-References ▶ BRaf-Signaling ▶ KRAS ▶ Ovarian Cancer ▶ Ovarian Cancer Pathology ▶ TP53

See Also (2012) Atypia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 303. doi:10.1007/978-3-642-16483-5_449 (2012) FIGO. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1407. doi:10.1007/978-3-642-16483-5_2188 (2012) Hematoxylin and eosin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1645. doi:10.1007/978-3-64216483-5_2623 (2012) Mitosis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2342. doi:10.1007/978-3-642-16483-5_3774 (2012) Psammoma bodies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3112. doi:10.1007/978-3-64216483-5_6927 (2012) Serous. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3389. doi:10.1007/978-3-642-16483-5_6936

Ovarian Small Cell Carcinoma Hypercalcemic Type Definition Small cell carcinoma of the ovary is a rare malignant ▶ ovarian cancer. It is the most common undifferentiated ovarian carcinoma in young women. Approximately two thirds of patients with ovarian small cell carcinoma have hypercalcemia, i.e., increased serum calcium level. The mechanism of development of hypercalcemia is unclear, although parathyroid hormone-related protein has been found in some of the cases, possibly suggesting that ectopic parathyroid hormone production by the tumor cells may be the cause of hypercalcemia. This tumor occurs predominantly in young women. The 1-year survival is only 50%, with an overall 5-year survival rate of approximately 10%. It is believed that the empirical treatment characterized by combination of radical surgery, chemotherapy, and radiotherapy results in the most favorable outcome in terms of survival. However, the

Ovarian Stromal and Germ Cell Tumors

outcome remains extremely poor despite this aggressive approach.

Cross-References ▶ Ovarian Cancer

Ovarian Stromal and Germ Cell Tumors Richard T. Penson Division of Hematology Oncology, Massachusetts General Hospital, Boston, MA, USA

Definition Cancers arising from the stromal and germ cell layers of the ovary are rare, heterogeneous, difficult to study, and require specialized multidisciplinary management. They more commonly present in younger patients and have a high cure rate. These tumors are associated with serum biomarkers that are informative for diagnosis and surveillance. Surgery is often part of primary treatment, with staging and preservation of fertility as important priorities. Most patients with germ cell tumors require adjuvant chemotherapy (▶ ovarian cancer chemotherapy) with ▶ bleomycin, ▶ etoposide, and ▶ cisplatin, as well as careful surveillance.

Characteristics Within the sex-cord-stromal and germ cell tumors, a variety of histopathological subtypes exist, and together they account for approximately 5% of ovarian neoplasms. They generally present unilaterally, at early stages, and have a relatively good prognosis, with the germ cell tumors being very The entry “Ovarian Stromal and Germ Cell Tumors” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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sensitive to chemotherapy. Patient age can be a predictive factor for diagnosis of non-epithelial ovarian cancers. Germ cell tumors are much more frequent among young women (▶ ovarian tumors during childhood and adolescence), with a median age of diagnosis at 26 years compared with median age at diagnosis for all ovarian cancers of 60 years. Median age at diagnosis for sex-cord-stromal tumors is 50 years. Normal ovarian stroma is responsible for hormone production, and sex-cord-stromal cancers are often hormonally active or secrete related substances that serve as useful tumor biomarkers. The introduction of chemotherapy with ▶ platinum complexes for germ cell tumors has revolutionized outcomes and has been one of the truly great success stories of modern oncology. Discovered serendipitously as an anticancer therapy during the investigation of electricity from platinum electrodes, it is the backbone of all systemic therapy for these classes of tumor. Sex-cord-stromal and germ cell tumors of the ovary are rare, but they are histologically parallel with ▶ testicular germ cell tumors, which are more common. Epidemiology Almost nothing is known about the etiology of stromal tumors. Evidence supports a familial or genetic component to testicular germ cell cancers, and there may be significant overlap with ovarian germ cell cancer etiology. The age-adjusted incidence rate of malignant germ cell tumors over the past 30 years has been 0.34/100,000. The peak incidence is between the ages of 15 and 19 years, with the majority being diagnosed before the age of 40. Individuals with ovarian dysgenesis (i.e., defective or abnormal development of an organ, especially of the gonads) are at such an elevated risk of developing stromal and germ cell tumors, especially dysgerminoma and gonadoblastoma, where prophylactic surgery is appropriate (Gershenson 2007). Pathology Table 1 summarizes the WHO classification of histologic subtypes. Review of pathology by an expert in gynecologic pathology is essential to ensure an accurate diagnosis of these unusual histologies (▶ ovarian cancer pathology).

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3320 Ovarian Stromal and Germ Cell Tumors, Table 1 Summary of the WHO classifications of ovarian sex-cord-stromal and germ cell malignancies Sex-cord-stromal tumors Granulosa stromal: granulosa cell tumor and thecafibroma tumor Sertoli-stromal cell tumors: Sertoli-Leydig cell tumor and adrenoblastoma Sex-cord tumors with annular tubules Gynandroblastoma Steroidal or Leydig tumor Unclassified Germ cell tumors Dysgerminoma Endodermal sinus tumor (yolk sac tumor) Teratoma: immature, mature monodermal, and mixed Non-gestational choriocarcinoma Embryonal carcinoma Polyembryoma

Sex-cord-stromal tumors represent neoplasia arising in the ovarian stroma. This group encompasses several histologies with the majority being ▶ granulosa cell tumors (adult and juvenile types), Sertoli-Leydig cell tumor, and unclassified sex-cord-stromal tumors. Granulosa cell tumors account for 90% of ovarian sex-cord-stromal malignancies and are usually characterized by indolent growth patterns (Shumer and Cannistra 2003). Dysgerminoma is the most common ovarian malignancy in adolescent and young women. Seventy-five percent are stage I, and they are more commonly bilateral than other non-epithelial ovarian tumors. These tumors can contain syncytiotrophoblastic giant cells and express placental alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Endodermal sinus tumors (yolk sac tumor) represent 25% of malignant germ cell tumors and express ▶ alphafetoprotein (AFP). Histopathologically they have a characteristic central vessel invagination (Schiller-Duval bodies; (Gershenson 2007)). Teratoma is most commonly divided into immature or embryonal, mature, and monodermal. Teratomas are graded according to the content of neural tissue, differentiation, and the presence of embryonal tissues. Mature

Ovarian Stromal and Germ Cell Tumors

teratomas, also called dermoids, represent 95% of all teratomas and are almost always benign. Monodermal teratomas have just one cell type and are extraordinarily rare. Disparate histologies from ▶ carcinoid and squamous cell to sarcoma and thyroid cancer have been described. They are treated according to the tissue type without regard to their ovarian origin (Gershenson 2007). Ovarian choriocarcinoma are non-gestational and associated with human chorionic gonadotropin (HCG) secretion. They are histologically analogous to choriocarcinoma arising in trophoblastic tissue, which occurs more commonly. Embryonal carcinoma is rare (4%) and the most malignant. Polyembryomas are rare, often mixed tumors, and characterized by “embryoid bodies.” The most common mixed germ cell tumors are combinations of dysgerminoma and endodermal sinus tumors. Rarer non-epithelial tumors include gonadoblastoma, tumors of rete ovarii, mesothelioma, and lymphoma. There are a number of incompletely characterized chromosomal and genetic abnormalities observed in germ cell cancers. Chromosome 12p abnormalities have been found highly prevalent in dysgerminoma and mixed germ cell tumors. Endodermal sinus tumors have been associated with gains in both 1q and chromosome 3, while malignant ovarian teratomas commonly had loss of 1p and gain of 1q and chromosomes 3, 8, 14, and 21. The clinical significance of these and other genetic aberrations has not been determined. New tissue markers, such as OCT4 and KIT for dysgerminoma, may be diagnostically helpful and open therapeutic options. Clinical Presentation Non-epithelial cancers of the ovary should be on the diagnostic differential when women present with incidental discovery of asymptomatic ovarian mass, hormonal symptoms, abdominal pain, or symptoms from rapidly progressing metastatic disease. Tumor Markers A variety of tumor markers have an important role diagnostically and in surveillance for tumor recurrence (Table 2). It is not cost effective to survey a

Ovarian Stromal and Germ Cell Tumors

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Ovarian Stromal and Germ Cell Tumors, Table 2 Ovarian germ cell and stromal tumor markers Granulosa cell Theca-fibroma Sertoli-Leydig Dysgerminoma Yolk sac Embryonal Choriocarcinoma Immature teratoma Gonadoblastoma

E2 +

Inhib +

MIS +

T

A

+

+

DHEA

AFP

hCG

pALP

LDH

VEGF +

+ +

+/ + +

+ + +

+

+

+

+ +

+

+

+ +

+

Isochromosome p12 [I(12)p] can be helpful diagnostically in tissue for diagnosis of germ cell tumors. Others: monodermal struma ovarii (T4), carcinoid (5-hydroxyindoleacetic acid positive). CA125 can be elevated in advanced disease. E2 ▶ estradiol, Inhib inhibin, MIS Müllerian inhibiting substance, T testosterone, A androstenedione, DHEA dehydroepiandrostenedione, AFP alpha-fetoprotein, hCG human chorionic gonadotropin, pALP placental alkaline phosphatase, LDH lactate dehydrogenase, VEGF ▶ vascular endothelial growth factor

panel of markers in all patients with suspected ovarian neoplasm, and their use should be selective. ▶ Granulosa cell tumors may express and secrete: (alpha and beta) inhibin, human chorionic gonadotropin (HCG), Müllerian inhibiting substance, CA125, ▶ estradiol, and occasionally testosterone (Shumer and Cannistra 2003). SertoliLeydig tumors often produce testosterone. Alphafetoprotein (AFP) is a 590-amino-acid glycoprotein produced by the fetal yolk sac. It is the serum marker identified as significantly elevated in patients with germ cell tumors with a component originating from the endodermal sinus. The halflife of AFP as a marker is approximately 5 days. Patients demonstrating adequate response to therapy will typically have a decline in AFP proportional to its half-life. Human chorionic gonadotropin (beta HCG) is a 244-amino-acid glycoprotein hormone produced by the placenta to maintain corpus luteal production of progesterone and maintenance of pregnancy. It is a heterodimer with a unique beta subunit and shares the same alpha subunit as luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroidstimulating hormone (TSH). The half-life of beta HCG as a marker is approximately 36 h. Isolated Adnexal Mass For patients with discovery of a solid or complex ovarian mass (adnexal mass), a non-epithelial

neoplasm may be on the differential diagnosis and must be considered in young women. Approximately one third of ovarian malignancies in women under 25 years of age are germ cell tumors. This situation demands surgical evaluation for the purposes of diagnosis, by a gynecological oncologist, for the purposes of diagnosis and staging. Appropriate staging in these tumors provides key prognostic information, as well as informing therapeutic decision making. In sex-cord-stromal tumors, stage has been the most important predictor of survival in a number of studies. Truly stage I tumors in several histologies may be appropriately managed with surgery alone, but without adequate staging surgeries, it is not possible to completely rule out occult metastatic disease (Gershenson 2007; Shumer and Cannistra 2003). The staging system used for sex-cord-stromal and germ cell ovarian cancers is the same as the International Federation of Gynecology and Obstetrics (FIGO) staging for epithelial ovarian cancers (ovarian cancer staging). This staging involves peritoneal cytology; total abdominal hysterectomy; bilateral salpingo-oophorectomy; biopsy of malignant peritoneal lesions or staging biopsies including left and right pelvic cul-de-sac, bladder, paracolic gutter, and diaphragm biopsies; and resection of positive lymph nodes or pelvic and para-aortic lymph node dissection. Staging

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3322 Ovarian Stromal Table 3 Staging

Ovarian Stromal and Germ Cell Tumors and

Germ

Cell

Tumors,

I FIGO staging for carcinoma of the ovary, germ cell, and stromal tumors I Tumor confined to ovaries IA Tumor limited to one ovary, capsule intact IB Tumor in both ovaries IC Tumor in one or both ovaries, with a capsule ruptured, surface involvement, cytologically positive ascites, or peritoneal washings II Tumor involves one or both ovaries with pelvic extension to the uterus (A), other structures (B), or with positive peritoneal washings (C) III Tumor involves one or both ovaries with microscopically (A) or macroscopically [(B), or >2 cm (C)] confirmed peritoneal metastasis outside the pelvis and/or regional lymph node metastases IV Distant metastasis beyond the peritoneal cavity Note: Liver capsule metastasis is stage III, liver parenchymal metastasis stage IV. Pleural effusion must have positive cytology Modified staging for testicular germ cell tumors IM Rising markers post gonadectomy II Abdominal lymph nodes (A) 5 cm III Supradiaphragmatic lymph nodes IV Extranodal disease Prognostic groups AFP bHCG LDH Good 54a

AFP   +++  / (+) / (+)b 

hCG (+)   +++ 

Inhibin     

Ca ++     



(+)a



++

n.d.a





++

++*

n.d.a













Abbreviations: n.d. no data a Under evaluation b Some poorly differentiated Sertoli-Leydig cell tumors may secrete AFP  May require high dose chemotherapy

tumors show a clinically benign behavior after complete resection, but serous adenocarcinoma, which represents the malignant form of papillary serous cystadenoma, must be distinguished histologically. In analogy, mucinous adenocarcinoma constitutes the malignant counterpart of the mostly huge but clinically benign mucinous cystadenomas. Ovarian adenocarcinomas rarely develop during adolescents; however, these tumors often take a malignant clinical course. Particularly in young patients and in patients with frank familial history, these tumors are associated with inherited BRCA mutations. CA125 can be used as tumor marker following tumor resection, but the vast majority of patients will require adjuvant chemotherapy (Table 1). Clinical Diagnosis Ovarian tumors are commonly detected as large tumors which may have led to a visible and palpable swelling of the abdomen or fatigue. In some patients, tumor-related torsion of the ovary or tumor rupture may result in severe acute abdominal pain and require emergency laparotomy. Yolk sac tumors secrete alpha1-fetoprotein (AFP) and choriocarcinoma-like human chorionic gonadotropin (hCG), which can be detected in the

serum. Pure embryonal carcinomas and teratomas are usually not associated with specific serum tumor markers. In ~20% of dysgerminomas, serum levels of placenta-like alkaline phosphatase may be elevated. Syncytiotrophoblastic cells in germinoma may also produce ß-hCG (▶ Serum Biomarkers). SCSTs often induce clinical symptoms that are related to the production of sex hormones by the tumor. Characteristically, infants and children present with signs of isosexual precocity, including breast enlargement, pubarche, and vaginal bleeding. In (post)pubertal girls, tumors may lead to primary or secondary amenorrhea and unspecific signs of virilization. As other steroid hormoneproducing cells, SCSTs also secrete inhibin. Thus, free inhibin can be measured in the serum and may serve as a serological tumor marker during follow-up. In rare but well-documented cases of poorly differentiated ovarian Sertoli-Leydig cell tumors, AFP (▶ Alpha-Fetoprotein) production has been reported, which may interfere with clinical diagnosis. Histologically, most of these tumors resemble SLCT with retiform, often hepatoid differentiation and heterologous elements. CA125 constitutes a rather unspecific marker of ovarian tumors that may be elevated in different

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histologic entities. CA125 may provide useful information regarding the response to treatment and for follow-up in tumors that are otherwise tumor marker negative. The clinical presence of hypercalcemia in approximately two-thirds of OSCC does not substitute the histologic diagnosis, since a variety of other ovarian neoplasms such as clear cell carcinoma or serous carcinoma may also be associated with hypercalcemia. The staging system of the World Health Organization and the International Federation of Gynecology and Obstetrics (FIGO) can be applied. Tumors confined to the ovary (stage I) are distinguished from tumors with locoregional spread in the small pelvis (stage II) and the abdominal cavity (stage III) and distant metastases (stage IV). Microscopic spread may have occurred even in stage I tumors, if malignant ascites is noted or if pre- or intraoperative violation of the tumor pseudocapsule occurred (stage Ic). Tumors with no microscopic spread on complete staging are categorized as stage Ia (unilateral tumor) or stage Ib (bilateral tumors). Peritoneal gliomatosis constitutes a specific phenomenon in teratomas and mixed malignant GCTs that refers to glial nodules in the peritoneal surface. Although gliomatosis imposes like widespread metastases, it represents a reactive and nonneoplastic disorder so that upstaging is inadequate. Surgical Therapy All ovarian tumors require surgical resection. In most patients, a primary tumor resection constitutes the standard approach and will result in complete tumor resection, because most tumors are encapsulated. In metastatic tumors with peritoneal spread, an up-front chemotherapy followed by delayed tumor resection is recommended as it may significantly facilitate complete tumor resection. Therefore, diagnosis is based on imaging and tumor markers, and only in tumor marker negative tumors, an initial biopsy will be required. A median laparotomy is considered the classical standard approach to ovarian tumors in childhood

Ovarian Tumors During Childhood and Adolescence

and adolescence, as it will allow for preparation and resection of the tumor in one piece. With the modern advances in laparoscopy, more patients will undergo laparoscopic surgery. However, the safety of this approach has not been prospectively evaluated in ovarian tumors of childhood. By no means, tumors should be punctured or resected in separate pieces in order to facilitate removal through the laparoscope, because this would not comply with the oncological criteria of complete resection and may thus be associated with an undefined increased risk of relapse. For the same reasons, organ-sparing surgery should be avoided and might only be reserved to those rare patients with bilateral tumors. Tumor resection may be restricted to ovariectomy, if surgical staging indicates a stage I tumor. Only in stage II or III tumors, the unilateral adenectomy is indicated. In general, hysterectomy is considered unnecessary. Surgical staging can be limited to resection of suspicious lymph nodes. Routine sampling of unsuspicious nodes is not recommended. In nonmetastatic tumors, omentectomy and appendectomy are also not required for oncological reasons, but in specific situations, e.g., adhesions may be performed on the basis of individual judging by the surgeon. Last, resection of glial nodules in gliomatosis peritonei is not generally recommended due to the nonmalignant nature of this disorder. However, resection of large nodules may become necessary if they result in local complications such as mechanical ileus. Adjuvant Therapy After resection, a choice regarding the indication for adjuvant therapy has to be made. ▶ Adjuvant therapy may be chosen from a broad and riskstratified panel of chemotherapeutic strategies, which may include two to four cycles of a twoor three-agent chemotherapy as well as strategies with locoregional or systemic treatment intensification. The adequate choice of treatment should always be based on a careful clinical and histopathologic assessment, preferably by a central reference pathologist. Therefore, all children

Ovarian Tumors During Childhood and Adolescence

and adolescents with ovarian GCTs should best be enrolled into cooperative protocols or clinical registries, which will provide the treating physician with the required infrastructure for diagnosis and risk assessment. Previously, adjuvant chemotherapy has been widely used even in completely resected stage I tumors. However, experience has shown that a careful watch-and-wait strategy may be justified in stage I tumors. In analogy to testicular GCTs, adjuvant chemotherapy should be considered in patients with histologic evidence of vessel invasion. A watch-and-wait strategy should include a careful and close follow-up schedule, which specifically includes the assessment of the para-aortic lymph nodes, the most frequent site of relapse. In addition, the patients should be informed that there is a general risk of relapse under a watchand-wait strategy that is 20–30%. However, with delayed chemotherapy, there is an excellent salvage rate, so that the overall survival exceeds 95%. Current modern platin-based chemotherapy usually consists of a three-agent combination including ▶ cisplatin (100 mg/m2/cycle) and ▶ etoposide (300–500 mg/m2/cycle) in combination with ▶ bleomycin (15–30 U/m2/cycle) or ifosfamide (7500 mg/m2/cycle). Cisplatin can be substituted by carboplatin (600 mg/m2/cycle) with similar cure rates. Intensification of cisplatin with 200 mg/m2/cycle results in significant ototoxicity; however, it also yields a slight survival advantage in high-risk patients. Etoposide is associated with an increased risk of secondary malignancies, in particular acute myelogenous leukemia. However, at the doses usually administered for GCTs and in the absence of concomitant radiotherapy, the risk is acceptably low and does not exceed 1%. In the different national protocols, different choices of the third drug have been made. In the UK and the USA, platin compounds and etoposide are combined with bleomycin, thus resembling the traditional chemotherapy for testicular GCTs. In contrast, the national groups in France, Brazil, and Germany are administering ifosfamide. The toxicity profiles of these

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two drugs differ significantly. Bleomycin in combination with cisplatin may cause pneumopathy, which in rare patients may be lethal. In contrast, ifosfamide has a more pronounced hematotoxicity and may result in chronic renal tubulopathy. Some tumors may show an only insufficient response to conventional three-agent chemotherapy and should therefore be selected for treatment intensification. In tumors with locoregional (i.e., peritoneal) spread, treatment intensification may be achieved by combination of chemotherapy with locoregional ▶ hyperthermia, with favorable long-term outcome. Metastatic tumors however may be selected for dose-intensified chemotherapeutic strategies (▶ Platinum-Refractory Testicular Germ Cell Tumors). Among metastatic GCTs, CNS metastases portend a particularly poor prognosis. In contrast to the more frequent GCTs, the experience regarding the adjuvant chemotherapy in the rare tumor entities such as SCSTs and OSCCs is much more limited and awaits further prospective evaluation. The overall prognosis of SCSTs is favorable and cure rates exceed 80%. Patients with stage I tumors are followed expectantly, while patients with peritoneal spread are selected for adjuvant chemotherapy (e.g., cisplatin, etoposide, ifosfamide). Patients with stage Ic tumors and malignant ascites of preoperative tumor rupture should all be selected for chemotherapy, since the outcome of this specific group is otherwise comparable to stage II and III tumors. In addition, Sertoli-Leydig cell tumors should be treated more aggressively than other SCSTs, because they may relapse even after only a minor tumor spread during surgery. Experience regarding the optimal treatment of OSCC is very limited and mostly derived from retrospective clinicopathological surveys and single case reports, indicating for an extremely aggressive natural course of this disease with an almost invariable fatal outcome. Regardless of stage, all patients with ovarian small cell carcinoma of hypercalcemic type require multi-agent chemotherapy (e.g., cisplatin, ifosfamide, and

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Adriamycin) during first-line treatment. Highdose chemotherapy can be used to consolidate the therapeutic success. The guidelines for the treatment of other epithelial tumors such as cystadenoma and adenocarcinomas follow those for the corresponding tumors in adults. If necessary, a combination of carboplatin and ▶ taxol is considered the current standard chemotherapy in ▶ ovarian cancer.

Cross-References ▶ Adjuvant Therapy ▶ Alpha-Fetoprotein ▶ Alpha-Fetoprotein Diagnostics ▶ Bleomycin ▶ Cisplatin ▶ Etoposide ▶ Granulosa Cell Tumors ▶ Hyperthermia ▶ Ovarian Cancer ▶ Ovarian Small Cell Carcinoma Hypercalcemic Type ▶ Peutz–Jeghers Syndrome ▶ Platinum-Refractory Testicular Germ Cell Tumors ▶ Serum Biomarkers ▶ Taxol ▶ Testicular Germ Cell Tumors

References Distelmaier F, Calaminus G, Harms D et al (2006) Ovarian small cell carcinoma of the hypercalcemic type in children and adolescents: a prognostically unfavorable but curable disease. Cancer 107:2298–2306 Göbel U, Schneider DT, Calaminus G et al (2000) Germcell tumors in childhood and adolescence. Ann Oncol 11:263–271 Schneider DT, Terenziani M, Cecchetto G, Olson TA (2012) Gonadal and extragonadal germ cell tumors, sex cord stromal tumors and rare gonadal tumors. In: Schneider DT, Brecht IB, Oslon TA, Ferrari A (eds) Rare tumors in children and adolescents. Springer, Heidelberg, pp 327–402 Schultz KA, Schneider DT, Pashankar F et al (2012) Management of ovarian and testicular sex cord-stromal tumors in children and adolescents. J Pediatr Hematol Oncol 34(Suppl 2):S55–S63

Oxidative Necrosis Wessalowski R, Schneider DT, Mils O et al (2013) Regional deep hyperthermia for salvage treatment of children and adolescents with refractory or recurrent non-testicular malignant germ-cell tumours: an openlabel, non-randomised, single-institution, phase 2 study. Lancet Oncol 14:843–852

See Also (2012) CA125. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 577–578. doi:10.1007/978-3-642-16483-5_761 (2012) Carboplatin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 641. doi:10.1007/978-3-642-16483-5_833 (2012) Choriocarcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 825. doi:10.1007/978-3-642-16483-5_6908 (2012) Dysgerminoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1177. doi:10.1007/978-3-642-16483-5_1759 (2012) FIGO. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1407. doi:10.1007/978-3-642-16483-5_2188 (2012) Germ cell tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905 (2012) Human chorionic gonadotropin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1752–1753. doi:10.1007/978-3642-16483-5_6914 (2012) Isochromosome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1917. doi:10.1007/978-3-642-16483-5_3155 (2012) KIT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1945–1946. doi:10.1007/978-3-642-16483-5_3228 (2012) Loss of heterozygosity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2075–2076. doi:10.1007/978-3642-16483-5_3415 (2012) Ovarian teratoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2721–2722. doi:10.1007/978-3-642-16483-5_4300 (2012) Sertoli-Leydig cell tumor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3390. doi:10.1007/978-3-642-16483-5_5266 (2012) Sex-cord stromal tumors. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3395–3396. doi:10.1007/978-3642-16483-5_5273 (2012) Teratoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3651. doi:10.1007/978-3-642-16483-5_5732

Oxidative Necrosis ▶ Ferroptosis

Oxidative Stress

Oxidative Stress Jérôme Alexandre, Carole Nicco and Frederic Batteux Faculté de Médecine Paris – Descartes, UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul, Paris, France

Definition Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. It is defined as a disturbance in the prooxidant–antioxidant balance in favor of the former leading to potential damage. Oxidative stress is believed to contribute to the development of several diseases, including cancer.

Characteristics The complex of oxidative stress involves a number of cell-chemical elements: Free radicals include any species that contain one or more unpaired electrons, generating electrons singly occupying an atomic or molecular orbital. The presence of unpaired electrons confers a considerable degree of reactivity upon a free radical. Dioxygen is the main source of intracellular free radicals. ▶ Reactive oxygen species (ROS) is a collective term that includes both oxygen free radicals such as superoxide anion (O2) and hydroxyl radical (OH•) and other reactive molecules as ▶ hydrogen peroxide (H2O2). H2O2 is not a free radical but is also reactive and easily converted into radicals. ROS are physiological by-products of normal aerobic metabolism. Ninety percent of intracellular superoxide anion comes from the mitochondrial electron transport chain. Other significant sources of superoxide anion include NADPH oxidase (NOX), xanthine oxidase, and NADPH–▶ cytochrome P450 reductase. NOX is a family of

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multimeric membrane oxidases tightly regulated by various stimuli including growth factors. Superoxide anion is considered as the “primary” ROS. The chemical reactivity of superoxide anion is weak in aqueous medium. Moreover, its negative electronic charge makes it unable to diffuse through lipid membranes. That is why superoxide anion by itself has little cellular effects. The conversion of O2 into H2O2 is catalyzed by enzymes of the superoxide dismutase (SOD) family. Three isoforms of SOD have been identified in humans: CuZn-SOD, which is located in the cytosol; Mn-SOD, located in the mitochondria; and extracellular (EC)-SOD. H2O2 is able to go through cellular membranes and can diffuse relatively far from its production origin. At physiological concentrations, H2O2 reacts mainly with sulfhydryl radicals, inducing reversible alterations of cysteines. Thus, H2O2 is able to specifically alter activity of proteins containing cysteine residues in their enzymatic sites, especially tyrosine phosphatases. The hydroxyl radicals are formed through the Fenton or Haber–Weiss reaction that converts O2 and H2O2 into OH• in the presence of Fe2+ or Cu2+. OH• is extremely reactive and induces severe cellular oxidative damages as DNA alterations, being thus highly mutagenic. It leads to polyunsaturated fatty acid peroxidation, increasing cellular membrane permeability. OH• is also able to react with proteins, inducing irreversible inactivation. Several antioxidant enzymes can detoxify the whole cascade of ROS and protect normal cells from the potential damages implicated by oxidative stress. SOD catalyzes the conversion of O2 into H2O2, which is further detoxified by catalase or by enzymes of the glutathione peroxidase family using reduced glutathione. This complex enzymatic antioxidant system is completed by exogenous compounds such as arginine; vitamins A, C, and E; b-carotene; glutathione; polyphenols; and minerals (selenium and zinc). Oxidative Stress is Involved in Oncogenesis There are several evidences that ROS play a key role in the oncogenesis process. In vitro exposure to chronic oxidative stress is associated with

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oncogenic transformation and tumor growth. Overexpression of NOX1 (the catalytic subunit of NOX) stimulates the generation of O2  and induces the transformation of mouse NIH 3T3 cells. Patients carrying specific polymorphisms on GSH-peroxidase 1 gene associated with decreased antioxidant activity present an increased risk of lung and breast cancers. An excessive production or a defective detoxification of ROS may favor the promotion and the development of cancer during chronic infection or ▶ inflammation which is considered to cause one third of world cancers. The tumoral transformation induced by ROS, especially by OH•, is at least in part consecutive to their mutagenic effects. They can act directly on DNA structures leading to single- or doublestrand breaks, to point and frameshift mutations, and to chromosome abnormalities. ROS can also promote proliferation and survival of cancer cells and tumor angiogenesis by means of ▶ epigenetic effects. By altering activity of several tyrosine phosphatases, including PTEN and MAPK phosphatases, H2O2 activates mitogenic and survival pathways. This molecule has also been reported to stimulate VEGF production by tumor cells. Increased Oxidative Stress in Cancer Cells

Under basal conditions of culture, various cancer cell lines produce more O2 and H2O2 than nontransformed cells. The presence of oxidative modifications of DNA in primary human cancer cells has also been demonstrated. The oxidative stress observed in cancer cells mostly results from the increase production of superoxide anion. The overexpression of MYC oncogene has been associated with increased production of superoxide by mitochondrial respiratory chain, while RAS oncogene mutations induce NOX activation. A decrease activity of antioxidant enzymes could also contribute to oxidative stress since tumor cells, compared with normal cells, often express lower levels of catalase, glutathione peroxidase, reductase, and their respective cofactors. The overproduction of ROS in cancer cells contributes to the genetic instability, leading to nuclear and mitochondrial DNA mutations and

Oxidative Stress

more aggressive phenotype. Thus, oxidative stress is part of a positive amplification loop where ROS induce malignant transformation and transformation is itself associated with more ROS production. Therapeutic Perspectives of Oxidative Stress Modulation

Differential Effects of Oxidative Stress in Normal and Cancer Cells Growing evidence suggests that ROS can induce a wide type of cellular responses from proliferation to senescence and cell death. Adding increasing amounts of exogenous H2O2 or increasing its intracellular levels by overexpression of SOD leads to a dose-dependent decrease in proliferation and tumor cell death. H2O2 can stimulate proapoptotic signal molecules such as apoptosis signal-regulating kinase 1, c-Jun-NH2-kinase, and p38, activation of the p53 protein pathway, and start-up of the mitochondrial apoptotic cascade. When adding ROS to cell culture media, differential cellular effects have been observed in normal and cancer cells. In normal cells, persistent ROS production leads to activation of the JNK pathway and apoptosis, whereas low concentrations or transient high levels of ROS induce the proliferation of those cells, through the activation of the ERK pathway. In sharp contrast, similar expositions to ROS cause tumor cell growth arrest and apoptotic death because of their high basal level of ROS that is close to the threshold of cytotoxicity. Thus, the fate of tumor cells exposed to oxidative stress is tightly correlated with the duration of ROS stimulation and depends on the basal redox status of the cell. Use of Antioxidant Agents in Cancer Patients Clinical data regarding the effects of antioxidant molecules in cancer patients have been controversial. Because oxidative stress induced DNA damages and is involved in malignant transformation, the hypothesis was made that antioxidant agents could have preventive effect against cancer. Numerous clinical trials were performed using various antioxidants (N-acetylcysteine (NAC), b-carotene, vitamin A,

Oxidative Stress

vitamin C, vitamin E, etc.), but failed to show any protective effect. Furthermore, a doubt has been cast on the possibility that some of these molecules, especially NAC, might trigger tumor growth. These results should be viewed in light of the biological effects of antioxidants in cancer cells. Indeed, we previously observed that NAC, which is endowed with catalase and glutathionelike activities, decreases intracellular H2O2 concentration and increases the proliferation of tumor cells in vitro and in vivo, probably because tumor cells are submitted to detrimental oxidative stress. Antioxidant molecules probably play a protective role against cancer in healthy individuals by preventing DNA damages linked to the oxidative stress. However, once cancer cells have emerged, a cancer-promoting effect could result from the administration of agents that decrease intracellular H2O2 level. Therefore, antioxidant should be used with caution in cancer patients. Anticancer Agents Increase Oxidative Stress in Cancer Cells The most commonly used anticancer agents, such as ▶ cisplatin, ▶ adriamycin, ▶ fluorouracil, ▶ irinotecan, or ▶ paclitaxel, are able to induce ROS production in cancer cells. Hydrogen peroxide and superoxide accumulation are observed within few hours of drug exposure and occur before the commitment of the cells into apoptosis. Moreover, antioxidants such as NAC and catalase are able to decrease their cytotoxicity. These results strongly suggest that oxidative stress is involved in the cytotoxicity of most anticancer agents. However, the underlying mechanisms seem to differ from an agent to the other. For example, 5-FU and anthracyclines induce ROS production by a p53-dependent pathway involving the activation of several mitochondrial oxidoreductases such as proline oxidoreductase and ferredoxin reductase. On the other hand, membrane NOX activation is induced by ▶ paclitaxel and arsenic trioxide. Tumor cells have higher levels of ROS than normal cells and could therefore be more sensitive to the additional oxidative stress generated by anticancer agents. This hypothesis offers a new explanation to the observation that

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anticancer agents are usually more toxic to cancer cells than to normal cells. Finally, cellular antioxidant enzymes may influence the sensitivity of tumor cells to anticancer agents. Thus, high levels of reduced glutathione, the cofactor of glutathione peroxidase, in tumor cells have been associated with a multidrug resistance phenotype. A correlation was found between resistance of breast cancers to docetaxel and the overexpression of several genes controlling the cellular redox environment. Using Oxidative Stress Modulators as Anticancer Agents The fundamental differences between normal and tumor cells in terms of responses to H2O2 overproduction provide new possibilities in the treatment of cancer, taking into account the tumor cell susceptibility to ROS-induced apoptosis. Overexpression of SOD in cancer cell lines induces an increase in H2O2 production and reduces tumor growth. However, the SODs are polypeptides of high molecular weight that are not able to cross the cellular membranes and therefore have a limited interest for clinical use. Several nonpeptidyl SOD mimics with lower molecular weight have been developed, such as (Cu(II)(3,5-diisopropylsalicylic acid)2) (CuDIPS), Mn(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP), or mangafodipir. The SOD mimics increase the concentration of H2O2, resulting in the abrogation of tumor cell proliferation in vitro. Similar results have been observed in vivo. Increasing the Therapeutic Index of Anticancer Agents by SOD Mimics Several reports have suggested that compounds that increase intracellular hydrogen peroxide concentration could enhance the activity of anticancer agents. For example, buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, can increase the cytotoxicity of melphalan by inhibiting glutathione peroxidase activity and increasing H2O2 level. Similarly, we previously showed that the antitumor activity of oxaliplatin and paclitaxel is enhanced by SOD mimics. A potential limitation to the clinical development of such compound is that they could also increase the toxicity of

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anticancer agents on normal cells. As an example, BSO depletes glutathione in both normal and cancer cells, increasing melphalan’s hematologic toxicity and thus abrogating any enhancement of the therapeutic index of this anticancer agent. Mangafodipir has SOD-, catalase-, and glutathione reductase-like properties, allowing it to act at multiple steps of the ROS cascade. Mangafodipir protects mice treated with paclitaxel from developing leukopenia (which is the reduction in the circulating white blood cell count to less than 4,000/mL) and also amplifies the antitumoral effect of paclitaxel on implanted tumor, increasing its therapeutic index. These opposite effects of mangafodipir in normal and cancer cells may be related to the differences in the redox status of these cells, as described above. Clearly, oxidative stress modulators, especially SOD mimics, warrant further development in anticancer treatment.

Cross-References ▶ Adriamycin ▶ Anthracyclines ▶ Antioxidant Enzymes ▶ Cisplatin ▶ Cytochrome P450 ▶ Epigenetic ▶ Fluorouracil ▶ Hydrogen Peroxide ▶ Inflammation ▶ Irinotecan ▶ MAP Kinase ▶ MYC Oncogene ▶ Paclitaxel ▶ RAS Genes ▶ Reactive Oxygen Species ▶ Vascular Endothelial Growth Factor

Oxygen Partial Pressure Distribution in Tumor Benhar M, Engelberg D, Levitzki A (2002) ROS, stressactivated kinases and stress signaling in cancer. EMBO Rep 3:420–425 Halliwell B, Gutteridge J (1999) Free radicals in biology and medicine. Oxford University Press, New York Laurent A, Nicco C, Chereau C et al (2005) Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 65:948–956 Nicco C, Laurent A, Chereau C et al (2005) Differential modulation of normal and tumor cell proliferation by reactive oxygen species. Biomed Pharmacother 59:169–174

See Also (2012) Antioxidant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 216. doi:10.1007/978-3-642-16483-5_328 (2012) Arsenic Trioxide. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 285. doi:10.1007/978-3-642-16483-5_404 (2012) Free Radicals. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1454. doi:10.1007/978-3-642-16483-5_2267 (2012) MAPK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) Multidrug Resistance. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2393. doi:10.1007/978-3-642-16483-5_3887 (2012) Mutagenic. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2412. doi:10.1007/978-3-642-16483-5_3909 (2012) NADPH Oxidase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2448. doi:10.1007/978-3-642-16483-5_3959 (2012) NIH-3T3 Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2520. doi:10.1007/978-3-642-16483-5_4084 (2012) Superoxide Dismutase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3563. doi:10.1007/978-3-642-16483-5_5579 (2012) Transformation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3757–3758. doi:10.1007/978-3-642-16483-5_5913 (2012) VEGF. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3906. doi:10.1007/978-3-642-16483-5_6174 (2012) Xanthine Oxidase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3963. doi:10.1007/978-3-642-16483-5_6270

References Alexandre J, Nicco C, Chereau C et al (2006) Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir. J Natl Cancer Inst 98:236–244

Oxygen Partial Pressure Distribution in Tumor ▶ Oxygenation of Tumors

Oxygen Sensing

Oxygen Sensing Zhong Yun Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA

Synonyms Hypoxia sensing

Definition Oxygen sensing refers to the ability of cells to detect and respond to changes of intracellular oxygen concentrations. Because cells often experience decreased oxygenation under both physiological and pathological conditions, oxygen sensing is also referred to as hypoxia sensing. This entry focuses on the common mechanisms of oxygen sensing in mammalian cells.

Characteristics Molecular oxygen (dioxygen O2) is a vital element for the majority of life forms on earth. The atmospheric O2 concentration is approximately 20.9% at the sea level. Under physiological conditions, oxygen concentrations are maintained approximately in the range of 2–10% depending on the tissue types in mammals. However, mammalian cells can be exposed to high levels (up to 20.9%) of oxygen concentrations due to physical wounding. On the other hand, oxygen concentrations in mammalian cells can decrease to 0–1% due to certain diseases including ischemia, cardiovascular diseases, and cancer. Through eons of evolution and natural selection, air-breathing animals have developed sophisticated mechanisms to monitor fluctuations in O2 concentrations and to regulate O2 consumption as well as cellular functions in accordance with different O2 environments. Generally speaking, mammals monitor changes in O2 concentrations at two levels in order to maintain O2 homeostasis: (1) at the

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whole-body level, carotid body, pulmonary artery, and adrenal chromaffin cells sense O2 concentrations and regulate the O2-carrying capacity of the blood and circulation of blood to different tissues in the body, and (2) at the cellular level, individual cells use a wide range of O2-utilizing enzymes either to give instructions for or to directly carry out specific cellular functions. O2-utilizing enzymes fall into two subclasses: (1) oxygenases (EC1.13) that catalyze the transfer of an oxygen atom from O2 to a substrate and (2) oxidoreductases (EC1.14) that catalyze oxidation/reduction reactions with O2 as an electron acceptor. Because they use O2 as a substrate, oxygenases and O2-utilizing oxidoreductases are considered the primary molecular sensors of O2. The best-known oxidoreductase is cytochrome-c oxidase (cytochrome aa3), a heme-containing protein that directly binds O2. It is an integral component of the mitochondrial complex IV, a large transmembrane protein assembly that converts O2 to water and simultaneously generates a transmembrane proton gradient to drive ATP production in mitochondria. In the universe of O2-utilizing enzymes, cytochrome-c oxidase has the highest affinity to O2 with a Km (0.5 mM at 25 C) that is much lower than the average O2 concentrations in virtually all tissues under physiological conditions. In this entry, we will focus on the expanding family of the nonheme oxygenases because of their role in the regulation of multiple important cellular functions via hydroxylation of proteins and/or nucleic acids. Prolyl Hydroxylase Domain-Containing Oxygenases (PHD) Prolyl hydroxylase domain-containing oxygenases (PHDs) are also known as hypoxiainducible factor prolyl hydroxylase (HIF-PHD). They use 2-oxoglutarate (2-OG) and divalent iron (Fe2+) as key cofactors among others to carry out hydroxylation of HIF-1a, HIF-2a, and HIF-3a proteins at the two specific proline residues [e.g., the proline residues at positions 402 (P402) and 564 (P564) human HIF-1a protein.] Among the three major PHDs, PHD2 appears to be the most abundantly expressed and is primarily located in

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cytoplasm. In contrast, PHD1 is mainly nuclear and PHD3 can be found in both cytoplasm and nucleus. PHDs are mainly involved in the O2-dependent regulation of protein stability of HIF-a proteins. Under most physiological O2 conditions (physiological normoxia), the enzymatically active PHDs hydroxylate two proline residues located in two separate O2-dependent degradation (ODD) domains of HIF-a proteins. The resultant hydroxylated HIF-a interacts with the von Hippel-Lindau protein (pVHL) in the multimeric E3 ubiquitin ligase complex and becomes ubiquitinated, which leads to degradation of the ubiquitinated HIF-a protein by proteasomes. As O2 concentrations decrease, PHD-mediated hydroxylation becomes progressively subdued. Unhydroxylated HIF-a proteins are stable and can translocate into the nucleus where a HIF-a protein forms a heterodimer with a HIF-b protein to initiate transcription of a wide range of genes including those involved in energy metabolism and cell survival among others. It is worth noting that the HIF-1 transcription factor can also increase the expression of PHD2 and PHD3, suggesting a negative feedback mechanism to prevent excessive accumulation of HIF-a proteins under hypoxia. Paradoxically, PHDs have a relatively low affinity to O2 with observed Km values of 100–250 mM, which may render them inefficient within the broad range of physiological normoxia (approximately 20–100 mM or 2–10% O2). It is possible that the O2 affinity and/or enzymatic activity of PHDs are regulated by yet unidentified posttranslational modifications, other cofactors, or PHD-interacting proteins. Factor-Inhibiting HIF (FIH) FIH is also a member of the 2-OG and Fe2+containing oxygenases. As compared to PHDs, FIH catalyzes hydroxylation of an asparagine residue located in the C-terminal transactivation domain of HIF-1a or HIF-2a protein (e.g., asparagine 803 of HIF-1a). Once hydroxylated at this C-terminal asparagine residue, HIF-a cannot recruit the transcription co-activator p300 for efficient transcription of HIF-regulated genes. The Km of FIH (Km = 90 mM) is lower than that of

Oxygen Sensing

PHDs (Km = 100–250 mM), suggesting that FIH has higher affinity to O2 and should be more active than PHDs at the same O2 concentration. However, functions of HIF-1a and HIF-2a proteins are more prominently regulated by PHDs than by FIH, suggesting that stabilization of HIF-a proteins is the most critical mode of regulation. Histone Demethylases Histones are DNA-binding proteins that help extended linear DNA strands assemble into compact chromosomes. Structures of chromosomes undergo dynamic changes during DNA replication and gene transcription, which involves posttranslational modification of histones such as acetylation of lysine residues and methylation of lysine or arginine residues. For a long time, protein methylation was thought to be an irreversible reaction until Ne-methyl lysine demethylases were identified in early 2000s. Histone demethylases are members of the JmjC domain-containing 2-OG oxygenases and catalyze the removal of Ne-methyl groups from lysine residues via hydroxylation. Two subfamilies of histone demethylases have been identified that possess differential activities toward tri-, di-, or monomethylated lysine residues. The tri- and dimethylated lysines on histone H3 are preferentially demethylated by members of the JmjC domain-containing 2 (JMJD2) oxygenases, whereas di- and monomethylated lysines are demethylated by oxygenases (e.g., FBXL and PHF8) that are incapable of accommodating the trimethylated lysines. The substrate selectivity of histone demethylases is likely determined by the sizes of their binding pockets as well as spatial positioning of amino acids of the binding pocket that mediate specific interactions with the methylated lysine residue inside the pocket. Detectable changes in histone methylation can be found below 3% O2. Furthermore, the majority of JmjC demethylases are upregulated by hypoxia. These findings suggest that chromatin structures may adapt to the changing O2 environments. DNA/RNA Demethylases Adding or removing methyl groups from DNA bases in promoter regions is an important

Oxygen Tension Distribution in Tumor

mechanism for regulating gene transcription. It has been shown that exposure of young rats to chronic hypoxia (10% O2 in breathing air) results in increased methylation of liver DNA. Several members of 2-OG oxygenases have been identified as demethylases for methylated DNA and/or RNA. ALKBH2 and ALKBH2, human homologues of the bacterial AlkB gene, catalyze demethylation of methylated nucleotide residues in DNA and RNA via hydroxylation as the intermediate reaction. Evidence suggests that ALKBH2 may play a role in repair of damaged DNA. On the other hand, ALKBH8 is capable of hydroxylating the 5-carboxymethyl-uridine of the anticodon of tRNAGly to 5-methoxycarbonylmethyl-uridine, a base modification that may affect protein synthesis from mRNA templates. However, exact substrates for most ALKBHs (ALKBH1-8) remain unknown. 5-Hydroxymethyl cytosine is a newly identified but naturally occurring modified base in human DNA. The ten-eleven translocation (TET) subfamily of 2-OG oxygenases has been identified as the enzyme responsible for the conversion of 5-methyl cytosine to 5-hydroxymethyl cytosine in DNA. Genetic evidence has shown that TET oxygenases are often mutated in malignant myeloid diseases, suggesting that loss of normal TET function might contribute to tumor development. Nucleic acid demethylases are also associated with obesity and diabetes in humans. Genetic studies have shown a strong correlation between type 2 diabetes and specific sequence variations in the fat mass and obesity-associated gene FTO that encodes a 2-OG oxygenase capable of demethylating methylated DNA, RNA, and nucleotides. Although it remains to be understood how demethylation regulates fat and/or glucose metabolism, inhibition of FTO can lead to weight loss. O2-Utilizing Enzymes in Other Biological Reactions O2 concentrations also exert profound impact on many types of biological reactions catalyzed by O2-binding oxidoreductases or oxygenases. Among the oxidoreductases, the well-known

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cyclooxygenases COX-1and COX-2 are involved in the synthesis of prostanoids such as prostaglandin, prostacyclin, and thromboxane; lipoxygenases catalyze incorporation of O2 into polyunsaturated fatty acids; and CYP450 mono-oxygenases can function as epoxygenases or hydroxylases for polyunsaturated fatty acids. On the other hand, Netrimethyl lysine hydroxylase and g-butyrobetaine hydroxylase, members of the 2-OG oxygenase family, are involved in carnitine biosynthesis.

Concluding Remarks With the identification of the increasing numbers of new classes of O2 sensors such as demethylases discussed above, we are just beginning to understand the broad and profound impact of O2 on many essential aspects of the aerial lives from energy production to regulation of gene expression and to maintenance of genomic integrity. As shown by a wealth of studies, specific O2 sensors play a significant role in tumorigenesis, tumor growth, and metastasis. Understanding the mechanisms of their biological actions would lead to the discovery of new and more effective cancer treatments.

O References Aragonés J, Fraisl P, Baes M, Carmeliet P (2009) Oxygen sensors at the crossroad of metabolism. Cell Metab 9(1):11–22 Loenarz C, Schofield CJ (2011) Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends Biochem Sci 36(1):7–18 Semenza GL (2011) Oxygen sensing, homeostasis, and disease. N Engl J Med 365:537–547 Vanderkooi JM, Erecinska M, Silve IA (1991) Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am J Physiol 260(6 Pt1): C1131–C1150

Oxygen Tension Distribution in Tumor ▶ Oxygenation of Tumors

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Oxygen Toxicity ▶ Antioxidant Enzymes

Oxygenation of Tumors Peter Vaupel and Arnulf Mayer Department of Radiooncology and Radiotherapy, University Medical Center Mainz, Mainz, Germany

Synonyms Oxygen partial pressure distribution in tumor; Oxygen tension distribution in tumor; Oxygenation status and tumor oxygen level

Definition Tumor oxygenation, which reflects the distribution of oxygen (O2) partial pressures (pO2 values) or O2 concentrations, results from O2 availability (O2 supply) to the tumor tissue, the diffusional flux of O2 from the microvessels to the cells, and the respiration rate (O2 consumption rate) of the parenchymal and stromal cells making up the tissue. O2 supply is mainly influenced by the efficacy of microcirculatory blood flow, diffusional O2 flux (predominantly influenced by diffusion distances), and blood hemoglobin concentration (cHb) in anemic patients.

Characteristics Whereas in normal tissues the O2 supply meets the metabolic demands, in many solid tumors, the respiration rate may outweigh an insufficient O2 supply and result in the development of tissue subvolumes with very low O2 levels (▶ hypoxia, O2-depleted tissue areas) or even areas completely lacking O2 (▶ anoxia).

Oxygen Toxicity

Considerable evidence demonstrates that in most human tumors, oxygenation is compromised as compared to normal tissues, which are characterized by “normal” O2 partial pressure distributions (normoxia). Oxygenation is extremely heterogeneous (both spatially and temporally, “4-D-heterogeneity”) within an individual tumor (intra-tumor heterogeneity). Furthermore, considerable heterogeneity of oxygen-depleted areas (hypoxic areas) has been shown between tumors of the same clinical size, stage, grade, or histological type (inter-tumor heterogeneity). Tumor oxygen supply is not regulated according to the metabolic demand, as is the case in the physiological situation. On average, the median pO2 values in tumors are substantially lower (25 mmHg) at the site of tumor growth (Fig. 1). In general, tumor oxygenation is independent of clinical size, stage, grade, histology, and various oncologic parameters or patient demographics. In some cancer entities, the oxygenation status significantly deteriorates in anemia (as a result of a decreased O2 capacity of the blood) and at Hb concentrations above the median Hb level (most probably due to rheological problems within the chaotic tumor microvessels). Metastatic lesions seem to have an oxygenation status comparable to that of the primaries, whereas local recurrences have a poorer oxygenation status than the primary tumors. Pathomechanisms of Tumor Hypoxia Tumor hypoxia results from an inadequate O2 delivery to the respiring neoplastic as well as stromal cells. Limited and even abolished O2 supply is due to severe structural and functional abnormalities of the microcirculation, as well as due to a deterioration of diffusion geometry. In addition, cancer-related and/or therapy-induced anemia and carboxyhemoglobin formation (in heavy smokers) can lead to a reduced O2 content of the blood. As a result, areas with very low (down to zero) O2 partial pressures exist in solid tumors. These very low pO2 microregions are heterogeneously distributed within the tumor mass and may be located next to regions with pO2 values corresponding to the normal tissue from

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Oxygenation of Tumors, Fig. 1 Oxygenation status of normal tissues (left panels) and solid tumors (right panels). Frequency distributions of measured pO2 values (pO2 histograms) for normal breast tissue and uterine cervix are compared to the respective cancer tissues (clinical stages Ib–IV). Oxygen partial pressures (pO2) in the tissue were

measured with a computerized polarographic microsensor technique which enables direct assessment of the pO2 data with an O2-sensitive needle electrode. Oxygen partial pressures were measured along several electrode tracks in each individual tumor. Pooled data presented here are derived from pretreatment measurements in conscious patients

which the tumor has been derived. In addition, significant temporal variations in the oxygenation status have been observed in tumors. Thus, tumor hypoxia is highly dynamic with complex, rapidly changing oxygen gradients. Based on underlying mechanisms and their duration, two main types of hypoxia have been described: chronic and acute hypoxia (see Table 1).

measurement of O2 partial pressures. With this invasive microtechnique, frequency distributions (histograms) of measured intra-tumor pO2 values can be obtained with a relatively high spatial resolution. Other direct procedures include fiberoptic O2 sensors and electron paramagnetic resonance (EPR) oximetry. This latter technique is minimally invasive requiring only application of the paramagnetic material. Measurement of intravascular oxyhemoglobin (HbO2) saturation is another method that has the potential to allow a characterization of the oxygenation status of human tumors (blood oxygen level-dependent magnetic resonance imaging, BOLD-MRI). However, it only provides information related to the vascular compartment, and thus,

Detection of Tumor Hypoxia Assessment of the tumor oxygenation status in experimental tumors and in the clinical setting has been performed using various techniques. So far, the most direct and often used method for description of oxygenation is the polarographic

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3344 Oxygenation of Tumors, Table 1 Causes of chronic and acute tumor hypoxia A. Chronic hypoxia Synonyms Continuous h., diffusion-limited h., used sustained h., long-term h. Causes 1. Diffusion-limited hypoxia (pathogenesis) Enlarged diffusion distances Adverse diffusion geometries (concurrent vs. countercurrent tumor microvessels, Krogh- vs. Hill-type diffusion geometry) Extreme longitudinal intravascular oxygen gradients Shunt perfusion 2. Hypoxemic hypoxia Tumor-associated anemia Therapy-induced anemia Small liver tumors supplied by portal vein HbCO formation in heavy smokers 3. Compromised perfusion of microvessels Disturbed Starling forces due to high interstitial fluid pressure (transmural coupling) Compression of microvessels due to solid-phase stress caused by non-fluid components Time frame Hours ! weeks (under experimental conditions) B. Acute hypoxia Synonyms Transient h., short-term h., perfusionused limited h., cyclic h., fluctuating h., intermittent h., periodic h. Causes 1. Temporary flow stop in (pathogenesis) microvessels Due to cell aggregates and/or fibrin clots Ischemic hypoxia due to vascular remodeling 2. Transient hypoxemia Temporal plasma flow in microvessels Fluctuating red blood cell fluxes Time frame Minutes ! 1 hour (not well defined; spontaneous hypoxia cycles show spatial and temporal irregularities)

the situation in the extravascular space can only be inferred. Another MRI-based technique is dynamic contrast-enhanced (DCE)-MRI, which provides only indirect information on the oxygenation status derived from the kinetics of the

Oxygenation of Tumors

exchange of a contrast agent between the blood vessels and the extravascular space. Tumor oxygenation can be measured in tomographic images in the clinical setting upon inhalation of radiolabeled 15O-gas in ▶ positron emission tomography (PET) studies. However, as with MRI procedures, limitations pertain to provide quantitative data the rather low spatial resolution of the method. The parameter measured with these noninvasive techniques is not directly interpretable as a tumor pO2 value or O2 concentration. Noninvasive methods for detection of tumor hypoxia include the binding and the retaining of radiolabeled bioreductive drugs, such as fluoromisonidazole (labeled with 18F and detected by positron emission tomography) and iodoazomycin arabinoside (labeled with 123I and detected with single-photon emission computerized tomography, SPECT). Several techniques for assessment of tumor oxygenation require the analysis of tumor biopsy specimens. Using immunohistochemistry with exogenous hypoxia markers (e.g., pimonidazole), detailed information concerning hypoxia at the cellular level can be obtained. Disadvantages include the need for injection of a hypoxia marker and possible sampling errors. The suitability of so-called endogenous hypoxia markers (e.g., HIF-1a, GLUT-1, CA IX) has been called into question due to a lack of correlation with direct pO2 measurements and the plethora of factors that impact on the expression levels of these markers besides hypoxia. Clinical Relevance Tumor hypoxia has been considered to be a therapeutic problem since it renders solid tumors resistant to sparsely ionizing radiation (X- and gamma rays), some forms of chemotherapy (e.g., cyclophosphamide, carboplatin), and photodynamic therapy. Oxygen levels may furthermore influence a series of biological parameters, which in turn may markedly increase the malignant potential of a tumor irrespective of tumor treatment modalities. Dynamic hypoxia is a common characteristic of locally advanced solid tumors that has been

Oxygenation of Tumors Oxygenation of Tumors, Fig. 2 Schematic representation of the mechanisms causing hypoxia-induced proteomic and genomic changes leading to tumor aggressiveness, malignant progression, treatment resistance, and poor prognosis

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Modulation of gene expression

Reversible proteome changes

Hypoxia-induced transcription factors (HIF-1α, NFkB, AP1)

Hypoxia ( 10.000 ng/ml LDH > 10x Norm

markers should decline according to their half-life (ß-hCG: 24–36 h, AFP: 5–7 days). Any plateau phase or any delay in decline is predictive for a poor outcome in terms of response to therapy. The prognostic significance of tumor markers at the time of diagnosis becomes evident for advanced disease only, adhering to the International Germ Cell Consensus Classification Group (IGCCCG) classification. The Lugano classification represents the most widely used clinical staging system for testicular cancer (Table 2) and describes the extent of metastatic involvement of the lymph nodes and visceral organs. The IGCCCG has introduced a new staging system for advanced TC defining three prognostic risk groups with regard to therapeutic outcome. Patients are classified to be at good risk (probability of cure 95%), intermediate risk (probability of cure 70%), or poor risk (probability of cure 50%). The IGCCCG classification gives high prognostic evidence and enables an individualized risk-adapted approach in patients with advanced TC. Therapy Once TIN is diagnosed, therapeutic intervention is recommended, since 70% of patients will develop invasive germ cell tumor within the next 7 years. Local radiation therapy with 18 Gy is the therapy of choice in patients with a contralateral invasive

Testicular Cancer, Table 2 Clinical Lugano – classification of TC Stage I

Stage IIA Stage IIB Stage IIC Stage IIIA Stage IIIB

Stage IIIC

Tumor markers normalized or decline according to their half-life after orchiectomy No detectable metastases by imaging studies Primary TC confined to the testicle Retroperitoneal metastases 5 cm Supraclavicular or mediastinal metastases Pulmonary metastases Minimal: 2 cm Extrapulmonary visceral metastases

T germ cell tumor. In patients with unilateral TIN and a contralateral normal testis, inguinal orchiectomy appears to be the preferred management, since local radiation bears the risk of damaging the healthy testicle. Non-seminomatous Germ Cell Tumors (NSGCT) Clinical stage I NSGCT represents a troublesome entity concerning recommendations for optimal management since about 30% of patients will

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exhibit microscopic lymph node disease. Several treatment options such as primary nerve-sparing retroperitoneal lymphadenectomy, primary chemotherapy, and active surveillance have been developed resulting in the same high cure rates of 98%. The European Germ Cell Cancer Consensus Group recommends an individualized, risk-adapted approach based on the results of prospective randomized trials considering the presence or absence of the risk factors vascular invasion (VI) and percentage of embryonal carcinoma (ECA). VI has been identified as the most powerful clinical predictor of lymph node metastasis with 48% of NSGCTs with VI developing metastases, compared to 14–22% of tumors without VI. A combination of VI and ECA might be even more powerful. Nowadays, nerve-sparing RPLND – if performed – is regarded as the standard approach. Up to 10% of patients will suffer from pulmonary relapse within the first 2 years and will be cured by platinum-based chemotherapy. Even in low-volume lymph node disease such as pathological stage IIA, the nerve-sparing RPLND can be performed as bilateral radical surgery without compromising the therapeutic outcome. Primary chemotherapy [two cycles of cisplatin, etoposide, and bleomycin (PEB)] and surveillance (absence of VI) result in relapse rates of only 7% and 14%, respectively. Low-Stage (IIA/B) NSGCT

Low-stage testicular disease comprises clinical stages IIA and IIB associated with a cure rate of 98%. Patients with low-volume disease and abnormal tumor marker levels of AFP, ß-hCG, or LDH are treated with two to three cycles of PEB chemotherapy; patients with negative markers might be offered nerve-sparing RPLND or surveillance; and patients with clinical stage IIB TC will undergo primary chemotherapy depending on the serum tumor marker concentrations with three or four cycles of PEB followed by secondary RPLND in about 30% of cases. Clinical Stages IIC and III

Inductive chemotherapy represents the therapy of choice, with the number of cycles applied

Testicular Cancer

depending on the IGCCCG-based prognostic classification. Patients with “good prognosis” face a long-term survival rate of >90% and are managed by three cycles of PEB. Patients with “intermediate prognosis” face a survival rate of 70–80% and are managed by four cycles of PEB or cisplatin, etoposide, and ifosfamide (PEI). Patients with “poor prognosis” have a survival rate of only about 50%; standard therapy consists of four cycles of PEB or PEI. A major advantage of primary high-dose chemotherapy has not been demonstrated, but this approach is currently being tested in prospective randomized trials. Seminomatous Germ Cell Tumors Clinical Stage I Seminoma

Despite negative CT scans, there is a risk of 12–32% of occult retroperitoneal lymph node metastases depending on the absence or presence negative prognostic markers. The cure rate of clinical stage I seminomatous germ cell cancer is close to 100% and can be achieved by the three different therapeutic options: active surveillance, radiation therapy, and carboplatin monochemotherapy. Adjuvant retroperitoneal radiation therapy to the para-aortic or paracaval region with 20 Gy or adjuvant chemotherapy with one cycle carboplatin AUC 7 is the standard approach for high-risk patients (tumor size >4 cm, rete testis invasion) and results in a relapse-free long-term survival of 97%. Active surveillance represents the most reasonable approach to patients with good prognostic markers associated with a low recurrence rate of about 12%. Treatment of relapses is more intense with systemic chemotherapy of three to four cycles PEB in most cases. Low-Stage (Clinical Stage IIA/B) Seminoma

Radiation therapy with 30 Gy (IIA) and 36 Gy (IIB), including the ipsilateral iliac and inguinal lymph nodes, is one standard therapeutic approach for low-stage seminomas. Relapse-free survival is as high as 92.5% in clinical stage IIA/B; relapse rates are about 5% in stage IIA and about 11% in stage IIB seminomas. Primary

Testicular Cancer

chemotherapy with two cycles of PEB is an alternative to radiation in clinical stage IIB seminoma. Clinical Stages IIC and III

As pointed out for advanced non-seminomatous germ cell tumors, therapy should be initiated according to the IGCCCG classification. For patients with good prognosis, three cycles of PEB chemotherapy are the treatment of choice; in patients with intermediate prognosis, four cycles of PEB chemotherapy are applied. Residual Tumor Resection (RTR) Following Chemotherapy for Advanced Testicular Cancer. RTR represents an integral part of the multimodality treatment of advanced testicular germ cell tumors. The rationale for RTR is to completely resect mature teratoma and vital cancer which will be found in 30–40% and 20% of the patients, respectively. Currently, all residual lesions independent on size should be resected in NSGCT since even small lesions 10% vital cancer cells or those with uncomplete resection might benefit from consolidation chemotherapy with two cycles. Post-chemotherapy or postradiotherapy RPLND in seminomas has only to be performed in lesions with a positive PET scan performed about 6 weeks after chemotherapy or radiation therapy in patients with residual lesions >3 cm. Salvage Chemotherapy, High-Dose Chemotherapy

In seminomas relapsing after first-line radiation therapy, a cure rate of >90% can be achieved by

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cisplatin-based chemotherapy according to the IGCCCG algorithm with regard to advanced seminomas. About 50% of relapsing seminomas following conventional chemotherapy can be salvaged with another combination chemotherapy consisting of platinol, etoposide, and ifosfamide (PEI) (VIP) or vinblastine, ifosfamide, and platinol (VeIP). Currently, a 10% benefit of highdose chemotherapy with regard to survival has been demonstrated; therefore, it seems advisable that all relapsing patients should be treated in a tertiary referral center. In NSGCT relapsing following conventional chemotherapy, salvage rates are as low as 15–40% using standard salvage protocols such as PEI–VIP or PEI–VeIP. In some institutions the addition of paclitaxel to ifosfamide and cisplatin has been favored due to a high response rate >50%. Conventional-dose cisplatin-based salvage chemotherapy can achieve long-term remission in 15–40% of patients. Early consideration of high-dose chemotherapy seems advisable: trials suggest a benefit for the use of high-dose chemotherapy and autologous bone marrow transfer, with 46% and 50% of the patients being alive and disease-free after a median follow-up of 31 months and 30 months, respectively. Options for third-line chemotherapy are combinations such as paclitaxel and gemcitabine, gemcitabine and oxaliplatin or paclitaxel, and gemcitabine and cisplatin, within clinical trials. Genetics With regard to predisposing genetic events, the locus Xq27 predisposes for bilateral TC and bilateral cryptorchidism. Other studies have reported the loci 1p36, 4p14–13, 5q21–21, 14q13–q24.3, and 18q21.1–21.3 to be highly associated with TC. It has been demonstrated that somatic mutations of exons 10, 11, and 17 of KIT occur significantly more often in patients with bilateral TC as compared to patients with unilateral disease. The results indicate that KIT might be involved in the development of familial and a minority of sporadic germ cell tumors and that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes.

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Currently, all molecular markers such as p53, Ki-67, bcl-2, cathepsin D, and E-cadherin have not been proven to be clinically useful prognosticators; only reverse transcriptase-polymerase chain reaction for AFP, hCG, and germ cell alkaline phosphate (GCAP) mRNA for the detection of circulating tumor cells appears to be an interesting approach, with 60% of clinical stage I testicular cancer patients exhibiting positive signals that turn into negative signals following adjuvant chemotherapy. Future Directions in TC Based on the excellent therapeutic outcome, there appear to be only a few developments possible that will have further impact on the survival of testicular cancer patients. However, there might be many options to improve quality of life either due to reduction of acute toxicity or due to the development of treatment regimes associated with a significantly reduced long-term toxicity. The risk of cardiovascular disease is significantly increased after standard chemotherapy with three to four cycles of PEB and/or salvage treatment (RR = 2.59). The increased risk is not due to an increase in classical cardiac risk factors but directly dependent on first-line therapy. For the future, attempts to minimize treatment should be undertaken especially in patients with good prognosis in whom this type of long-term toxicity might be a greater risk to long-term survival than testicular cancer itself. Elucidation of those mechanisms involved in the development of intrinsic and extrinsic chemorefractoriness in testicular cancer will be a major issue in the future, to apply effective chemotherapeutic protocols and to save even more lives. There are some promising approaches using modern molecular techniques such as gene expression profiling to explore the role of mismatch repair genes, multidrug-resistance genes, and potentially unknown genes. Despite the high cure rates, it will be necessary for testicular cancer to be treated by clinicians and institutions with sufficient experience in diagnosis and management of germ cell tumors. Specific problems such as extended tumor masses, relapsing tumors, or

Testicular Cancer

poor prognosis at initial diagnosis must be referred to tertiary centers having the ability of an interdisciplinary approach.

Cross-References ▶ Carcinoma in Situ

References Albers P, Albrecht W, Algaba F, Bokemeyer C, CohnCedermark G, Fizazi K, Horwich A, Laguna MP, Nicolai N, Oldenburg J (2015) Guidelines on testicular cancer: 2015 update. Eur Urol 68(6):1054–1068 Beyer J, Albers P, Altena R, Aparicio J, Bokemeyer C, Busch J, Cathomas R, Cavallin-Stahl E, Clarke NW, Claßen J, Cohn-Cedermark G, Dahl AA, Daugaard G, De Giorgi U, De Santis M, De Wit M, De Wit R, Dieckmann KP, Fenner M, Fizazi K, Flechon A, Fossa SD, Germá Lluch JR, Gietema JA, Gillessen S, Giwercman A, Hartmann JT, Heidenreich A, Hentrich M, Honecker F, Horwich A, Huddart RA, Kliesch S, Kollmannsberger C, Krege S, Laguna MP, Looijenga LH, Lorch A, Lotz JP, Mayer F, Necchi A, Nicolai N, Nuver J, Oechsle K, Oldenburg J, Oosterhuis JW, Powles T, Rajpert-De Meyts E, Rick O, Rosti G, Salvioni R, Schrader M, Schweyer S, Sedlmayer F, Sohaib A, Souchon R, Tandstad T, Winter C, Wittekind C (2013) Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer. Ann Oncol 24 (4):878–888 Cavalli F, Manfardini S, Pizzocaro G (1980) Report on the international workshop on staging and treatment of testicular cancer. Eur J Cancer 6:1367–1372 Daneshmand S, Albers P, Fosså SD, Heidenreich A, Kollmannsberger C, Krege S, Nichols C, Oldenburg J, Wood L (2012) Contemporary management of postchemotherapy testis cancer. Eur Urol 62(5):867–876 Fung C, Fossa SD, Williams A, Travis LB (2015) Longterm morbidity of testicular cancer treatment. Urol Clin North Am 42(3):393–408 Heidenreich A, Srivastava S, Moul JW et al (2000) Molecular genetic parameters in pathogenesis and prognosis of testicular germ cell tumors. Eur Urol 37:121–135 International Germ Cell Consensus Classification Group (1997) A prognostic factor-based staging system for metastatic germ cell cancers. J Clin Oncol 15:594–603 Oldenburg J, Fosså SD, Nuver J, Heidenreich A, Schmoll HJ, Bokemeyer C, Horwich A, Beyer J, Kataja V; ESMO Guidelines Working Group (2013) Testicular seminoma and non-seminoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 24(Suppl 6):vi125–32

Testicular Germ Cell Tumors Schmoll HJ, Souchon R, Krege S et al (2004) European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG). Ann Oncol 15:1377–1399 Skakkebaek NE, Bertlesen JG, Giwercman A et al (1987) Carcinoma in situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytic seminoma. Int J Androl 10:19–28

See Also (2012) Germ Cell Tumors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905

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Definition Testicular cancer represents a group of histologically heterogeneous neoplasms typically arising in gonadal tissue and, uncommonly, arising in extragonadal sites such as the retroperitoneum or mediastinum. Tumors of the testis include germ cell tumors (which include seminomas and nonseminomas) or sex cord tumors (includes (▶ Leydig Cell Tumor, Sertoli, and ▶ granulosa cell tumors). With improvement of care, even metastatic disease is now curable in many men with testicular cancer.

Characteristics

Testicular Feminization (TFM) ▶ Androgen Receptor

Testicular Germ Cell Tumor ▶ Testicular Cancer

Testicular Germ Cell Tumors Kamran Zargar-Shoshtari1, Craig Kovitz2, Phillippe E. Spiess3 and Nizar M. Tannir4 1 Department of Urology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Department of Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Genitourinary Oncology, Moffitt Cancer Center, Tampa, FL, USA 4 Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Dysgerminoma; Gonadal neoplasms; Testicular tumors

Incidence Testicular cancers represent only 1% of male tumors and 5% of genitourinary malignancies; however, germ cell tumors are the most common malignancy in men between the ages of 20–40 years. These tumors demonstrate a bimodal distribution in occurrence, and they are most often seen in 15–25 year-old men with a second, smaller peak at about age 60. Overall incidence of seminoma peak is in the 40s and nonseminoma in 30s. It is estimated that in the Western society, 3–10 new cases per year occur per 100,000 males. Over the past few years, the incidence of testicular cancer has significantly increased, with the highest rise reported in the Great Britain, United States, and Northern Europe. Risk Factors Cryptorchidism has been identified as a major risk factor for development of testicular cancer, although only about 10% of cases are associated with this phenomenon. Data suggest that orchidopexy prior to puberty may reduce the risk of testicular cancer. This increased risk is true for the contralateral testicle even if it is descended normally. Additional risk factors include a previous history of testicular tumor as well as the presence of a first degree relative with the disease. Testicular dysgenesis syndrome (cryptorchidism, hypospadias, reduced spermatogenesis, and subfertility) is also linked to the development of

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testicular cancer. Scrotal trauma and toxic exposures have no proven association with the occurrence of germ cell tumors. Histological Classification The main histological categories of germ cell tumors are seminomas (seminomatous germ cell tumor) and nonseminomas (nonseminomatous germ cell tumor). Nonseminomas are further subcategorized as embryonal carcinomas, endodermal sinus tumors (also known as yolk sac tumors), choriocarcinoma, and teratoma. Tumors that contain more than one histological subtype are termed mixed germ cell tumors. For classification and treatment purposes, any tumor not histologically a pure seminoma is classified as a nonseminoma. Non–germ cell tumors are known as sex cord or stromal tumors and include Leydig cell, Sertoli cell, Granulosa cell, as well as some other rare types of stromal tumors. Clinical Presentation and Diagnosis Painless testicular swelling or a palpable testicular mass is the most common form of presentation for testicular malignancy. Associated pain is often related to infarction or bleeding within the tumor. Systemic symptoms at presentation such as abdominal and back pain, anorexia with or without associated weight loss, night sweats, chest pain, and shortness of breath or hemoptysis usually indicate an advanced stage of disease or an extragonadal primary tumor. Testicular ultrasound is the ideal form of radiological examination for assessment of scrotal masses, with sensitivity approaching 100%. Magnetic resonance imaging (MRI) is also an option, although this remains less practical. Additionally, the tumor markers human chorionic gonadotropin (hCG), ▶ alpha-fetoprotein (AFP), and lactate dehydrogenase (LDH) should be obtained at diagnosis. These have value in diagnosis, staging, and evaluation of response to therapy as well as follow up assessment of germ cell tumors. Although not specific for germ cell tumors, AFP is produced by tumors with endodermal sinus or embryonal components as well as by immature teratomas. HCG is a hormonal product of syncytiotrophoblasts and can be expressed

Testicular Germ Cell Tumors

by choriocarcinoma, mixed germ cell tumors and, sometimes, by seminomas. LDH is a cellular protein expressed in numerous tissues and can be produced by nonseminomas. Additional preoperative workup typically includes a chest radiograph and discussion of sperm banking with the patient. When a testicular mass is found on ultrasonography, a radical inguinal orchidectomy is the standard of care. However, in certain circumstances when the patient has extensive systemic disease, systemic chemotherapy should be started prior to an orchidectomy. In this instance, it would be appropriate to proceed with chemotherapy without tissue diagnosis, if the markers are elevated and the clinical picture is compatible with the diagnosis of germ cell tumor. Postoperatively (or preoperatively if this will not delay surgery) an abdominal and pelvic computed tomography (CT) scan should be performed and, if clinically indicated, brain magnetic resonance imaging (MRI) and a bone scan. CT scans can reveal clinically significant lymphadenopathy that will be important in staging and clinical decision-making. The preferred sites of initial spread for right-sided tumors are typically the infrarenal paracaval, interaortocaval, and possibly paraaortic nodes. In contrast, left-sided tumors preferentially spread to the infrarenal paraaortic nodes initially. Direct spread from right side of the retroperitoneum to the left side, but not vice versa, is also possible. Transscrotal biopsies should be avoided as they can disturb lymphatic channels, potentially changing the typically predictable lymphatic spread of these tumors. Organ sparing orchidectomy is possible in certain clinical situations, including synchronous bilateral testicular tumors or in tumor in a solitary testis. Organ sparing surgery can only be considered when there is normal preoperative testosterone levels and when the tumor volume is less than 30% of the total testicular volume and clear surgical margins can be achieved. Staging and Risk Stratification The aim of staging is to classify patients with respect to prognosis and to allow for standardized

Testicular Germ Cell Tumors

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Testicular Germ Cell Tumors, Table 1 International germ cell cancer consensus group classification prognostic risk stratification Good risk

Intermediate risk

Poor risk

Seminoma Any disease primary site No nonpulmonary visceral metastases Normal markers

82% 5-year PFS; 86% 5-year OS Any primary site Nonpulmonary visceral metastases Normal AFP Any hCG Any LDH 67% 5-year PFS; 72% 5-year OS –

Nonseminoma Testis or retroperitoneal primary No nonpulmonary visceral metastases S0 AFP < 1,000 ng/mL S1 hCG < 5,000 mIu/mL LDH < 1.5  (ULN) 86% 5-year PFS; 90% 5-year OS Testis/retroperitoneal primary No nonpulmonary visceral metastases S2 AFP 1,000–10,000 ng/mL hCG 5,000–50,000 mIu/mL LDH 1.5–10  ULN 75% 5-year PFS; 80% 5-year OS Mediastinal primary Nonpulmonary visceral metastases S3 AFP > 10,000 ng/mL HCG > 50,000 mIu/mL LDH > 10  ULN 41% 5-year PFS; 48% 5-year OS

ULN upper limits of normal range, PFS progression free survival, OS overall survival

treatment planning. Primary tumor characteristics, extent and size of lymphadenopathy, presence of visceral metastases as well as post orchidectomy tumor markers are important factors in determining accurate disease stage. The standard risk stratification used for these tumors is that developed by the International Germ Cell Cancer Consensus Group (IGCCCG). Through a retrospective multivariate analysis of nearly 6,000 patients with germ cell tumors, this group identified a number of clinical features which are strongly associated with prognosis: primary disease site, the presence of nonpulmonary visceral metastases, and tumor marker levels after orchidectomy. Based on these characteristics, IGCCCG divided nonseminomatous germ cell tumors into good-, intermediate-, and poor-risk categories and seminomas into good- and intermediate-risk categories (Table 1). This risk stratification system provided the basis for the American Joint Commission on Cancer (AJCC) TNM staging system for germ cell tumors. In general, stage I disease is confined to the testis, stage II disease is limited to the retroperitoneum, and stage III disease involves the nodal disease beyond the retroperitoneum or

nonnodal metastatic disease. Elevated serum tumor markers can help to define higher stages of disease. Management As the biology and management of seminomas and nonseminomas are quite different, we will consider the management of these distinct histologies separately. Seminomas

By IGCCCG risk stratification, all patients with seminomas without nonpulmonary visceral metastases are categorized as having good-risk disease. Amongst these patients are those with stages I and II disease and some with stage III disease. The majority of patients with stage I disease (TxN0M0, based on CT scan staging) will be cured with radical inguinal orchidectomy alone; however, at 5 years, 15–20% of patients with stage I seminoma will relapse, mainly in the infradiaphragmatic lymph nodes. As a result, attempts have been made to identify those features of stage I disease which would indicate a higher

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risk of relapse. It has been reported that there is a subset of patients with stage I seminoma with a primary tumor smaller than 4 cm in size and without rete testis involvement who have a relapse-free survival of 88%, and thus may constitute a group of patients most appropriate for surveillance. Although, the latter finding has not been validated by other studies. At present, surveillance is considered a reasonable option for highly motivated patients with stage I seminoma after orchidectomy. Adjuvant chemotherapy is an alternative choice, with one or two cycles of carboplatin reducing relapse rates to 1–3%. Seminoma is also extremely radiosensitive and adjuvant 20 Gy of radiotherapy to a paraaortic field will reduce the relapse rate to 1–3%. Some have demonstrated that single dose of carboplatin may be less toxic than radiotherapy in stage I seminoma. Patients with stage II disease (infradiaphragmatic nodal disease only) are divided into those with nonbulky disease (those with nodal metastases less than 5 cm) and those with bulky disease (those with nodal metastases greater than 5 cm). For patients with nonbulky disease (stage IIA-B), infradiaphragmatic radiation therapy (30 Gy stage IIA AND 36Gy stage IIB) to include the paraaortic and ipsilateral iliac nodes is the standard therapy. This technique yields a relapse-free survival in stage IIA and IIB of 92% and 90%, respectively. Residual abnormalities are sometimes encountered following radiation therapy, but observation is generally recommended. Alternatively, primary chemotherapy with four cycles of EP (eposide/cisplatin) or three cycles of BEP (▶ Bleomycin/▶ Etoposide/▶ Cisplatin) is an option. There are no phase III trials comparing chemotherapy with radiation in this setting, although chemotherapy offers similar disease-free rates as radiation therapy. Some authorities recommend chemotherapy as the first choice for stage IIB, and radiation therapy as the preferred choice for stage IIA. Retroperitoneal node dissection or surveillance is not recommended in this clinical situation. For patients with advanced seminoma, defined as having stage IIC or stage III disease, chemotherapy is the treatment modality of choice. For

Testicular Germ Cell Tumors

those patients with good-risk disease, chemotherapy with four cycles of EP is generally offered. Bleomycin is generally excluded as the risk of pulmonary toxicity outweighs the small incremental benefit afforded by its use. In the same light, carboplatin has been demonstrated to be inferior to cisplatin and is thus not substituted in this scenario. For those patients with intermediate-risk disease, chemotherapy is usually administered with four cycles of BEP. After completion of chemotherapy, patients with advanced seminoma are restaged with chest, abdominal, and pelvic CT scans as well as serum tumor markers. If no residual mass is found, or the residual mass measures 3 cm in size, a ▶ positron emission tomography (PET) scan is performed 6 weeks after chemotherapy to assess for the presence of viable tumor. In the presence of a positive PET scan, second line chemotherapy or retroperitoneal lymph node dissection can be considered. Patients who are found to have progressive disease after initial therapy are generally treated with salvage chemotherapy. These include VeIP (vinblastine, ifosfamide, cisplatin), TIP (paclitaxel, ifosfamide, cisplatin), or high dose chemotherapy. Nonseminomatous Germ Cell Tumors (NSGCT)

NSGCT are risk stratified according to the IGCCCG classification schema based on the location of the primary tumor, the presence of nonpulmonary visceral metastases, and the level of tumor marker elevation. Treatment options vary by stage and can include observation, chemotherapy, and/or retroperitoneal lymph node dissection (RPLND). Patients with stage IA NSGCT (tumor limited to the testis and epididymis without lymphovascular invasion and normal postorchidectomy tumor markers) are generally managed with either surveillance (in reliable patients) or RPLND. The data from largest surveillance studies suggest a relapse rate of about 30%, with 80% of relapses seen within the first 12 months, 12% seen in the second year, and 6% during the third year, with

Testicular Germ Cell Tumors

1% occurring during the fourth and fifth years. Patients who relapse while on surveillance can be treated with chemotherapy with excellent longterm outcomes. RPLND is often used because it usually leads to accurate staging and can be curative in the majority of patients. The presence of pN1 or pN2 disease at RPLND can then be managed either with surveillance (pN1) or two cycles of EP or BEP chemotherapy (pN2). Patients with N3 disease found at RPLND are typically managed as good-risk advanced stage patients and are treated with EP for four cycles or BEP for three cycles. Patients with stage IB NSGCT can be managed with RPLND, although either active surveillance (for T2 disease only) or chemotherapy with two cycles of BEP is also appropriate. One or two cycles of BEP for stage I disease has reported relapse rates of 1.6–3.1%. Patients with Stage IS disease (persistent marker elevation after orchidectomy) are managed with chemotherapy (either four cycles of EP or three cycles of BEP). Stage IIA NSGCT in the presence of negative postorchidectomy tumor markers are generally approached with either RPLND or primary chemotherapy with EP for four cycles or BEP for three cycles. Those patients with stage IIA disease who have persistent tumor marker elevations are managed with chemotherapy alone. Patients with stage IIB disease and negative markers may undergo RPLND as long as lymph node metastases are within lymphatic drainage sites. PostRPLND management is the same as for those patients who have RPLND for stage I disease. If the patient has multifocal lymph node metastases, adjuvant chemotherapy is the preferred management option. Patients with advanced disease (stage IIC and III) are risk stratified by the IGCCCG into three categories (good, intermediate, and poor). Those with good-risk disease (testis or retroperitoneal primary; no nonpulmonary visceral metastases as well as AFP 2 cm but 4 cm in greatest dimension Tumor >4 cm in greatest dimension Tumor invades adjacent structures (e.g., through cortical bone, into deep extrinsic muscle of tongue, maxillary sinus, skin; superficial erosion of bone/tooth socket by gingival primary is not sufficient to classify as T4) Regional lymph nodes cannot be assessed No regional lymph node metastasis Metastasis in single ipsilateral node, 3 cm in greatest dimension Metastasis in single ipsilateral node, >3 cm but 6 cm Metastasis in multiple ipsilateral nodes, all 6 cm Metastasis in bilateral or contralateral nodes, all 6 cm Metastasis in lymph node >6 cm in greatest dimension Distant metastasis cannot be assessed No distant metastasis Distant metastasis (continued)

Tongue Cancer 0 I II III IV

Tis N0 M0 T1 N0 M0 T2 N0 M0 T3 N0 M0, T1–2 N1 M0, T3 N1 M0 T4 N0–1 M0, anyT N2–3 M0, anyT anyN M1

It has been shown by many studies that tumor thickness, but not the largest dimension, is a significant independent prognostic factor in predicting subclinical nodal metastasis, local recurrence, and survival of oral tongue carcinoma. The thicker the tumor, the higher would be the risk of local recurrence, subclinical nodal metastasis, and treatment failure. It is however still unresolved yet on the best cutoff thickness value for staging purpose. The proposed cutoff thickness values for prognosis or staging by various studies vary between 3 and 9 mm. The other important prognostic factor is histologic feature of perineural spread which is a significant risk factor of local recurrence after surgical treatment. Pretreatment Assessment Preoperative endoscopy and biopsy should be done to confirm the diagnosis and evaluate the extent of local tumor infiltration. Small neck nodes less than 1 cm along the jugular chain may not be palpable with fingers. Ultrasonography of the neck and ultrasound-guided fine-needle aspiration for cytology should be done to screen for presence of small nodal metastasis that may not be palpable. Tumor thickness is an important factor for prognosis evaluation and treatment planning. Preoperative assessment of tumor thickness cannot be done with palpation. Intraoral ultrasonography using 7.5 MHz probe can be used to document the tumor thickness. MRI can also be used to assess the tumor thickness for preoperative evaluation of prognosis. Both T1- and T2-weighted MRI images can show the tumor thickness satisfactorily. MRI images in three-dimensional planes can also help the surgeon in the planning of surgical resection. Treatment Options The treatment of oral tongue carcinoma remains controversial. Brachytherapy alone or surgery

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alone is each commonly used as primary treatment for early carcinomas by radiation oncologists and surgeons. The curative results of brachytherapy and surgery are similar for early stage carcinoma with over 90% of local control. Either surgery or brachytherapy alone is not effective for stage III and IV carcinomas; combined surgery and postoperative chemoradiotherapy are recommended for advanced-stage carcinomas. Other less commonly practiced alternative treatment options include laser surgery or photodynamic therapy for early stage carcinomas. Concurrent intra-arterial regional chemoperfusion of high dose of cisplatin and radiotherapy may be considered as alternative treatment option for advanced T4 stage carcinoma. Controversy of Elective Neck Dissection Versus Observation of N0 Neck Since there is high risk of nodal recurrence of observed N0 neck of early tongue carcinoma, elective neck dissection is commonly practiced in many cancer centers worldwide. In retrospective studies of elective selective neck dissection versus observation of N0 neck, the regional recurrence rates could be reduced from 30 to 50% of observation to 10–15% after for elective selective I, II, III neck dissection. The regional recurrence related mortality rates could be reduced from 20% to 25% for observation to 4–10% for elective selective I, II, III neck dissection group. From the results of retrospective studies, elective neck dissection can reduce both initial regional recurrence rate and regional recurrence related mortality, and the reduction of noderelated mortality contributes to long-term survival benefit. There is however no prospective randomized study comparing the long-term benefit of elective neck dissection compared with observation in the treatment of N0 neck of early stage I and stage II oral tongue carcinoma. There is risk of mortality and morbidity of elective neck dissection. Both elective neck dissection and observation have their proponents in different cancer centers. Instead of performing elective neck dissection for all patients with early tongue carcinoma, patients should be informed of the

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possible choice of both treatment options of either observation or elective neck dissection of N0 neck. Observation is particularly suitable for patients with thin carcinomas of less than 3–4 mm. The risk of nodal recurrence of patients with thin tumors of 3–4 mm is in the range of 10–15%. Patients choosing observation treatment of N0 neck should be advised to have regular follow-up after primary treatment for early detection of nodal recurrence. Early nodal recurrence can be salvaged with modified or radical neck dissection successfully. Of those patients who cannot be closely followed up, elective neck dissection is a more suitable treatment of choice. Outcome of Treatment Local or regional lymph node recurrences account for over 90% of recurrences. Majority of local recurrences cannot be salvaged. Over 90% nodal recurrences of closely observed neck can be successfully salvaged with neck dissection. Of those patients with elective selective neck dissection of cN0 neck, the nodal recurrence rate of pN0 neck is less than 5% and is 30–40% for pN+ neck. Radiotherapy of pN+ neck is therefore advised. The overall 5-year disease-free survival rates are in order of 90–100% for stage I, 60–80% for stage II, 30–50% for stage III, and less than 20% for stage IV.

Topoisomerases

▶ Osteopontin ▶ p21 ▶ Podoplanin ▶ Retinoid Receptor Cross-Talk ▶ Vascular Endothelial Growth Factor

References Tsantoulis PK, Kastrinakis NG, Tourvas AD et al (2007) Advances of the biology of oral cancer. Oral Oncol 43(6):523–534 Yuen APW (2004) Cancer of the tongue: operative techniques in otorhinolaryngology. Head Neck Surg 15:234–238 Yuen APW, Wei WI, Wong YM et al (1997) Elective neck dissection versus observation in the surgical treatment of early oral tongue carcinoma. Head Neck 19:583–588

See Also (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331

Topoisomerases Wenjian Ma National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA

Cross-Reference Definition ▶ Adhesion ▶ Akt Signal Transduction Pathway ▶ Angiogenesis ▶ Apoptosis ▶ Bcl2 ▶ Cyclin D ▶ E-Cadherin ▶ Epidermal Growth Factor-like Ligands ▶ Ets Transcription Factors ▶ Furin ▶ Maspin ▶ Matrix Metalloproteinases ▶ Motility ▶ Oral Cancer

DNA topoisomerases are a class of enzymes required for the regulation of DNA folding and supercoiling to maintain chromosome integrity and cellular function. They are essential for relaxing entangled DNA and modulating DNA topology by transiently breaking and rejoining DNA single or double strands. Topoisomerases play a key role during DNA replication, transcription, and chromosome segregation. They are also involved in resolving specific DNA structures and intermediates arising from other cellular processes such as DNA repair and recombination.

Topoisomerases

Characteristics To fit into the tiny nucleus, cellular DNA, which has a combined length of several meters, is compacted and bound by proteins to form chromatin, which is twisted and folded into a space of only a few microns. However, excess folding of DNA poses a unique challenge to living organisms and generates topological problems when the DNA helix must be unwound to make its genetic information accessible. This highly supercoiled DNA, in the form of condensed chromatin, must be relaxed during the cell cycle in order for cellular processes such as transcription and replication to occur. In addition, as long as both strands of the double helix are covalently continuous, progressive unwinding of one region of DNA increases overwinding and therefore tension, in adjacent regions. This presents an impediment to further unwinding. These types of topological problems were recognized immediately after the discovery of DNA helical structure, but how cells deal with these problems had been a mystery until the discovery of the first topoisomerase in E. coli by James C. Wang in 1971. Afterward, it was shown that topoisomerases bind to either singlestranded or double-stranded DNA and generate transient DNA strandbreaks, allowing the DNA to be untangled or unwound. Topoisomerase Families Multiple topoisomerases, specializing in different types of DNA manipulation and cellular function, have been found in organisms ranging from virus to human. They can be divided into two major categories based on their DNA cleavage property. Type I enzymes wrap around DNA to make a transient break in only one DNA strand, which is important for the release of underwinding or overwinding forces of the DNA helix. Type II enzymes cleave both strands at the same time to untangle supercoiled DNA. Topoisomerases with an odd number after their names belong to type I (e.g., Top1), and those with even numbers belong to type II (e.g., Top2a). The letters A, B or a, b following Roman numbers are used to distinguish between subfamily members that are different in their sequence/structure, polarity of

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strand cleavage, etc. So far, six topoisomerases have been identified in mammalian cells, namely, TOP1 and TOP1mt (mitochondria form), TOP2a and b, and TOP3a and b. Molecular Mechanism The catalytic reactions of all topoisomerases share some common features. These enzymes contain a nucleophilic tyrosine to promote DNA strand scission and covalently bind to either the 30 - or 50 -end of the broken DNA. Formation of such transient enzyme-DNA adducts is believed to prevent the exposure of the broken DNA ends that may lead to genome instability. Following breakage of the DNA strands, unwinding/relaxation of DNA is through one of the following two mechanisms: Rotation – The nicked DNA strand spins around the phosphodiester bond of the other intact DNA strand. After releasing the overwinding force, the topoisomerase catalyzes religation of the nick to restore the DNA double helix. This reaction does not require ATP or divalent metal binding, and the spin is controlled by friction between the DNA and the enzyme. Strand passage – A single- or double-stranded DNA is nicked and physically opened. A homodimer of the enzyme binds at the break ends to serve as a “gate” allowing the passing of the other intact DNA strands. Finally, it rejoins the broken DNA to finish the unwinding/untangling procedure. This mechanism uses ATP to power strand passage. Depending on their cellular function and the DNA structures to resolve, topoisomerases of the same category may have different enzymatic mechanisms. For example, TOP1 and TOP3 are both type I topoisomerases, but Top1 enzymes unwind/relax DNA through the controlled rotation mechanism, whereas TOP3 uses “strand passage.” These differences are reflected in their cellular function. While TOP1 is required for DNA replication and transcription dealing with the DNA helix, TOP3 plays a role in resolving Holliday junctions during recombination, where two DNA molecules are intertwined. Both Top2

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and Top3 enzymes modulate DNA topology by the strand passage mechanism, but with different targets. Top2 enzymes cleave both strands of a DNA duplex and pass a second intact duplex through the transient break. Top3 enzymes generate a single-strand break in double-stranded DNA, which allows another single-stranded DNA to go through the break. The difference also depends on cell function as Top2 enzymes act on DNA supercoils, intertwined chromosomes, and DNA catenanes. Cellular Functions Topoisomerases recognize specific DNA structures associated with different cellular processes. Their major activity includes modulating the under- or overwound DNA helix, resolving supercoiled or entangled DNA, facilitating decatenation, and participating in chromatin remodeling. They have shown to play important roles in the following cellular processes: Replication – Chromosomal DNA needs to be unwound in order for the replication machinery to gain access. Topoisomerases are needed at a very early stage of replication for replisome assembly. During strand synthesis, they are indispensable since the DNA helix would become either overwound or underwound following the progress of the replication fork, which generates positive supercoils ahead of the replication machinery and negative supercoils behind it. This function requires type 1 topoisomerases. Transcription – Topological problems need to be dealt with when transcribing genetic information from DNA to RNA, which produces DNA supercoiling with the movement of RNA polymerase. If left unchecked, this supercoiling can impede the progress of the transcription protein machinery. Type I topoisomerases are implicated in the removal of supercoils as well as suppressing loop formation. Chromosome segregation – At the G2 phase of the cell cycle, sister chromosomes are catenated or looped around each other. Removal of these catenations is vital for proper chromosome segregation. In addition, organisms with a circular

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genome are also in need of decatenation since the DNA strands are topologically linked or knotted. Topoisomerases II and IV (bacteria) are implicated in these processes. DNA repair and recombination – Topoisomerases also play a role during the repair of DNA damage, especially the repair of DNA doublestrand breaks. Repair of the broken DNA ends often proceeds through a pathway called homologous recombination, which leads to the formation of interlinked intermediates. Resolution of the intermediates was shown to require the function of topoisomerase III, which can introduce single-strand breaks to facilitate the strand exchange between homologous sequences. Other types of recombination events, such as sister chromosome exchange before cell division and the insertion of viral DNA into host chromosomes, also require topoisomerases. Chromosome packaging and condensation – Topoisomerases not only help in relaxing or disentangling DNA, they also participate in chromosome condensation to form the intertwined DNA duplexes and assemble the highly ordered chromatin structure. Topoisomerase II is the major player in this process. It promotes intense supercoiling of the DNA helix to aid in chromosome condensation. This occurs at specific stages of the cell cycle and is regulated by cell cycle control and checkpoint proteins such as p53 and Cdc2. Topoisomerase Inhibition and Cancer Therapy As part of the reaction mechanism, topoisomerases generate transient DNA strand breaks in DNA, which, if not efficiently resealed, can lead to genome instability. Disruption or inhibition of topoisomerase often causes serious problems for DNA metabolism and may even trigger cell death. However, it also generates significant clinical benefits. Some inhibitors targeting bacterial topoisomerases II and IV have been developed as antibiotics to eliminate pathogens and to treat diseases such as tuberculosis and malaria. Most importantly, topoisomerases are especially relevant in cancer.

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Interfering with topoisomerase function is one of the most effective strategies for anticancer therapy. On the one hand, topoisomerases play a central role in various DNA metabolic pathways such as replication that are critical for the fast-growing tumor cells. On the other hand, cancer cells are often defective in one or more of the DNA repair pathways that cannot efficiently deal with topoisomerase-mediated DNA strand breaks. Some chemicals serving as topoisomerase inhibitors prevent enzymatic function by recognizing structural motifs present in the enzymes or by intercalation into specific DNA sequences bound by the topoisomerase. These inhibitors often prevent the nick-resealing step and the dissociation of topoisomerase from DNA, which results in persistent single- or double-strand breaks. In other words, the enzyme is converted into a cellular poison trapped at the ends of DNA strand breaks. While normal cells are capable of repairing such damage to restore cellular function, tumor cells are killed due to gene mutations or deficiency required to repair certain damages, especially DNA double-strand breaks. Most of the inhibitors/drugs selectively target either topoisomerase I or II, and both types of inhibitors have been exploited in cancer therapy. TOP1 inhibitors, such as camptothecin, block DNA replication and interfere with cell proliferation to kill rapidly dividing cancer cells. They have been used to treat ovarian cancers and small-cell lung cancers (SCLC). Topoisomerase II targeting drugs can be classified into two groups, topoisomerase II poisons and topoisomerase II catalytic inhibitors. Topoisomerase II poisons that traps the enzyme onto double-strand breaks are among the most successful anticancer drugs currently in clinical use. So far six top II-related anticancer agents have been approved for use in the United States, more in other countries. For example, the widely used anthracycline drug doxorubicin is highly effective in the treatment of a wide range of cancers including hematological malignancies and many types of carcinoma. Despite their efficacy for many cancers, the use of topoisomerase-inhibiting agents for cancer therapy is limited by common negative effects, such as chronic cardiotoxicity, hematological toxicity, and secondary malignancies.

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References Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 9(5):327–337 Vos SM, Tretter EM, Schmidt BH, Berger JM (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12(12):827–841 Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3(6):430–440

Torisel™ ▶ Temsirolimus

Toxicity Testing ▶ Preclinical Testing

Toxicogenomics Scott Auerbach Biomolecular Screening, National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA

Definition Toxicogenomics was first described in 1999 as the application of omic technologies (genomics, transcriptomics, proteomics, and metabolomics) to the study of toxicology. Today the definition should be expanded to include epigenomics. The omic disciplines represented a continuum of biological information starting with the genome, which is then regulated by the epigenome to give rise to the transcriptome, which in turn encodes the proteome whose actions give rise to the metabolome (Fig. 1). Since its inception, The entry “Toxicogenomics” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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Toxicogenomics, Fig. 1 Toxicogenomics involves the application of multiple omic disciplines to the study of toxicity and its related diseases. These omics domains represent a continuum of functionally related biological information. The biological entities studied in each domain are distinct; therefore, each omic domain requires distinct measurement techniques to generate quantitative and qualitative information

toxicogenomics has primarily focused on the identification of biomarkers that diagnose or predict disease. In addition, it has been used extensively to shed light on the molecular mechanisms and key events that lead to toxicity.

Characteristics Genomics Genomics is the study of DNA variants (e.g., single nucleotide polymorphisms (SNPs), genomic copy number variations (CNVs)) and their causal role in the manifestation of phenotypes such as disease and chemical toxicity. Approaches, Technology, and Data Analysis

For many years the field of pharmacogenetics/ ecogenetics has focused on single gene genetic association studies to identify the genetic basis for susceptibilities to toxicity. The genes that

served as a focal point of studies were selected based upon biochemistry and molecular biology studies that indicated they play a role in detoxifying/metabolizing xenobiotics. In the broader field of genetics, the first approach developed for querying the entire genome for genotype-phenotype relationships was linkage analysis, which traces transmission of alleles and phenotypes through families to identify regions of the genome that carry an allelic variant which confers a phenotype. Subsequently, with the sequencing of the human genome and the formulation of the human haplotype map, case-control-based genome-wide association (GWA) studies became possible. Finally, in the last 5 years, it has become economically feasible for labs to sequence an entire genome and to identify allelic variants that underlie phenotypes using integrated bioinformatics approaches. The two major technologies that are currently employed for genome-wide genotype to phenotype mapping are SNP genotyping arrays and whole

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exome/genome-wide sequencing. Most SNP arrays work by hybridization methods in which probes representing distinct alleles across the genome are hybridized to label genomic DNA. In the case of exome sequencing, genomic DNA is first enriched through hybridization-based methods for exonic sequence; the enriched DNA is then integrated into sequencing libraries and applied to any number of next-generation sequencing platforms. The output from these machines is a collection of millions of random reads across the genome, which are either de novo assembled into a genome or mapped to a reference genome. Once the mapping is done, variant genomic positions can be identified. Whole genome sequencing is similar with the exception that there is no enrichment for exonic sequence. A number of critical variables go into designing and then subsequently analyzing GWA studies including considerations for statistical power (i.e., population size necessary to detect a statistical association between an allele and a phenotype), genetic stratification between cases and controls, and the potential genic and allelic diversity that underlies the phenotype under study (which relates back to statistical power to detect an association). The data from a GWA study is typically analyzed using a chi-square test to determine if any of alleles are significantly associated with the phenotype under study. Unlike GWA studies, genomic sequencing data has the added complexity of rare mutations. These rare mutations although potentially biologically meaningful create a significant statistical challenge; hence, alternative methods that incorporate prior knowledge of biology (i.e., disease pathways, protein-protein interactions, and evolutionary conservation of the mutated positions) are taken into consideration when determining if a mutation has a plausible causal role in the phenotype under study.

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findings, a number of different resources are often used to provide biological interpretation of associated genes including network-level analysis (i.e., gene coexpression and/or protein-protein interaction networks). In addition extensive literature mining is often performed to piece together plausible biological relationships between the phenotype under study and the associated gene(s). Applications in Toxicology

It is notable that some of the first discoveries in human genetics related to drug toxicity (e.g., isoniazid toxicity and N-acetyltransferase, quinine antimalarial toxicity, and glucose-6-phosphate dehydrogenase). Genome-wide association studies of the side effects of select drugs have identified genetic variation in the HLA class I and class II genes, HLA-A, SLC01B1, CERKL, and CYP2C8, as determinants of susceptibility for drug-induced liver toxicity, skin hypersensitivity reactions, myotoxicity, QT prolongation, and osteonecrosis of the jaw, respectively. It is notable that the genes identified by these studies are disproportionately associated with inflammatory processes and drug metabolism and distribution. In addition to studies in humans, a number of groups are leveraging the power of genetically defined inbred strains of mice to identify genetic determinants of susceptibility to toxicity. One example study was published in 2009 that identified allelic variants in Cd44 as being a determining factor for the susceptibility to acetaminophen-induced liver toxicity. Epigenomics Epigenomics is the study of the complete set of epigenetic modifications (DNA methylation, posttranslational histone modification, and nucleosome positioning) on the genome of a cell. Approaches, Technology, and Data Analysis

Data Interpretation

It is often the case in GWA studies that the genes identified do not have a documented biological link to the disease or phenotype under study. This general observation underscores our limited understanding of gene-phenotype relationships and the complex network biology that underlies phenotype. In order to contextualize the GWA

There are a variety of techniques used to analyze the epigenome. They vary based upon the epigenetic modification that is under study and by the degree to which they resolve the genomic location where changes in an epigenetic mark occur. One of the lowest-resolution assessments involves cleavage of chromatin using nuclease digestion. In this assay regions that are hypersensitive to

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nuclease digestion reflect areas where nucleosomes have been removed and the chromatin structure is open. A slightly higher-resolution analysis involves cross-linking proteins to DNA, shearing the DNA, performing immunoprecipitation with antibodies targeted against any number of epigenetic marks, and then labeling and hybridizing immunoprecipitated DNA to microarrays tiled with probes that survey the landscape of the genome. Alternatively, the immunoprecipitated DNA can be incorporated into sequencing libraries in a technique called ChIP-seq and then sequenced using next-generation sequencing technologies. The resultant data is then mapped to the genome to identify areas of that exhibit differing levels of the epigenetic mark under study. Another approach to mapping one particular type of epigenetic mark (DNA methylation) involves the use of restriction endonucleases that are sensitive to the methylation state of cytosine. With this approach differentially digested DNA is either labeled in hybridized to the genome-wide CpG array, or alternatively fragments are evaluated by next-generation sequencing. Still another technique for evaluating DNA methylation is the use of sodium bisulfite which converts unmethylated cytosine to uracil, but has no effect on methylated cytosine. The converted DNA can then be hybridized to arrays that contain probes that are complementary to either the cytosine or uracil-containing sequence. Alternatively, the bisulfite-treated DNA can be analyzed using next-generation sequencing where the methylation state can be identified for individual nucleotides by mapping sequence reads to a virtual bisulfate-treated or untreated genome sequence. Data Interpretation

Currently there is a limited understanding of how the various epigenetic marks translate into gene regulatory processes. This is particularly true for DNA methylation as the degree of methylation does not correlate with gene expression. Hence, it is often challenging to translate epigenomic findings at a gene level to clear molecular mechanism or biological significance. However, as with other functional genomic data, pathway and gene ontology analysis tools can potentially be

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used (assuming epigenetic marks can be mapped to individual genes) for formulating hypotheses on the role of alterations in the epigenome. Applications in Toxicology

Toxicity-related epigenomics has become an area of significant interest among researchers. One of the primary reasons for this interest in the epigenomics relates to concerns about early life exposure and its impacts on epigenetic patterning, which may lead to altered risk of disease later in life. In addition, studies of identical twins have shown significant epigenetic drift over a lifetime suggesting a strong environmental influence on the epigenome. Studies in rodents have suggested chemicals that alter the epigenome during development may do so through alteration of the methylation associated with parasitic DNA (e.g., retrotransposons) which can lead to expression of these elements and subsequent related genomic instability. In addition, alterations in repeat element DNA methylation have been demonstrated to alter expression of genes proximal to the repeat elements. Still further studies have demonstrated that target organ toxicity at a level that produces cell proliferation causes global DNA hypomethylation, therefore suggesting a dynamic role for epigenomics in tissue repair and adaptation to toxic stress. Transcriptomics Transcriptomics is the genome-wide study of RNA (i.e., mRNA, miRNA, ncRNA) expression levels. Approaches, Technology, and Data Analysis

There are two primary approaches to highdimensional transcriptomic analysis; hybridizationbased microarrays, and direct sequencing of cDNA fragments using next-generation sequencing (RNA-seq or digital gene expression). In a typical microarray experiment, RNA is extracted from a biological sample, reverse transcribed into cDNA, labeled with fluorescent dyes, and then hybridized to a high-density microarray which contains antisense oligonucleotides that correspond to most, if not all, annotated genes for the species under study. Hybridized arrays are then

Toxicogenomics

scanned to determine the degree of hybridization to each spot, and the intensity data that is collected is normalized using any number of algorithms. There are a variety of microarray types; the most common is a 30 in which the probes hybridize with 30 end of a transcript. Arrays have been developed that cover a wider range of the transcriptome. The arrays contain probes that hybridize each exon allowing for the identification of alternative splicing. In the case of RNA-seq, one starts with a similar RNA preparation to that used for microarrays; however, depending upon which RNA species an investigator desires to evaluate, different purification steps are employed. The purified RNA is then sheared to a size that is appropriate for the sequencing technology that will be employed, and then the fragmented RNA will then be incorporated into a cDNA library using primer ligation PCR. The library is then applied to a next-generation sequencer and individual fragments are sequenced and recorded. In a typical RNA-seq experiment, many millions of individual reads per sample are generated that can range in size from 50 to over 500 base pairs in length. The sequences are then mapped back to the genome in essence creating histograms across the genome for every nucleotide, which when normalized and compiled at the gene level corresponds to a detailed picture of transcript expression across the genome. The added value of RNA-seq is most notable when it is performed with whole transcript protocol as it allows for the sequence level identification of alternatively spliced transcripts and fusion transcripts that arise in cancer due to rearrangement of the genome. As with other high-dimensional data types discussed here, there are a number of multivariate approaches that can be used to analyze transcriptomic data. In general these analyses break down into two categories, supervised and unsupervised. The distinction between these approaches relates to the integration of data labels into the analysis (e.g., cancer vs healthy). In unsupervised analysis relationships in the data are driven by the data itself in the absence of sample context, whereas supervised analysis takes into consideration sample type and will

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often prefilter data to identify genes that differentiate phenotypes under study which in turn can be used to generate predictive/diagnostic, machine learning-based models. Another critical concept that needs to be addressed in multivariate data analysis is the issue of multiple testing corrections. This issue arises when there are a large number of statistical tests (e.g., all genes on a microarray; all SNPs on SNP array) in a single comparison (e.g., treated vs control). Just by chance alone 5% of all genes on an array will exhibit significant differential expression if a P 105. One of the primary strengths of NMR-based metabolomics is the robust reproducibility of the method. Further NMR-based metabolomics does not involve fractionation or destruction of the sample, therefore allowing for further study of biospecimens. Its primary liability is its low sensitivity although developments using so-called cryoprobes and low-volume probes have pushed the limits of detection for NMR into the 105 M range. Mass spectral-based metabolomics involves a wide variety of platforms and methodologies. There are two basic approaches to MS-based metabolomics, those that fractionate complex mixtures prior to MS analysis and those in which complex mixtures are directly injected into the mass spectrometer. The inherent challenge with mass spectral-based metabolomics is the simultaneous ionization of multiple analytes. In order to detect an analyte using mass spectrometer, the analyte must be ionized. In metabolomics this is most often achieved by electrospray ionization which applies enough energy to induce ionization without destroying the analyte. In the case of metabolomics, one is trying to apply a charge to many metabolites all it wants; hence, there is competition for applied energy which may favor a subset of metabolites. To limit this competition, fractionation techniques have been coupled with mass spectrometry. The fractionation methods that are used include gas chromatography, highperformance liquid chromatography, and capillary electrophoresis. Gas chromatography provides high chromatographic resolution; however, since this technique can only fractionate volatile chemicals, it is necessary to derivatize many metabolites prior to analysis which can introduce experimental variability. High-performance liquid chromatography has lower resolution than gas chromatography; however, with the development of ultrahigh-pressure liquid chromatography systems that use small particle stationary phases, it is possible to significantly enhance chromatographic resolution of HPLC systems. Capillary electrophoresis theory has greater separation efficiency than high-performance liquid chromatography

Toxicogenomics

and is amenable for use with a greater variety of metabolites than gas chromatography. In order for the analytes to be detected following separation by one of the above techniques, they need to undergo ionization before injection into the mass spectrometer. In GC-MS, electron impact ionization is often used that breaks each analyte into numerous ionized fragments which in turn produces a fragment pattern in the subsequent mass spectral analysis. Fragmentation patterns can then be compared to those of known metabolites therefore allowing for metabolite identification. In LC-MS the most common method of ionization is electrospray ionization (ESI). This form of ionization preserves the molecular ion therefore making the spectral patterns slightly less complex. Direct injection or surface-based metabolomics was developed primarily to avoid the analytical variability introduced by chromatography; however, the lack of fractionation introduces some challenges including salts, charge competition, and the inability to distinguish compounds with the same molecular weight. Matrix-assisted laser desorption/ionization (MALDI) was the first technology used in direct injection metabolomics. When performing MALDI a matrix is applied to the biological sample to allow for desorption and ionization of the metabolites following laserbased energy transfer. Due to matrix-associated challenges with MALDI, such as high background in the low-molecular-weight range and crystal formation that leads to challenges with tissue imaging, a number of other technologies that are matrix independent have been developed. Nanostructure-initiator mass spectrometry (NIMS), secondary ion mass spectrometry (SIMS), and desorption electrospray ionization (DESI) are all matrix-free techniques used for direct injection metabolite studies. Each analytical approach and platform requires distinct approaches to data processing and analysis. In the case of fingerprinting methods, various types of supervised and unsupervised multivariate analysis is performed in the case of NMR following parsing of spectra into frequency bins or in the case of GC/LC-MS following the collection of data features such as chromatographic retention

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time, mass charge (m/z) pairs, and peak intensity values. Analysis of data using multivariate approaches allows for the development of classification models of different biological states which can then be applied to subsequent studies. Often times simple fingerprints are not satisfactory to biologists and specific identification/quantification of analytes is critical to understanding biological processes. In the case of NMR, annotation of spectral peaks is done by comparison to spectral standards that are available through several commercial and publicly available databases. Analyte identification in MS-based metabolite mix is faced with the challenge of deconvoluting complex spectral patterns that arise due to the process of analyte ionization. As with NMR the identification of spectral patterns and assignment to specific molecular species is dependent upon repositories of spectra generated from standards. With respect to quantification, NMR peak integrals are directly proportional to the concentration of the nucleus under study; hence, quantification of metabolites is relatively straightforward following deconvolution of the data. Quantification of metabolites from MS data is slightly more challenging because ionizability varies between metabolites in addition to the challenges associated with ion suppression that arise due to competition for ionization energy. This makes absolute quantification only possible when spectra are calibrated against different concentrations of standards. Hence, it is often the case that quantification occurs in targeted follow-up studies.

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toxicity. This premise was the basis of the COMET consortium which generated spectral profiles from over 100 short-term rodent toxicity studies (Lindon et al. 2005). These profiles were then used to train a number of predictive models. In addition to the efforts of the COMET consortium, metabolomics has been used to identify biomarkers of the hepatic and renal toxicity and to identify diseases that are commonly associated with toxic exposure (e.g., alcoholic liver disease). Metabolomics has also been employed in mechanistic toxicology to characterize a number of processes including microsomal enzyme induction, oxidative stress, myotoxicity, and inflammationassociated hepatotoxicity.

References Afshari CA, Hamadeh HK, Bushel PR (2011) The evolution of bioinformatics in toxicology: advancing toxicogenomics. Toxicol Sci 120(Suppl 1):S225–S237 Daly AK (2012) Using genome-wide association studies to identify genes important in serious adverse drug reactions. Annu Rev Pharmacol Toxicol 52:21–35 Lindon JC, Keun HC, Ebbels TM, Pearce JM, Holmes E, Nicholson JK (2005) The Consortium for Metabonomic Toxicology (COMET): aims, activities and achievements. Pharmacogenomics 6(7):691–699 Merrick BA, Witzmann FA (2009) The role of toxicoproteomics in assessing organ specific toxicity. EXS 99:367–400 Robertson DG, Watkins PB, Reily MD (2011) Metabolomics in toxicology: preclinical and clinical applications. Toxicol Sci 120(Suppl 1):S146–S170 Szyf M (2011) The implications of DNA methylation for toxicology: toward toxicomethylomics, the toxicology of DNA methylation. Toxicol Sci 120(2):235–255

Data Interpretation

Biological interpretation of metabolomics results often involves performing biochemical pathway enrichment analysis. In order to understand where critical perturbations occur in biochemical system, it is often necessary to know the levels at which feedback control function and where rate limiting steps in a biochemical pathway occur.

T Toxicological Carcinogenesis Takuji Tanaka Department of Oncologic Pathology, Kanazawa Medical University, Kanazawa, Japan

Application to Toxicology

One of the initial applications of metabolomics was in the area of toxicity prediction. It was hypothesized that analysis of biofluids would provide metabolite signatures (fingerprints) of

Synonyms Chemical carcinogenesis; carcinogenesis

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Definition In the broadest possible sense, ▶ carcinogenesis is a process of generation of benign and malignant neoplasm. Agents such as viruses, radiation, and chemicals are able to induce ▶ cancer in humans and experimental animals. However, the importance of chemicals as a cause of cancer has long been recognized in basic and clinical studies and is emphasized by the epidemic of tobacco-related lung cancer in the twentieth century. Carcinogenesis may be considered as a form of toxicity in which cells achieve a different steady state from the normal and do not respond normally to homeostatic mechanisms. Carcinogenesis induced by chemicals is called “toxicological (chemical) carcinogenesis.” Basic and clinical research in the field of toxicological carcinogenesis has led to many major advances, ranging from the fields of epidemiology and international human studies to laboratory research on mechanisms involved in the complex processes that are associated with the initiation and development of malignant disease (cancer). Many chemical carcinogens have been identified, and their effects documented in experiments in which animals exposed to the agents at the maximum tolerated dose develop neoplasm. Toxicological carcinogenesis and human cancer epidemiology studies have clearly identified specific chemicals that can act as human carcinogens in both occupational and environmental settings. The main groups of relevance to human disease include ▶ polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, ▶ alkylating agents, and heterocyclic amines. Cancer resulting from exposure to chemicals in the environment has taken on new importance. Knowledge about the mechanisms and natural history of cancer development from toxicological carcinogenesis as well as epidemiology of human cancer is critical in the control and prevention of human neoplastic disease.

Characteristics Mutagens are agents that can permanently alter the genetic constitution of a cell. The most widely

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used screening test, the Ames test, uses the appearance of mutants in a culture of bacteria of the Salmonella species. Approximately 90% of known carcinogens are mutagenic in this system. Moreover, most, but not all, mutagens are carcinogenic. This close correlation between carcinogenicity and mutagenicity presumably occurs because both reflect ▶ DNA damage. The in vitro mutagenicity assay is a valuable tool in screening for the carcinogenic potential of chemicals. Cultured human cells are also being increasingly used for assays of mutagenicity. Chemical carcinogens may cause development of neoplasm either directly or indirectly. They can be grouped into two main classes according to the mechanism by which they stimulate development of neoplasm: (i) Genotoxic carcinogen causes direct damage to DNA by forming chemical/ ▶ DNA adducts. The abnormal areas of DNA are prone to damage in replication and some adducts are resistant to normal ▶ DNA repair mechanisms. (ii) Non-genotoxic carcinogen is a carcinogen for which there is no evidence of direct interaction with cellular DNA. This type of carcinogen can be divided into two subgroups. Mitogenic carcinogen binds to receptors on or in cells and stimulates cell division without causing direct DNA damage. In experimental skin carcinogenesis such agents have been shown to bind to and activate protein kinase C, causing sustained epidermal hyperplasia. Cytotoxic carcinogen produces tissue damage and leads to hyperplasia with cycles of tissue regeneration and damage. In some cases it is believed that cytokines generated in response to tissue damage act as mitogenic factors. Chemical carcinogens can be further divided into two groups: (i) direct-acting carcinogen (the agent is capable of directly causing neoplasia) and (ii) procarcinogen (the agent requires conversion to an active carcinogen). This conversion takes place through normal metabolic pathways. In procarcinogens the ▶ cytochrome P450 (CYP) monooxygenase system plays an important role in conversion in many instances. ▶ Detoxification reactions also occur, the accumulation of carcinogen being determined by a balance between (i) dose of procarcinogen, rate of

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Toxicological Carcinogenesis, Fig. 1 Toxicological carcinogenesis as a multistep process

detoxification and elimination, and (ii) rate of conversion to the active form. Three stages have been defined in toxicological carcinogenesis (Fig. 1). Studies of toxicological carcinogenesis among experimental animals have shed light on the individual stages in the progression of normal cells to cancer. From these studies, one can define three stages (▶ multistep development) of toxicological carcinogenesis: 1. Initiation is the first stage and likely represents mutations in a single cell. The nature of the initial changes in cells is still uncertain. In experimental toxicological carcinogenesis in the skin, the Harvey-▶ RAS gene has been identified as being frequently mutated. This gene is involved in epidermal proliferation, and when it becomes abnormal, epidermal cells are less responsive to signals that

normally cause terminal differentiation. Only relatively few genes have been identified as being mutated in other animal models of toxicological carcinogenesis. 2. Promotion follows initiation and is characterized by clonal expansion of the initiated cell. Induction of cell proliferation takes place at this stage. The altered cells do not exhibit autonomous growth, but remain dependent on the continued presence of the promoting stimulus, including an exogenous chemical or physical agent or an endogenous mechanism, such as hormonal stimulation. In this phase of carcinogenesis, a promoting agent brings about increased cell proliferation. Promotion is initially reversible if the promoting agent is withdrawn. 3. Progression is the third stage, in which growth becomes autonomous and is independent of the

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carcinogen or promoter. At this stage, additional genomic changes presumably endow cells with a relative growth advantage that, in turn, results in their further clonal expansion. Cancer is the end result of the entire sequence and is established when the cells acquire the capacity to invade and metastasize. If there is persistent cell proliferation, initiated cells acquire secondary genetic abnormalities in oncogenes, which first lead to dysregulation and eventually to autonomous cell growth. The ultimate end point of progression is development of an invasive neoplasm. The various tests that have been applied to identifying agents with carcinogenic potential may be classified into several general areas on the basis of the time involved in the assay: short, medium, and long. These include short-term tests for mutagenicity (e.g., the Ames test), gene mutation assays in vivo (e.g., the LacZ mouse, the LacI mouse, the LacI rat), assay for chromosomal alterations (e.g., ▶ micronucleus assay, sister chromatid exchange), measurement of primary DNA damage in vitro and in vivo, and chronic bioassays for carcinogenicity (e.g., chronic 2-year bioassay, medium-term bioassays-Ito model, multistage models of neoplastic development, transgenic and knockout mice as models of carcinogenesis). History It is widely recognized that exposure to chemicals in the workplace and the environment can contribute to human cancer risk. This was first indicated in 1775 by Dr. Pott, who attributed scrotal skin cancers to prolonged exposure to soot in London chimney sweeps. In 1914, Dr. Boveri first hypothesized that cancer was a genetic disease, prior to the discovery of the genetic material. In 1915, Dr. Yamagiwa and co-workers successfully induced skin cancer in rabbits by painting their ears continuously with benzene solutions of tar. In the 1930s Dr. Kenneway and co-workers demonstrated that pure chemicals isolated from coal tar could also produce tumors in animals. In the 1950s there were parallel discoveries of the structure

Toxicological Carcinogenesis

of the DNA double helix and its establishment as the hereditary material and mutagenic potential of ionizing radiation and certain chemical carcinogens in humans and experimental systems and extensive investigations into the relationship between chemically induced mutations and human cancer. The 1980s saw the elucidation of the first oncogenes that appeared to be responsible for the initiation of cancer as first predicted by Dr. Boveri. This era also saw the development of the Ames Salmonella bacterial mutagenesis assay (the Ames test) and similar genetic toxicology assays. These developments firmly established the basic paradigm for the field of toxicological carcinogenesis: chemicals capable of induction of mutations are presumed to be carcinogens. It was predicted that any chemical or physical agent that can covalently damage DNA could also cause mutations through its DNA-damaging mechanism and hence can be a carcinogen. The data that followed in the 1990s appeared to strongly support this central assumption, as numerous chemicals that were initially tested for DNA damage or mutations were also carcinogens in experimental animals. Since then, our understanding of the molecular basis of cancer has improved substantially. In addition, investigations into the molecular basis of toxicological carcinogenesis, as well as more extensive human cancer epidemiology studies using modem molecular tools, have greatly expanded our knowledge in this area.

Cross-References ▶ Adducts to DNA ▶ Alkylating Agents ▶ Cancer ▶ Carcinogenesis ▶ Cytochrome P450 ▶ Detoxification ▶ DNA Damage ▶ Micronucleus Assay ▶ Multistep Development ▶ Polycyclic Aromatic Hydrocarbons ▶ Repair of DNA

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References

Definition

Clayson DB (2001) Toxicological carcinogenesis. CRC Press LLC, Boca Raton Ito N, Tamano S, Shirai T (2003) A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 94:3–8 Sugimura T, Ushijima T (2000) Genetic and epigenetic alterations in carcinogenesis. Mutat Res 462:235–246 Tanaka T (1997) Effect of diet on human carcinogenesis. Crit Rev Oncol Hematol 25:73–95 Williams GM, Iatropoulos MJ, Weisburger JH (1996) Chemical carcinogen mechanisms of action and implications for testing methodology. Exp Toxicol Pathol 48:101–111

The TP53 ▶ tumor suppressor gene is located on chromosome 17p13.1 and encodes a ubiquitous phosphoprotein of molecular mass 51,000–53,000, essentially expressed in the nucleus. This gene is frequently inactivated by somatic mutation or by loss of alleles in many common human cancers. More than 25,000 such mutations have been described so far. Inherited, heterozygous mutations have been identified in about 400 families with ▶ Li-Fraumeni syndrome and Li-Fraumeni-like syndromes (LFS and LFL), characterized by the early occurrence of cancers at multiple organ sites. TP53 belongs to a ▶ p53 family that also includes TP73 (1p36) and P63 (3p28). In contrast with TP53, these two genes have a restricted, tissue-specific, and developmental expression pattern and are not frequently mutated in cancer. The p53 protein is a latent transcription factor that is activated in response to multiple forms of physical and chemical stress to exert diverse, complementary effects in the regulation of cell proliferation, genetic integrity, and survival. These effects include:

See Also (2012) Cytotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1058. doi:10.1007/978-3-642-16483-5_1498 (2012) Direct-Acting Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1124. doi:10.1007/978-3-642-16483-5_1641 (2012) DNA Repair. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1141. doi:10.1007/978-3-642-16483-5_1687 (2012) Genotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1540. doi:10.1007/978-3-642-16483-5_2394 (2012) Human Cancer Epidemiology. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1752. doi:10.1007/978-3-642-164835_2846 (2012) Mitogenic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2336. doi:10.1007/978-3-642-16483-5_3772 (2012) Non-Genotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4109 (2012) Procarcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2989. doi:10.1007/978-3-642-16483-5_4746

TP53 Pierre Hainaut International Prevention Research Institute, Lyon, France

Synonyms p53

• Induction of ▶ apoptosis • Control of cell division through regulation of cell cycle progression in G1 and G2, ▶ centrosome duplication, and mitosis • Modulation of DNA replication and ▶ repair of DNA The main function of the p53 protein is to act as an “emergency brake” to prevent the proliferation of cells with damaged genetic material, caused by exposure to genotoxic agents (Fig. 1). In a broader context, the protein acts as an integrator of multiple exogenous and intracellular signals to regulate cell proliferation during replicative senescence, differentiation, and development. Inactivation of TP53 in mice resulted in accelerated development of multiple tumors, while a fraction of p53-deficient embryos displayed a lethal defect in neural tubule closure, resulting in exencephaly.

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4602 TP53, Fig. 1 The p53 pathway. The p53 protein is induced in response to various forms of stress and mediates a set of coordinated, antiproliferative responses including cell cycle arrest, control of replication, transcription, repair, and apoptosis. Blue: factors that bind to p53 and that are regulated by protein interactions. Red: factors that are regulated by p53 at the transcriptional level

TP53

Genotoxic stress γ and X rays UV chemical carcinogens

p14arf

p21

Non-genotoxic stress Hypoxia depletion of ribonucleotides or microtubules

JNK

mdm2

14-3-3σ Gadd45

CDK PCNA

CDC25

G1 G1/S

G2/M

Cell cycle arrest

Characteristics The TP53 gene spans 20 kb and contains 11 exons, the first one being noncoding. The coding sequence contains five regions showing a high degree of conservation in vertebrates, located in 2, 5, 6, 7, and 8. An orthologue has been described in Drosophila. Several gene polymorphisms are identified in the human population, with allele frequencies that vary with ethnic origin. However, there is only limited evidence to prove that these polymorphisms play a role in tumor susceptibility. The TP53 gene does not contain a conventional TATA box but is under the control of several ubiquitous transcription factors, including NFkB, Sp1, and Jun. It is expressed in the form of one major transcript of 2.8 kb and several isoforms generated by alternative splicing or use of an alternative promoter, intron 4.

XPB XPD RPA

Control of DNA replication, transcription and repair

Bax Apol/Fas Killer/DR5 Pig 3, 6, 12 IGF-BP3

Apopstosis

The protein contains 393 residues and is organized in a hydrophobic, central core (residues 110–296, encoded by exons 5–8) flanked by an acidic N-terminus and a basic C-terminus (Fig. 2 top). The N-terminus contains two complementary transcriptional activation domains, with a major one at residues 1–42 and a minor one at residues 55–75, specifically involved in the regulation of several proapoptotic genes. The central core is made of a scaffold of two b-sheets supporting a set of flexible loops and helices stabilized by the binding of an atom of zinc. These loops and helices make direct contact with DNA sequences containing inverted repeats of the motif RRRC(A/T). The C-terminus contains the main nuclear localization signals and oligomerization domains (residues 325–366). The active form of the protein is a tetramer (in fact, a pair of dimers). The extreme C-terminus has multiple regulatory functions and exerts a negative control on

TP53 TP53, Fig. 2 Diagram of the p53 protein structure. c: linear structure, showing the three main structural domains. Codon numbers of the main mutation hotspots are shown as colored boxes. Sites of posttranslational modifications are shown as “P” (phosphorylations), “A” (acetylations), and “Z” (zinc binding sites). bottom: 3-D structure of the central core of p53 in complex with target DNA. Hotspot residues are shown in the same color code as above

4603 N-terminus

Core domain

C-terminus

1-42

96-296

325-393

A

P P P

sequence-specific DNA-binding activities. Both N- and C-terminal regions contain multiple posttranslational modification sites, while only a few have been identified so far in the central core (see Table 1). Upstream of p53: Signaling of DNA Damage The p53 protein is constitutively expressed in most cells and tissues as a latent factor. Due to its rapid turnover (5–20 min), the protein does not accumulate unless it is stabilized in response to a variety of intracellular and extracellular stimuli. Signals that activate p53 include diverse types of ▶ DNA damage (strand breaks, bulky adducts, oxidation of bases), blockage of RNA elongation, ▶ hypoxia, depletion of microtubules, ribonucleotides or growth factors, modulation of cell ▶ adhesion, and alteration of polyamine

A P P

P

Z Z ZZ

175

A

273

245 248

249

282

P

Phosphorylation sites

A

Acetylation sites

Z

Zinc-binding sites

metabolism. Most of the current knowledge of p53 protein activation is derived from studies using DNA strand breaks as inducing signals. The main regulator of p53 protein activity is ▶ MDM-2, a protein which binds p53 in the N-terminus (residues 17–29); it conceals its transcription activation domain, redirects p53 from the nucleus to the cyclasm, and acts as a ubiquitin ligase to target p53 for degradation by the ▶ proteasome. The MDM-2 gene is a transcriptional target of p53, thus defining a regulatory feedback loop in which p53 controls its own stability. The p53/mdm-2 complex is regulated by Arf (alternative reading frame), a 14 kD protein encoded by the p16/CDKN2A gene. The kinetics, extent, and consequences of p53 activation vary according to the nature and intensity of the inducing signals. In response to

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TP53

TP53, Table 1 Factors involved in the activation and posttranslational modification of p53 Factor PARP HMG-1 E6AP Hif-1 14-3-3 s p300/CBP c-abl mdm-2 NO Cdc2/Cdk2Cyclin A/B cdk7-cyclin H CKII

Biochemical function/activated by p53 ADP-ribose polymerase/DNA strand breaks, nucleotide depletion High mobility group 1/? E6 accessory protein/ubiquitin-mediated degradation Hypoxia-inducible factor/hypoxia Cell cycle regulator/ionizing radiations Histone acetyltransferases/co-activators of transcription Tyrosine kinase/irradiation, DNA strand breaks Oncogene/negative control of p53 Nitric oxide/oxidative stress, inflammation, irradiation Cell cycle-dependent kinases Component of TFIIH Kinase/UV

MAPK ATM DNA-PK Chk-2 JNK/p38 PKC CKI

Mitogen-activated protein kinase/UV? Kinase/ionizing radiations Kinase/UV Cell cycle-dependent kinase Stress-activated kinases/UV Protein kinase C Kinase/?

p19arf

Cell cycle inhibitor, alternative product of CDKN2A Redox-repair enzyme/oxidative stress, hypoxia

Ref-1

ionizing radiation, activation of the p53 protein is thought to proceed through several consecutive steps, with first phosphorylation of p53 in the N-terminus by kinases involved in the sensing of DNA damage such as Atm (the product of the ataxia telangiectasia mutated gene) and Chk-2 (a cell cycle regulatory kinase). These phosphorylations contribute to the dissociation of the p53/ mdm-2 complex and stabilize the protein. Second, p53 binds co-activators with acetyltransferase activity such as ▶ P300/CBP co-activators and pCAF. These factors acetylate p53 in the C-terminus. These processes, as well as other coordinated posttranslational modifications of the C-terminus, induce conformational changes that turn the protein into an active form with a

Interaction with p53 ADP-ribose polymers bind to p53 Binding to N-terminus or to DNA-binding domain Binding to p53 Binding to p53 Binding, C-terminus (Ser-376) Binding, N-terminus acetylation, C-terminus Binding, proline-rich region Binding, residues 13–29 Oxidation of cysteines in DNA-binding domain Phosphorylation of Ser-315; forms complexes with p53 Phosphorylation of Ser-33 Phosphorylation of Ser-389; forms complexes with p53 Phosphorylation, Thr-73, and 83 (mouse p53) Phosphorylation, Ser-15 Phosphorylation, Ser-15 and Ser-37 Phosphorylation, Ser-20 Phosphorylation, Ser-34, mouse p53 Phosphorylation, Ser-378 Phosphorylation, several N-terminal serines (including Ser-6 and Ser-9) Prevents p53-mdm-2 interactions Reduction of cysteine in DNA-binding region binding to C-terminus

high affinity for specific DNA-binding sites. The third step involves redox regulation of sensitive cysteines within the DNA-binding domain of the protein. This three-step mechanism may account for p53 induction in response to most forms of DNA damage. Downstream of p53: Cell Cycle Control, Apoptosis, and DNA Repair Once activated, p53 exerts its effects through two major mechanisms: transcriptional control (activation or repression of specific genes) and complex formation with other proteins. Important downstream effectors of p53 (Table 2) include regulators of ▶ cell cycle checkpoints (in G1/S, G2, and during mitosis), factors involved in the

TP53

4605

TP53, Table 2 Some important downstream effectors of p53 functions Factor Apo-1/Fas/CD95

Activity Death signaling receptor

Bax-1

Dominant negative inhibitor of Bcl-2 Repressor of apoptosis Inhibitor of IGF-I Death signaling receptor Regulatory subunit of PI3 kinase Glutathione transferase homologue Quinone oxidase homologue Proline oxidase homologue Growth factor Survival factor Inhibitor of angiogenesis Binding to PCNA Inhibitor of proliferation Inhibitor of CDK2–4 and 6

Bcl-2 IGF-BP3 Killer/DR5 P85 Pig-12 Pig-3 Pig-6 IGF-I IL-6 Thrombospondin-1 Gadd45 BTG2 p21waf-1 Cyclin A Cyclin G GPx NOS2/iNOS COX2 Pig-1 PCNA RPA

Cell cycle regulation, S phase Cell cycle regulation Glutathione peroxidase Inducible nitric oxide synthase Inducible cyclooxygenase Galectin-7 Auxiliary subunit of polymerase d Replication protein A

ERCC2/ERCC3

Helicases, TFIIH complex

P53RR2 TBP

Ribonucleotide reductase homologue TATA box-binding protein

Mdm-2 MDR-1

Oncogene Multidrug resistance

signaling of apoptosis, and components of the transcription, replication, and repair machineries. At the cellular level, activation of p53 most frequently results in either cell cycle arrest (mostly in G1 and/or G2/M) or apoptosis. How a given cell “chooses” between cell cycle arrest and apoptosis in response to specific stimuli may depend upon many factors, such as the nature and intensity of the stress, as well as the cell type. In many tissues, p53 plays a role in drug-induced apoptosis and is thus

Mode of regulation Transcriptional activation? Transcriptional activation

Function Apoptosis

Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation

Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis

Transcriptional activation Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation

Apoptosis Apoptosis Apoptosis? Apoptosis? Apoptosis? Cell cycle arrest? Cell cycle arrest, G1 Cell cycle arrest, G1, and G2/M Cell cycle arrest, G1/S Cell cycle arrest? Control of oxidative stress Control of oxidative stress Control of oxidative stress? Differentiation? DNA repair/replication

Transcriptional repression Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation

Apoptosis

Inhibition by protein binding Activation by protein binding Transcriptional activation

DNA repair/replication

Inhibition by protein binding Transcriptional activation Transcriptional repression

Inhibition of transcription

DNA repair/transcription DNA repair?

Repression of p53 Resistance to chemotherapy

an important effector in the response of cancer cells to chemo- or radiotherapy. In addition, loss of p53 function results in deficient cell cycle arrest, inefficient mitotic spindle checkpoint, aberrant centrosome duplication, premature reentry into S phase, ▶ genomic instability, and ▶ aneuploidy. Clinical Relevance The TP53 gene is often inactivated by missense mutations, in contrast with many other tumor

T

4606

TP53

GC to TA

GC to AT(CpG)

Colon Thyroid Digestive organs Haematopoietic Brain Urinary system Female gential organs Skin Bladder Breast Prostate Connective tissue Rectum Head and neck Kidney Esophagus Liver Bronchus and lung 50

40

30

20

10

5

10

15

20

25

30

35

40

TP53, Fig. 3 Prevalence of two common mutation types: G to T transversions and C to T transitions at dipyrimidine (CpG) repeats in tumors of various organs. Tumors with high prevalence of G to T transversions often have a low

prevalence of transitions and vice versa. G to T transitions are a common molecular signature of many environmental carcinogens, such as tobacco smoke components (lung and esophageal cancers) or dietary mycotoxins (liver cancer)

suppressors such as APC, RB1, ▶ BRCA1, or p16/▶ CDKN2A that are inactivated by gene deletion or truncation. The mutations described to date mostly occur in the region of the gene encoding the DNA-binding domain. Most of these mutations impair DNA binding by disrupting the structure of the domain or crucial contact points between the protein and target DNA. About 30% of missense mutations affect six “hotspot” codons (175, 245, 248, 249, 273, and 282) (Fig. 2 bottom). The other mutations are scattered over 300 different codons. Mutations are very common in the invasive stages of many

epithelial tumors. A database of all published mutations is available at the International Agency for Research on Cancer (▶ IARC TP53 Database, http://www-p53.iarc.fr/). In many cancers, the patterns of mutations show variations, revealing clues about the mechanisms responsible for the formation of the mutations. Specific carcinogen-induced mutations have been identified in ▶ hepatocellular carcinoma (mutations induced by ▶ aflatoxins in sub-Saharan Africa and in Southeast Asia), in skin tumors (double transitions at adjacent cytosines, a typical signature of mutagenesis by UV in

TRA-8

squamous and in basal cell carcinomas), and in lung cancers (G to T transversions associated with exposure to tobacco smoke; ▶ tobacco carcinogenesis; (Fig. 3)). The usefulness of TP53 mutation detection in molecular pathology is still a matter of debate. As mutation often results in the accumulation of the protein, ▶ immunohistochemistry (IHC) has often been used as a criterion to detect TP53 abnormalities. However, positive IHC does not always correlate with mutation as several common missense mutants, as well as most frameshift and nonsense mutants, do not result in protein accumulation. Several well-established methods have been described for the detection of mutations in the TP53 gene, including SSCP (single-stranded conformation polymorphism) analysis, TTGE (temporal temperature gradient electrophoresis), yeast-based functional assays, and microarray hybridization assays. TP53 gene mutations are good markers for the clonality of tumor lesions. In many tissues, mutation correlates with bad prognosis and poor response to therapy, but TP53 mutation has been shown to behave as an independent marker of prognosis only in rare cases such as breast and head and neck cancers. Evidence suggests that the nature and position of the mutation may help to predict poor response to treatment. Detection of circulating anti-p53 antibodies as well as of free plasmatic DNA-containing mutant TP53 may be of interest in the early detection of cancer lesions. TP53 is the target of several experimental therapeutic approaches. Gene transfer of wild-type TP53 into cancer cells has been tested in several human tumors. However, the effects reported to date are limited and, at best, transient. Another approach is based on the use of cytolytic viruses selectively replicating in TP53-deficient cells (ONYX vectors). Several preclinical studies have investigated the use of small lipophilic compounds or peptides to activate TP53 function or to restore the activity of mutant proteins.

4607

References Foster BA, Coffey HA, Morin MJ et al (1999) Pharmacological rescue of mutant p53 conformation and function. Science 286:2507–2510 Levine AJ, Hu W, Feng Z (2006) P53 pathway: what questions remain to be explored. Cell Death Differ 13:1027–1036 Lowe SW, Bodis S, McClatchey A et al (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807–810 Olivier M, Hussain SP, Caron de Fromentel C et al (2004) TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci Publ 157:247–270 Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310

TP63 ▶ p53 Family

TP73 ▶ p53 Family

TP73L ▶ p53 Family

T TPI ▶ Cystatins

Cross-References

TRA-8

▶ p53 Family

▶ TRAIL Receptor Antibodies

4608

Trabectedin

Trabectedin Federico Gago1 and Sergio Moreno2 1 Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain 2 Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain

Synonyms Ecteinascidin Yondelis

743;

ET-743;

NSC-684766;

Definition A potent antitumor tetrahydroisoquinoline alkaloid in clinical development originally derived from a marine tunicate and now obtained by a synthetic process developed by PharmaMar starting from microbially produced cyanosafracin B.

Characteristics Crude aqueous ethanol extracts of the ascidian, or sea squirt, Ecteinascidia turbinata were shown to have powerful immunomodulating and antiproliferative properties as early as 1969, but the active principles were not identified until the early 1990s. The first six alkaloids that were characterized received the names ecteinascidins 729, 743 trabectedin (Fig. 1), 745, 759A, 759B, and 770, in accordance with the molecular masses ascribed to these compounds. They revealed a unique chemical structure consisting of a novel pentacyclic skeleton composed of two fused tetrahydroisoquinoline rings (subunits A and B) linked to a ten-member lactone bridge through a benzylic sulfide linkage and attached through a spiro ring to an additional ring system (subunit C) made up of either tetrahydroisoquinoline (as in trabectedin) or tetrahydro-b-carboline (as in ET-736). The first two subunits bear a clear structural resemblance to

microbially derived safracins and saframycins and also to sponge-derived renieramycins, all of them less potent anticancer agents than ecteinascidins. On the other hand, a reactive a-carbinolamine or hemiaminal (N-C-OH) group is also present in naphthyridinomycins, quinocarcins, and pyrrolo [4]benzodiazepine antibiotics such as anthramycin, sibiromycin, and tomaymycin. By analogy to these related antibiotics, the potent biological activity of trabectedin and other ecteinascidins was rapidly associated with their ability to form covalent adducts to DNA, following in situ dehydration of the carbinolamine group to an iminium intermediate that is covalently attached to the amino group of guanine in the minor groove. Ecteinascidia turbinata was first harvested from the wild and then successfully grown by the Spanish pharmaceutical company, PharmaMar, in aquaculture facilities in Spain (near Formentera Island, in the Mediterranean Sea). Subsequently, several synthetic schemes were developed to produce the multigram quantities required for clinical studies worldwide and overcome the limitation of the very low yield (0.0001%) of trabectedin in its natural source. The first enantioselective total synthesis of trabectedin was achieved in 1996, but industrial manufacturing was made possible through a synthetic route involving the conversion of cyanosafracin B, readily available by fermentation of the bacterium Pseudomonas fluorescens, to trabectedin in a very short and straightforward way developed by PharmaMar. Structural and Biophysical Characterization of Trabectedin-DNA Adducts Direct evidence that trabectedin alkylates duplex DNA at the exocyclic amino group of guanines was provided by a variety of experiments including gel electrophoresis, DNA footprinting, nuclear magnetic resonance (NMR) spectroscopy, and band shift assays, as well as molecular modeling studies. As a result of this work, it was found that trabectedin was protonated on N12 at physiological pH, and a role for hydrogen bonding in sequence recognition and orientation in the DNA minor groove was demonstrated, with TGG, CGG, AGC, GGC, and AGA being established

Trabectedin

a

4609

b

HO C NH MeO

OMe

O HO

O H3C

O

A

S O

H3C

CH3

12

N B

O

CH3

N 21

O

OH

Trabectedin, Fig. 1 Chemical formula and three-dimensional stick representation of the X-ray crystal structure of trabectedin

as the preferred DNA triplets for stable adduct formation and much higher rates of reversibility being measured for site-directed AGT- versus AGC-containing adducts. The proposed mechanism for activation takes advantage of the increased strength of the hydrogen bond between the proton on N12 and the hydroxyl group on C21 as the trabectedin molecule approaches the minor groove and is desolvated. This proton, which is essential for both sequence recognition and adduct stabilization, would then catalyze the dehydration of the carbinolamine, yielding the reactive iminium intermediate that undergoes nucleophilic attack at C21 by the exocyclic amino group of the guanine. Since a similar mechanism operates in the activation of pyrrolo[4]benzodiazepine antibiotics, it appears that nature ensures the reactivity of these carbinolamine-containing molecules by the inclusion of an internal catalytic proton adjacent to the leaving hydroxyl group. As a consequence of trabectedin bonding, the double helical structure is only minimally perturbed except for the widening of the minor groove and a net smooth bending toward the major groove due to the introduction of positive roll. This latter feature was novel among minor groove DNA monoalkylating agents as covalent modification of N3 of adenine in AT-rich regions by (+)-CC-1065 and related compounds is accompanied by the bending of the DNA into the minor rather than the major groove. Furthermore, if

multiple binding sites for trabectedin are properly phased in a relatively short stretch of DNA, the imposed cumulative curvature could bring closer together specified fragments not contiguous in primary sequence, and the drug could be serving a surrogate protein function. On the contrary, binding of three trabectedin molecules in a headto-tail fashion to three adjacent optimal binding sites would result in no net DNA curvature because the localized bends, brought about by the increase in roll at the sites of covalent attachment, would cancel out over virtually one turn of the helix. In fact, the DNA structure in one such complex (containing the sequence TGGCGGCGG) was shown to be intermediate between the canonical A and B forms of DNA, thereby strongly resembling the conformation that DNA adopts when bound to the consecutive C2H2 zinc fingers that are present in transcription factors (transcription factor) such as EGR-1 and Sp-1 (which bind to the major groove of GC-rich regulatory sequences in many gene promoters) or that observed in the hybrid double helix of template DNA paired to nascent RNA in the active site of RNA tumor viruses (RNAPII) elongation complex. The close contacts and the hydrogen-bonding interaction network that are established between trabectedin and DNA on both sides of the covalent adduct involve both DNA strands and therefore give rise to a significant increment in the stability of the resulting drug-DNA complexes. As a consequence, notable increases in the temperature of

T

4610

thermal denaturation of duplex DNA and substantial blockade of the helicase activities of both simian virus (SV40) large tumor antigen (T-antigen) and bacterial UvrABC and RecBCD enzymes have been reported for DNA oligonucleotides containing trabectedin adducts. This hampering or prevention of strand separation is also expected to result in stalled replication and transcription forks, as observed for a variety of conventional interstrand cross-linkers (e.g., nitrogen mustards, mitomycin, or cisplatin). An added advantage in the case of trabectedin would be the minimal distortions inflicted on the normal DNA structure that could help evade some of the recognition and repair mechanisms used for the processing of cross-links produced in the major groove by these other agents. Biological Activity In vitro cytotoxicity studies with trabectedin and other ecteinascidins established subnanomolar potencies against L1210 and P388 mouse leukemia cells, as well as human A549 lung cancer, HT29 colon cancer, MEL-28 melanoma cells, and human tumors explanted from patients. Tumorspecific responses and concentration-dependent relationships were observed when a soft agar cloning assay was used to determine the effects of a continuous exposure of trabectedin at different concentrations. These experiments clearly indicated that the duration of exposure to trabectedin was an important factor in human tumors, thereby pointing to preferential administration schedules in clinical trials. In vivo activity was then evaluated in several mouse tumor models and a variety of human tumors xenografted into nude mice, including melanoma, non-small cell lung carcinoma, and ovarian cancer. Long-lasting, complete, or partial regressions were observed in both chemosensitive and marginally cisplatin-resistant xenografts at the maximum tolerated dose (MTD), but no activity was seen in highly chemoresistant tumors such as MNB-PTX-1, MEXF 514, and LXFA 629. Importantly, the absence or incomplete cross-resistance with cisplatin and the comparable efficacy against the ovarian carcinoma xenografts justified the clinical assessment of trabectedin in ovarian cancer.

Trabectedin

The activity parameters for trabectedin in the panel of 60 human tumor cell lines of the National Cancer Institute (NCI) Anticancer Drug Screen revealed a rather unique profile that encouraged further development as an anticancer agent. The COMPARE algorithm (COX inhibitors) established a very high correlation coefficient (0.96) with chromomycin A3, an aureolic acid derivative shown to give rise to a pattern of distinct bands in human metaphase chromosomes, thus suggesting similarities in their apoptotic mechanisms. Despite the fact that these two compounds display very different modes of binding to DNA (i.e., covalent versus noncovalent, carbinolamine activation versus ion-mediated dimerization, etc.), both share a strong binding affinity for some common DNA sites, such as the self-complementary hexanucleotide TGGCCA, to which two trabectedin molecules can bind in a tail-to-tail fashion, each covalently bonded to a different strand. Furthermore, these two natural products are known to exert at least part of their cytotoxicity by interfering with DNA replication and transcription. Thus, at physiologically relevant concentrations (1–100 nM), trabectedin has been shown to effectively inhibit intracellular DNA synthesis by decreasing replication origin activity and by inducing unusual replication intermediates that may be blocked in fork progression. In addition, trabectedin is able to abrogate the transcriptional activation of a number of genes, including those encoding the multidrug resistance P-glycoprotein (MDR1), heat shock protein 70 (hsp70), the cell cycle inhibitor p21 Cip1 (p21), and collagen a1(I) (COL1A1). Nevertheless, global gene expression profiling of trabectedin-treated cancer cells has revealed rather complex patterns of both up- and downregulation. The reported effects on MDR1 and additional in vitro data showing enhancement by trabectedin of the cytotoxicity exerted by other chemotherapeutic agents that are substrates for P-gp/MDR1 suggest that combination treatment may be valuable in the clinic. The extremely low concentrations of trabectedin that are necessary to cause cell cycle arrest and cell death are suggestive of a trans-acting mechanism that probably operates

Trabectedin

through one or more cellular DNA damage response pathways or checkpoints. In this respect, it is notable that cell sensitivity to trabectedin appears to be somehow dependent on a proficient transcription-coupled ▶ nucleotide excision repair (TC-NER) machinery and more specifically on the presence of selected components that are implicated in ▶ xeroderma pigmentosum and Cockayne syndromes. Thus, the initial observation that hamster cells deficient in XPB, ERCC1, or CSB, as well as human XPA and XPC cells, had reduced sensitivity to trabectedin was followed by the report that a human colon carcinoma cell line selected for increased (20-fold) resistance to trabectedin (following continuous exposure to increasing concentrations of this drug for 1 year) had a truncated and inactive form of the XPG structure-specific endonuclease. Furthermore, drug sensitivity was restored in all cases upon complementation with the respective wild-type protein. These intriguing effects were recapitulated and expanded using yeast as a simpler eukaryotic model system. It was seen that trabectedin activates the G2-M and S-phase DNA damage checkpoints, in good agreement with the G2/M block and S-phase delay reported in human cells. Likewise, cells deficient in the XPG orthologue (rad13 in Schizosaccharomyces pombe) were shown to be much more resistant to trabectedin and underwent much less DNA damage than the corresponding isogenic wild-type strains. However, it became clear that it was not the missing endonuclease activity of this protein that conferred resistance to trabectedin but the lack of part of its DNA-binding domain in the COOHterminal region. Furthermore, on the basis of a homology model suggesting that the rad13/ DNA/trabectedin ternary complex could be stabilized through the direct interaction of subunit C of the drug with a highly conserved arginine residue, an S. pombe strain carrying an Arg961!Ala point mutation in rad13 was generated. This mutant displayed normal endonuclease and NER activity but was found to be strongly resistant to the drug. Haploid yeast mutants with deletions in the RAD52 epistasis group of genes encoding proteins responsible for homologous recombination

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(HR) and hence most double-strand break (DSB) repair in eukaryotic cells (e.g., rad51, rad22 (the fission yeast counterpart of mammalian rad52), and rad54) were found to be extraordinarily sensitive to trabectedin. This result reinforced other indications that the drug is giving rise (directly or indirectly) to DSBs that need to be repaired by homologous recombination, and the fact that the absence of rad13 partially rescued rad51D cells supports the view that a rad13-containing complex is somehow involved in the induction or irreparability of lethal DSBs. These results may have important implications for the optimal use of trabectedin in cancer therapy because patients harboring tumor cells with proficient NER and deficient HR systems would be expected to respond best to the treatment. Clinical Studies Trabectedin was selected for clinical development in preference to other related ecteinascidins because of its outstanding potency and greater relative abundance in the tunicate. Among the criteria that were taken into account for bringing it into clinical trials in early 1996 both in Europe and in the United States, we can summarize the following: (i) a novel chemical entity harboring a potential new mode of action, (ii) evidence for a positive therapeutic index, (iii) lack of complete cross-resistance with conventional chemotherapeutic agents, and (iv) feasibility of supply for clinical development. Toxicity to trabectedin so far has been shown to follow a transientreversible pattern and to be predictable, doserelated, and mostly limited to bone marrow and liver. Following a favorable opinion adopted by the Committee for Orphan Medicinal Products (COMP) of the European Agency for the Evaluation of Medicinal Products (EMEA), trabectedin was granted by the European Commission’s Orphan Medicinal Product Designation for the treatment of soft tissue sarcoma (STS) in April 2001 and for P-glycoprotein family (OC) in October 2003. The US Food and Drug Administration (FDA) awarded Orphan Drug Designation to trabectedin in the indication of STS in October 2004 and in OC in April 2005.

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Extended phase II trials and comparative studies have produced evidence of long-lasting responses and tumor control in advanced pretreated sarcomas, breast carcinoma, ovarian carcinoma, and prostate cancer. In July 2007, Yondelis received a positive opinion from the EMEA for the treatment of metastatic or advanced soft tissue sarcoma after failure to anthracyclines and ifosfamide. In 2009, trabectedin also received a positive opinion from EMEA, in combination with liposomal doxorubicin, for the treament of patients with relapsed platinum-sensitive ovarian cancer. Finally, in 2015, the FDA approved Yondelis for the treament of specific soft tissue sarcomas (liposarcomas and leiomyosarcomas) that are unresectable or metastatic.

Trace Elements

Traditional Chinese Medicine ▶ Chinese Versus Western Medicine

Traffic ATPases ▶ ABC-Transporters

TRAIL ▶ TNF-Related Apoptosis-Inducing Ligand

References

TRAIL Receptor Antibodies Feuerhahn et al (2011) XPF-dependent DNA breaks and RNA polymerase II arrest induced by antitumor DNA strand interstrand crosslinking-mimetic tetrahydroisoquinoline alkaloids. Chem Biol 18:988–999 Gago F, Hurley LH (2002) Devising a structural basis for the potent cytotoxic effects of ecteinascidin 743. In: Demeunynck M, Bailly C, Wilson WD (eds) Small molecule DNA and RNA binders: from synthesis to nucleic acid complexes. Wiley-VCH, Weinheim, pp 643–675 Herrero AB, Martín-Castellanos C, Marco E et al (2006) Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin. Cancer Res 66:8155–8162 Manzanares I, Cuevas C, García-Nieto R et al (2001) Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anticancer agents. Curr Med Chem Anticancer Agents 1:257–276 Martínez et al (2013) Inhibitory effects of marine-derived DNA-binding antitumour tetrahydroisoquinolines on the fanconi anemia pathway. Br J Pharmacol 170:871–882 Rinehart KL (2000) Antitumor compounds from tunicates. Med Res Rev 20:1–27 Sainz-Diaz CI, Manzanares I, Francesch A et al (2003) The potent anticancer compound ecteinascidin-743 (ET-743) as its 2-propanol disolvate. Acta Crystallogr C 59:o197–o198

Trace Elements ▶ Mineral Nutrients

Claus Belka Department of Radiation Oncology, University of Tübingen, Tübingen, Germany

Synonyms DR4 antibodies; DR5 antibodies; Lexatumumab; Mapatumumab; TRA-8

Definition TRAIL induces ▶ apoptosis preferentially in malignant tissues. Therefore, TRAIL is considered to be a potential antineoplastic drug. Agonistic TRAIL receptor antibodies have been developed as alternative pharmacological tool for apoptosis induction via the TRAIL receptors.

Characteristics After having identified TRAIL as a member of the family of cell death-inducing ligands, it became obvious that TRAIL has a strong propensity for transformed or malignant tissues.

TRAIL Receptor Antibodies

Therefore, TRAIL is considered to be a candidate anticancer drug. TRAIL exerts its apoptosisinducing activity via the two respective agonistic TRAIL receptors DR4 and DR5. TRAIL itself is able to induce cell death in a wide array of cancer cells in vitro or when grown in xenograft settings. The efficacy of TRAIL is increased whenever the ligand is combined with conventional cytostatic agents or ionizing radiation. Depending on the production process, there was a concern that TRAIL, like CD95-L, could exert considerable hepatotoxicity. Released data from phase I trials suggest that TRAIL can be safely administered in patients up to serum concentrations consistent with those demonstrating efficacy in tumor xenograft models. In parallel to the development of TRAIL as anticancer drug, agonistic antibodies directed against both death-inducing TRAIL receptors were developed. Up to now relevant data on three agonistic TRAIL antibodies are available. The signaling pathways triggered by agonistic antibodies have not been reported to differ from the cascades triggered by TRAIL. Treatment of susceptible cells with agonistic antibodies results in the activation of ▶ caspase-8, caspase-9, and cleavage of PARP. As shown for TRAIL, agonistic TRAIL antibodies also induce cleavage of the antiapoptotic MCL-1 protein. How far key regulatory molecules including FADD, c-FLIP, and caspase-10 are involved in the regulation of cell death induction via agonistic TRAIL antibodies has not been tested in detail. However, the fact that there is a cross-resistance between TRAIL and the agonistic antibodies in such a way that cells being resistant toward TRAIL cannot be killed by either antibody indicates that the signaling pathways are identical or at least highly similar. The agonistic TRA-8 antibody directed against DR5 has been developed by Sankyo together with researchers from the University of Alabama (Birmingham, USA) and was the first agonistic antibody being described. TRA-8 was generated by immunizing BALB/c mice with a fusion protein containing the extracellular domain of DR5 and the Fc proportion of human IgG1. The antibody does not cross-react with murine DR5. The

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Kd values for TRAIL or TRA-8 binding to DR5 were estimated at 59 and 3 nM, respectively. The high specificity of TRA-8 for DR5 was documented by competition assays showing that TRA-8 efficiently competed with TRAIL for binding to DR5 but not for binding to DR4. In addition, these results indicate that TRA-8 potentially recognizes an epitope within the TRAILbinding site on DR5. In general, TRA-8 induces apoptosis in tumor cell systems in vitro as well as in murine xenograft model systems (Jurkat and 1321N1 astrocytoma cells). In contrast to the TRAIL preparation used for comparison, the TRA-8 antibody did not induce any signs of hepatopathy in mice. In subsequent studies, the increased efficacy of multimodal approaches combining either TRA-8 with radiation, chemotherapy, or other response modifiers was documented. In this regard it is important to notice that the efficacy of the tested combinations in terms of growth delay was shown in xenograft models for ▶ cervical cancer, ▶ breast cancer, and pancreal cancer. The overexpression of Bax using an adenoviral vector system increased the efficacy of TRA-8 in a wide array of glioma cells suggesting that Bax is involved in the efficacy of the combined treatment. The increased cell death induction translated into an increased growth delay. Besides the TRA-8 antibody, Sankyo also develops an agonistic DR4 antibody 2E12. However, considerably less data are available regarding the pharmacology and efficacy of this antibody. The second group of antibodies was developed by Cambridge Antibody Technology in conjunction with Human Genome Sciences (Rockville, USA). HGS-ETR1 (mapatumumab) is directed against DR4 and HGS-ETR2 (lexatumumab) is directed against DR5. HGSI also develops a third agonistic TRAIL antibody (HGS-TR2, targeting DR5) that was initially developed by Kirin Brewery Ltd. Up to now only data on mapatumumab and lexatumumab are available. Mapatumumab is a fully humanized monoclonal antibody and was isolated from 102 different antiTRAIL receptor mAbs that were generated by phage display technology. The antibody has a high affinity for the DR4 receptor and exerts

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antitumor activity (EC50 values of 3.4 nM) in diverse preclinical tumor models including breast, gastrointestinal, lymphoma, ovarian carcinoma, and uterine cancers. Mapatumumab was shown to specifically recognize the TRAIL-R1 protein without any relevant interactions with the TRAIL decoy receptors. The efficacy of mapatumumab is increased by combinations with various cytostatic drugs including carboplatin, cisplatin, camptothecin, topotecan, paclitaxel, as well as radiation. Xenograft models for breast cancer, colorectal cancer, non-small cell lung cancer, as well as uterine cancer revealed a high activity of either the drug alone or in combination with other cytotoxic treatment approaches including radiation. Like mapatumumab, lexatumumab is a fully humanized IgG1g antibody. The antibody was also generated using a phage display and screening with 383 short chain fragments for DR5 binding properties. In contrast to mapatumumab, no data on specificity, selectivity, and affinity of the antibody are publicly available. Similar to TRA-8, lexatumumab exert pronounced apoptotic reactions in a wide array of malignant cell systems when used alone. Importantly the drug has proven efficacy on tumor growth in xenograft systems from renal cell carcinoma, non-small cell lung cancer, breast cancer, and glioma. As already shown for TRA-8, the combination of lexatumumab with various chemotherapeutic agents (camptothecin, cisplatin, carboplatin, paclitaxel, doxorubicin, bortezomib) or radiation increased the efficacy in cell lines and xenografts. The underlying mechanisms of sensitization are still not completely understood. However, it seems likely that the presence of the proapoptotic Bax molecule as well as the upregulation of the respective receptor participates in the increased efficacy of the combined approach. Clinical Aspects Mapatumumab (Anti-DR5)

Data from several early clinical trials are available and allow a cautious judgment regarding

TRAIL Receptor Antibodies

pharmacological and toxicological aspects of mapatumumab. An open label phase Ia/b trial was conducted in 39 patients with various advanced solid tumors. During the first phase, dose escalation of mapatumumab was performed (0.01, 0.03, 0.1, 0.3, 1.0, or 3.0 mg/kg). The second phase of the trial involved administration of mapatumumab (10 mg/kg) once every 28 days or once every 14 days. The i.v. administration of mapatumumab produced dose-proportional pharmacokinetics up to a dose of 1.0 mg/kg, with a half-life of 15 days for 1.0 mg/kg. The pharmacokinetic data indicate that distribution and clearance follow a two-compartment model, with first-order elimination from the central compartment. The best clinical responses reported so far are stable diseases in a proportion of the heavily pretreated patients. No data from ongoing trials combining chemotherapy with TRAIL are available at present. In addition to various phase I trials, mapatumumab was tested in a multicenter phase II trial in patients with relapsed or refractory non-Hodgkin lymphoma. Patients (n = 40) received either (3 or 10 mg/kg mapatumumab once every 21 days). Partial responses were observed in three patients, and one patient with relapsed follicular mixed-cell lymphoma demonstrated a more pronounced regression. Two other phase II trials with mapatumumab are ongoing. One trial was conducted in patients with relapsed or refractory colorectal cancer. No data on safety, tolerability, pharmacokinetics, tumor response, time to response, duration of response, and progression-free survival from this trial are available. The second phase II trial including patients with solid tumor was performed in 32 patients with non-small cell lung cancer (median of three previous treatment cycles). These patients received 10 mg/kg mapatumumab every 21 days until disease progression. Mapatumumab was well tolerated with no treatment discontinuations due to drug-related toxicity. In 29% of these patients, a stable disease (median duration of 2.3 months) was observed. The most

Transabdominal Metastasis

common mapatumumab-related adverse events were nausea, fatigue, hypotension, myalgia, pyrexia, peripheral sensory neuropathy, diarrhea, constipation or abdominal pain, rash, hypertension, and thrombocytopenia. A clear maximumtolerated dose had not been achieved. No antibodies to mapatumumab had been observed. The phase II trial in NSCLC patients revealed that mapatumumab administration was generally safe and well tolerated. In 97% of these patients at least one adverse event was reported; however, only 44% of the patients experienced an adverse effect that was considered to be drug related. Again, no immunogenic responses were observed. Lexatumumab (Anti-DR5)

Data from several phase I trials using lexatumumab are available. Results from a US dose escalation trial (0.1, 0.3, 1.0, 3.0, and 10 mg/kg i.v. lexatumumab every 2 weeks) revealed that dose responses were linear up to the 10 mg/kg level with a mean half-life of 11 day at the 10 mg/kg dose level. The analysis of a similar trial performed in the UK (37 patients with advanced cancer of different organs sites treated with doses of 0.1, 0.3, 1.0, 3.0, 10, or 20 mg/kg every 3 weeks) revealed linearity over the whole dose range and a distribution model consistent with a two-compartment model with first-order elimination from the central compartment (reported pharmacological values: mean parameters for the 1 and 10 mg/kg groups were C max = 24.0 and 195.9 mg/ml; AUC = 190.8 and 2,379 mg/days/ml; half-life = 12.0 and 15.3 days; plasma clearance = 5.7 and 4.8 ml/kg/day; V1 = 44.1 and 50.8 ml/kg; and VdSS = 83.7 and 87.1 ml/kg). Clinical results from both trials have been reported with no major toxicity. Of 31 patients entered in the US trial, 10 experienced disease stabilization and 20 had disease progression. Of 37 patients entered in the UK trail, 11 experienced disease stabilization and 26 had disease progression. Data from the studies indicate that lexatumumab was well tolerated at doses up to 10 mg/kg. The most frequently reported

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toxicities were fatigue, nausea, anorexia, constipation, diarrhea, tachycardia, and vomiting. The DLT for lexatumumab was defined as 10 mg/kg. No data from phase II trials are available at present. TRA-8 (CS1008)

Up to now no results from clinical trials have been reported with the humanized version of TRA-8 (CS1008). Perspectives TRAIL receptor-based treatment strategies are currently entering clinical trials. The feared liver toxicity of TRAIL and agonistic compounds has not been documented in any of clinical trials currently available. No judgment on the definitive clinical anticancer activity of agonistic TRAIL receptor antibodies can be made although the phase I and phase II data revealed some clinical activity.

TRAM1 ▶ Steroid Receptor Coactivators

TRAM-1 ▶ Amplified in Breast Cancer 1

T Transabdominal Dissemination ▶ Transcoelomic Metastasis

Transabdominal Metastasis ▶ Transcoelomic Metastasis

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Transcoelomic Metastasis David S. P. Tan1 and Stanley B. Kaye2 1 Department of Medical Oncology, National University Cancer Institute, Singapore (NCIS), National University Hospital, and Cancer Science Institute, National University of Singapore, Singapore, Singapore 2 Drug Development Unit, Institute of Cancer Research, The Royal Marsden Hospital, Sutton, UK

Synonyms Peritoneal dissemination; Transabdominal dissemination; Transabdominal metastasis

Definition Dissemination or spread of malignant tumor throughout the peritoneal (abdominal and pelvic) cavity.

Characteristics Transcoelomic (meaning “across the peritoneal cavity”) ▶ metastasis refers to the dissemination of malignant tumors throughout the surfaces and organs of the abdominal and pelvic cavity covered by \peritoneum. Transcoelomic metastasis can occur as a result of ▶ invasion into the peritoneal cavity by (i) a primary cancer arising from within the abdominal/pelvic cavity, e.g., ▶ ovarian cancer; (ii) as a manifestation of systemic metastasis following hematogenous or lymphatic invasion by a primary cancer, e.g., advanced ▶ breast cancer; or (iii) following intraperitoneal seeding during surgical manipulation, e.g., during surgical resection of a colorectal tumor. The incidence of transcoelomic metastasis is higher with tumors that arise from the peritoneal cavity, e.g., ovarian (up to 70% of patients at presentation) and colorectal (up to 28% of patients at presentation). In contrast, extraperitoneal

Transcoelomic Metastasis

cancers, e.g., breast and lung, are associated with a much lower overall incidence of transcoelomic metastasis, although certain histological subtypes, e.g. infiltrating lobular breast cancers, have demonstrated a greater predilection for metastases to the gastrointestinal tract, gynecological organs, and peritoneum/retroperitoneum. This suggests that while the location of the primary tumor may be a key determinant in the development of transcoelomic metastasis, the tumor phenotype is also an important factor. Hence, it appears that a combination of anatomical and tumor-specific factors is involved in the transcoelomic metastatic process. Transcoelomic metastases contribute considerably to the morbidity associated with carcinomatosis because they have the capacity to affect multiple vital organs within the abdomen. Common examples include bowel obstruction caused by lesions along the gastrointestinal tract and renal failure caused by obstruction of the ureters. In addition, transcoelomic metastases are frequently associated with the formation of malignant ascites resulting in raised intra-abdominal pressure with consequent abdominal distension and discomfort. This results in early satiety, leading to dietary deficiency, impaired circulation of blood and ▶ lymphatic vessels, and respiratory compromise secondary to diaphragmatic splinting. Hence, there are potentially significant therapeutic advantages to be gained in understanding the process of transcoelomic metastasis. Mechanisms of Transcoelomic Metastasis Models of Metastasis

Two models have been hypothesized for the genetic origins of tumor metastases. The first model, often referred to as the seed-and-soil hypothesis, is that tumors are genetically heterogeneous and metastases arise from clones with a genetically acquired metastatic phenotype, which determines the final site of metastasis. The alternative hypothesis, the stochastic model, is that metastatic cells do not represent a genetically selected clone distinct from the primary tumor but arise as a stochastic event from tumor cell clones genetically identical to the primary tumor.

Transcoelomic Metastasis

Studies exploring this question using in vivo models have suggested a combination of both models of metastasis. Regardless of the metastatic model, there are certain observed characteristics that appear to be important for transcoelomic metastatic progression, in which complex cellular adaptations need to occur after cell detachment from the primary tumor mass to ensure survival within the peritoneal cavity. Cell Detachment Anchorage-independent growth and the ability to resist ▶ anoikis is a vital step for the initiation of metastasis. This process appears to involve the increased expression of ▶ survivin and X-linked inhibitor of ▶ apoptosis (XIAP), members of the inhibitor of apoptosis protein (IAP) family, which suppress apoptosis by inhibition of ▶ caspases. Other mediators of anoikis resistance include the family of extracellular matrix (ECM) to cell adhesion molecules known as integrins. Alterations in levels of integrin-mediated ECM-ligand binding have been found in many different tumor types and can result in decreased cell adhesion, changes in cell morphology and increased ▶ migration in vitro, and activation of ECM degrading enzymes including ▶ Matrix Metalloproteinases (MMP). Peritoneal Fluid and Anatomy The peritoneal cavity is normally empty except for a thin film of fluid that keeps surfaces moist. Peritoneal fluid arises primarily from plasma transudate and ovarian exudate. Other sources of peritoneal fluid include fallopian tubal fluid, retrograde menstruation, and macrophage secretions. The volume of peritoneal fluid is usually 5–20 ml and varies widely depending on physiological or pathological conditions. Peritoneal fluid contains a variety of free-floating cells, including ▶ macrophages, natural killer (NK) cells, lymphocytes, eosinophils, mesothelial (peritoneal surface epithelial) cells, and ▶ mast cells, which are all involved in immunological surveillance. Intraperitoneal fluid flow is directed by gravity to its most dependent sites and then drawn via the paracolic gutters to the diaphragm by the generation of negative intra-abdominal pressure in the upper abdomen during respiration. There is

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preferential flow along the right paracolic gutter, liver capsule, and diaphragm. Therefore, a natural flow of peritoneal fluid exists within the abdominal cavity, providing a route for the transcoelomic dissemination of detached tumor cells. As the epithelial surfaces of the female genital tract (i.e., ovaries, fallopian tubes, and endometrium) share a common embryological lineage with the peritoneal epithelium, it has been suggested that transcoelomic metastases from gynecological malignancies, such as fallopian tube and ovarian tumors, are not true metastases but a result of malignant transformation at multiple foci throughout the peritoneum, i.e., peritoneal metaplasia. If the metaplasia hypothesis is correct, then one might expect metastatic lesions to be randomly distributed throughout the peritoneum. Alternatively, if the theory of dissemination via peritoneal/ascitic fluid is true, then one might expect that detached tumor cells would, by virtue of gravity, be more frequently implanted in the floor of the pelvis, e.g., the pouch of Douglas (the space between the rectum and back wall of the uterus), followed by the organs in the paracolic gutters, and finally on the diaphragm, i.e., along the normal route of peritoneal fluid circulation. Studies have shown that a high incidence of metastatic implants for all cancers, including ovarian malignancies, within the peritoneal cavity is found on organs where peritoneal fluid resorption occurs (omentum and omental appendages). In addition, the colon, greater omentum, and pouch of Douglas are most often affected, with a reduced incidence of implants seen on the small bowel and its mesentery, which is free to move by peristalsis, compared to the ileocecal area (the junction between the ileum and cecum), which is fixed to the retroperitoneum. Hence, location and topography with regard to the flow of peritoneal/ascitic fluid appear to be key determinants in the process of transcoelomic dissemination for all cancers. As such, in the case of gynecological cancers, peritoneal metaplasia alone appears unable to fully account for the peritoneal distribution of carcinomatosis. Ascites: A Metastatic Milieu The development of transcoelomic metastasis is often associated with the formation of excess

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peritoneal fluid known as malignant ascites. It is hypothesized that, in addition to hypoalbuminemia (low plasma albumin levels) secondary to dietary deficiency, at least three other pathological events can cause ascites: (i) reduced lymphatic drainage from the peritoneal cavity caused by the obstruction of lymphatic vessels by tumor cells, (ii) increased vascular permeability of the peritoneal cavity, and (iii) tumor neo-▶ angiogenesis. While lymphatic obstruction is a well-recognized cause of ascites, the fact that massive amounts of fluid can accumulate in patients despite relatively little tumor burden suggests the involvement of other nonobstructive factors. These include ▶ Vascular Endothelial Growth Factor (VEGF), a glycoprotein which induces angiogenesis, and increased vascular permeability in response to hypoxia. Other immune modulators, vascular permeability factors, and MMPs secreted by both tumor cells and mesothelial cells also contribute significantly to ascites formation and stimulate tumor growth, invasion, and angiogenesis. Immune Evasion Many immune cells, such as macrophages, are present in peritoneal fluid and accumulate in so-called milky spots within the omentum. These omental macrophages have been found to be cytotoxic against tumor cells ex vivo. Consequently, omental macrophages might play an important role in killing tumor cells, thereby preventing development of transcoelomic metastasis and local peritoneal recurrences. Paradoxically, however, in vivo studies have shown that cancer cells seeded intraperitoneally specifically infiltrate the milky spots in the early stage of peritoneal metastasis. These studies suggest that omental milky spots are insufficient to prevent tumor progression and that intraperitoneal metastasis requires tumor cells to possess or acquire mechanisms for evasion of immunological surveillance. Tumor-infiltrating and malignant ascitesderived lymphocytes, in particular gamma delta T cells, from patients with metastatic ovarian and colorectal cancer, have also been shown to possess antitumor activity. Hence, it appears that metastatic tumor cells have also developed strategies

Transcoelomic Metastasis

to evade T cell-mediated cytotoxicity. Fas ligand (FasL) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following binding to its receptor, Fas. Expression of FasL has been observed in renal, ovarian, colorectal, and head and neck tumors and may be responsible for the immune privilege of tumor cells by inducing apoptosis of antitumor immune effector cells within the tumor microenvironment – the “Fas counterattack.” Studies have also shown that tumor progression and metastasis are associated with increased expression of FasL. Other examples of immune evasion include the recruitment of regulatory T (Treg CD4+CD25+) cells to suppress tumor-specific T-cell immunity; the presence of high concentrations of soluble forms of the complement pathway inhibitors C1 inhibitor, factor H, and FHL-I on isolated metastatic ovarian cancer cells in ascitic fluid; and the phenomenon of spheroid formation observed in breast, colorectal, and ovarian cancer where tumor cells clump together by upregulating cell adhesion molecules, thus resulting in increased complement resistance due to insufficient penetration of antibodies and complement into the spheroids. Tumor Implantation Although topography appears to be a key determinant in the final site of metastatic implantation within the peritoneum, the actual mechanisms behind tumor implantation remain unclear. However, there is evidence to suggest the involvement of a dynamic regulation of the tumor cell’s adhesiveness and its interaction with the underlying peritoneal mesothelium. Potential mechanisms for the attachment of tumor cells to the peritoneal mesothelium include binding to ECM proteins like collagen type I and IV, laminin, and fibronectin via tumor cell surface integrins and to hyaluronan expressed on the surface of human peritoneal mesothelial cells via the ▶ CD44 tumor cell surface protein, of which there are ten alternative exon splice variants (v1–v10). Upregulation of certain CD44 variants have been associated with distant metastasis in breast, colorectal, and ovarian cancer. Tumor antigen/marker CA125, a glycoprotein overexpressed on the cell

Transcoelomic Metastasis

surface and secreted by ovarian tumor cells in the majority of ovarian cancer patients, has been shown to bind to mesothelin, a glycosylphosphatidylinositol-linked cell surface molecule expressed by mesothelial cells. Upregulation of the cell adhesion molecule ▶ Ecadherin may also mediate adhesion of circulating tumor cells to metastatic sites. Adhesion onto the peritoneal surface may be followed by haptotactic migration in which coordinated anti- and pro-migratory signals mediated by ECM proteoglycans confer directionality to tumor cell motility, effectively laying the tracks until a “stop” signal is encountered. Once attached to the peritoneal surface, metastatic cells proliferate and invade into the subjacent epithelium. The MMP family of proteinases and the urokinase-type ▶ plasminogen activator (uPA) system appear to be major contributors to this process. Human peritoneal epithelial cells and their associated immune and stromal cells have been shown to release regulatory ▶ chemokines and cytokines, such as IL-1, IL-6, and IL-8, in response to serosal inflammation and injury induced by tumor implantation, which in turn facilitate tumor angiogenesis and ascites formation (via increased secretion of VEGF), and enhanced tumor migration, attachment, proliferation, and invasion. Finally, just as extraperitoneal tumors can metastasize to the peritoneum, intraperitoneal tumors can also metastasize extraperitoneally. Apart from the rich intraperitoneal network of blood and lymphatic vessels which can be invaded by tumors, peritoneal fluid is also continually being returned to the systemic circulation via the subdiaphragmatic lymphatic network and thoracic duct into the left subclavian vein, thus providing a direct “metastatic expressway” for peritoneal metastases to gain access into the lymphatic and circulatory system. Clinical Aspects Patients with transcoelomic metastasis often present with signs and symptoms of abdominal pain, abdominal distension secondary to an enlarging tumor or ascites, constipation or diarrhea, shortness of breath, fatigue, loss of appetite, and weight loss. A careful clinical history followed by

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thorough clinical examination is required to ascertain the likely source of the primary tumor. Investigations should include routine blood tests, including relevant tumor markers, followed by radiological investigations including ultrasound and computer tomographic (CT) scans of the chest, abdomen, and pelvis to confirm the likely source of tumor and disease stage. In all cases, particularly those in which there is no obvious source of primary tumor (i.e., carcinoma of unknown primary origin), a biopsy of an accessible lesion should be obtained for histopathological and immunohistochemical confirmation and diagnosis. In the past, clinical situations involving transcoelomic metastasis were treated mainly with palliative intent. Increasingly, studies have shown that an aggressive approach to peritoneal surface malignancy involving peritoneal debulking (cytoreductive) procedures, combined with optimal perioperative or postoperative systemic or intraperitoneal ▶ Chemotherapy in carefully selected patients, can result in long-term survival. Clinical assessment parameters that need to be considered include the patient’s performance status; preoperative abdominal and pelvic CT scans to define the extent and operability of disease, including the presence of extraperitoneal metastases; and tumor histopathology. Key prognostic indicators following surgery include the completeness of peritoneal debulking surgery, the presence of intraperitoneal lymph node and visceral metastases, and tumor type. Of the various scoring systems used to assess the extent of peritoneal carcinomatosis, the most frequently quoted is the peritoneal cancer index (based on the intraoperatively observed distribution and size of intraperitoneal metastasis) and the completeness of cytoreduction score (based on the amount of residual disease following peritoneal debulking surgery), which have been found to correlate well with prognosis in colorectal, gastric, and ▶ ovarian cancer. A meta-analysis of studies comparing combined peritoneal debulking surgery and perioperative intraperitoneal chemotherapy with systemic chemotherapy alone for the treatment of peritoneal carcinomatosis from colorectal carcinoma has demonstrated improved survival in the

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combination therapy group. In patients with ovarian cancer and peritoneal metastasis, 2-year survival following radical resection of all macroscopic tumors is 80%, in contrast to less than 22% for the patients with residual lesions larger than 2 cm. Early aggressive treatment of minimal peritoneal surface dissemination appears to confer the most benefit. In patients with inoperable tumors at presentation, primary systemic or intraperitoneal chemotherapy is recommended, following which reassessment for surgical intervention may be possible if a good treatment response is observed. Palliative measures in the management of malignant ascites include repeated paracentesis (drainage of ascites), which provides relief in up to 90% of patients, and permanent percutaneous drains. The creation of a peritoneovenous shunt (which allows ascitic fluid to drain from the peritoneal cavity into the superior vena cava) prevents the need for repeated paracentesis. Promising experimental approaches in the treatment of transcoelomic metastasis include the use of intraoperative hyperthermic intraperitoneal chemotherapy, antiangiogenic agents such as the MMP inhibitors and the VEGF antagonists, as well as ▶ immunotherapy approaches including antibody targeted T-cell therapy and combinations of intraperitoneal immunotherapy and thermochemotherapy.

Cross-References ▶ Angiogenesis ▶ Anoikis ▶ Apoptosis ▶ Breast Cancer ▶ Caspase ▶ CD44 ▶ Chemokines ▶ Chemotherapy ▶ Colorectal Cancer ▶ Colorectal Cancer Premalignant Lesions ▶ E-Cadherin ▶ Gastric Cancer ▶ Immunotherapy ▶ Invasion

Transcoelomic Metastasis

▶ Lymphatic Vessels ▶ Macrophages ▶ Mast Cells ▶ Metastasis ▶ Migration ▶ Ovarian Cancer ▶ Plasminogen-Activating System ▶ Survivin ▶ Vascular Endothelial Growth Factor

References Becker G, Galandi D, Blum HE (2006) Malignant ascites: systematic review and guideline for treatment. Eur J Cancer 42(5):589–597 Baratti D, Kusamura S, Pietrantonio F, Guaglio M, Niger M, Deraco M (2016) Progress in treatments for colorectal cancer peritoneal metastases during the years 2010-2015. A systematic review. Crit Rev Oncol Hematol. pii: S1040-8428(16)30017–30018. doi:10.1016/j.critrevonc.2016.01.017. [Epub ahead of print] Review Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12(2):89–96 Koppe MJ, Boerman OC, Oyen WJ et al (2006) Peritoneal carcinomatosis of colorectal origin: incidence and current treatment strategies. Ann Surg 243(2):212–222 Seimetz D1, Lindhofer H, Bokemeyer C (2010) Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) as a targeted cancer immunotherapy. Cancer Treat Rev 36(6): 458–467. doi:10.1016/j.ctrv.2010.03.001. [Epub 2010 Mar 27] Tan DS, Agarwal R, Kaye SB (2006) Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol 7(11):925–934

See Also (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Lymphatic System. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2120. doi:10.1007/978-3-642-16483-5_3450 (2012) Metaplasia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2259. doi:10.1007/978-3-642-16483-5_3670 (2012) Omentum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2609. doi:10.1007/978-3-642-16483-5_4213 (2012) Palliative. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2759. doi:10.1007/978-3-642-16483-5_4350

Transduction of Oncogenes (2012) Performance Status. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2814. doi:10.1007/978-3-642-16483-5_4444 (2012) Peritoneal Debulking Surgery. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2818. doi:10.1007/978-3-642-16483-5_4459 (2012) Peritoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2819. doi:10.1007/978-3-642-16483-5_4467 (2012) Retroperitoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3292. doi:10.1007/978-3-642-16483-5_5078 (2012) Stage. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3499. doi:10.1007/978-3-642-16483-5_5478

Transdifferentiation ▶ Stem Cell Plasticity

Transduction of Oncogenes Jaquelin P. Dudley Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

Synonyms Oncogene transduction; Retroviral transduction

Definition Retroviruses are RNA-containing viruses that replicate through a DNA intermediate (provirus) using the enzyme reverse transcriptase. During retroviral replication, which requires integration into the host chromosomal DNA for efficient transcription of viral RNA, some retroviruses have acquired specific cellular ▶ oncogenes, usually with multiple modifications and often with the loss of trans-acting viral functions. Inclusion of one or more oncogenes in the viral genome then imparts transforming activity on the recombinant virus independent of the site of integration in the cellular genome.

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Characteristics Identification of Cellular Oncogenes In 1911, Peyton Rous described the isolation of a virus that caused fibrosarcomas in chickens. The Rous sarcoma virus (RSV) subsequently was shown to transform chicken embryos and the surrounding membranes and formed small tumors on the chorioallantoic membrane in proportion to the number of virus particles. Further quantitative assays were developed when RSV and other retroviruses were shown to transform or induce morphological and growth behavior changes in cultured cells that mimicked tumor formation in the animal. The ability of RSV to transform cells in culture led to the conclusion that the virus encoded a gene responsible for such changes. Isolation of transformation-defective variants of RSV allowed comparisons with wild-type RSV and the discovery of the viral oncogene, v-src. Experiments from the laboratories of Harold Varmus and Michael Bishop revealed that the v-src gene is highly related to a specific cellular gene or proto-oncogene, c-src, which encodes a protein tyrosine kinase. Unlike the v-src gene in RSV, the cellular homologue contained introns, which could be alternatively spliced in various cell types to give different mRNAs. Further characterization showed that the product of the v-src gene, v-Src, had substitutions within several functional domains that prevented the normal regulation of the kinase activity during the process of ▶ signal transduction. Shortly after the discovery of v-src, other transforming viruses were isolated and characterized. Some of these viruses were recovered by treatment of normal cells with halogenated pyrimidines to induce endogenous retrovirus expression, by coculture of primary cells with chemically transformed cells that express non-transforming retroviruses, whereas others were isolated by in vivo passage of nontransforming retroviruses. In each case, the transforming virus appears to be the result of recombination between a non-transforming retrovirus and one or more cellular genes. The ability of retroviruses to acquire cellular sequences in their genome and transmit these genes to other

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cells is known as retroviral transduction or, in the case of proto-oncogenes, oncogene transduction. Examples of transforming viruses and the acquired proto-oncogenes are listed in Table 1. Deregulation can occur at many steps of ▶ signal transduction, leading to oncogenesis. Nevertheless, recombination events leading to the generation of a transforming retrovirus appear to be rare in nature. Most of the resulting viruses are defective for replication because the acquisition of cellular proto-oncogenes is accompanied by the deletion of viral structural genes, which are necessary to produce viral particles. Such defective transforming viruses are not transmissible unless they successfully coinfect a cell with a related retrovirus that provides the missing gene products in trans. Thus, most transforming viruses are more interesting as research tools for the identification and functional characterization of oncogenes and the process of transduction than as a major cause of disease in animals and humans. Mechanism of Oncogene Transduction The majority of transforming retroviruses are defective for viral replication. Since these viruses are isolated after acquisition of transforming activity, the exact steps required to form these retroviruses are unknown nor is it clear whether every transforming virus has been generated by the same mechanism. However, a general model has emerged for the formation of such viruses (Fig. 1). First, a non-transforming retrovirus integrates upstream of a cellular gene, an event known to occur at a reasonable frequency. Many retroviruses integrate preferentially within coding sequences or near sites of active transcription. Non-transforming retroviruses often cause tumors by insertion in or near proto-oncogenes, resulting in the activation of transcription or production of abnormal transcripts. These transcripts arise due to enhancer activation of the cellular promoter or activity of the viral promoter on the cellular gene. If the proviral integration results in cellular transformation, cells containing the integration site will be selected for growth. However, some retroviruses integrate upstream or downstream and in the opposite orientation relative to the

Transduction of Oncogenes Transduction of Oncogenes, Table 1 Oncogenes transduced by retroviruses Transforming virus Abelson murine leukemia virus AKT8 Cas NS-1 virus Avian sarcoma virus CT10 Avian erythroblastosis virus-ES4 Avian erythroblastosis virus-ES4 Avian myeloblastosis virus-E26 Avian retrovirus RPL30 Snyder-Theilen feline sarcoma virus Gardner-Rasheed feline sarcoma virus McDonough feline sarcoma virus Finkel-Biskis-Jinkins murine sarcoma virus Fujinami avian sarcoma virus Avian sarcoma virus 17 Hardy-Zuckerman-4 feline sarcoma virus Avian retrovirus AS42 (sarcoma) Mill-Hill virus 2 (avian myelocytoma virus) Moloney murine sarcoma virus Mouse myeloproliferative leukemia virus Avian myeloblastosis virus-E26 Myelocytomatosis virus 29 Avian retrovirus ASV31 (sarcoma) 3611 murine sarcoma virus Harvey murine sarcoma virus Kirsten murine sarcoma virus Avian reticuloendotheliosis virus T UR2 avian sarcoma virus S13 avian erythroblastosis virus Simian sarcoma virus SKV770 avian sarcoma virus Rous sarcoma virus Y73/Esh avian sarcoma virus

Acquired proto-oncogene abl akt cbl crk erbA erbB ets eyk fesa fgr fms fos fpsa jun kit maf milb mos mpl myb myc qin rafb HRAS KRAS rel ros sea sis ski src yes

a

fes and fps are the same oncogene derived from feline and avian genomes, respectively b mil and raf are the same oncogene derived from avian and murine genomes, respectively

proto-oncogene, and it is believed that the generation of transforming retroviruses requires proviral integration upstream and in the same orientation as the oncogene. Such events can result in cancer induction without transduction of

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4623 U3RU5 5’LTR

3’LTR

Proviral integration

c-onc DNA 3’LTR

5’LTR

RNA Readthrough transcription of truncated provirus

Readthrough transcription of full-length provirus

5’LTR

5’LTR

3’LTR

DNA RNA RU5

RU5

U3RU5

Splicing and co-packaging of normal and hybrid oncogene transcripts RU5

RU5 Retroviral particles

(-) strand DNA RU5

U3RU5

(-) strand DNA RU5

U3R

U3R

Reverse transcription: recombination and mutagenesis

v-onc 5’LTR

3’LTR

Transducing provirus with mutations

Transduction of Oncogenes, Fig. 1 Mechanism of retroviral oncogene transduction (See text for details)

oncogenes. Thus, many proviral insertion events may lead to cancer, but not formation of transforming retroviruses. Second, transcription initiating in the 50 LTR generates a transcript that would read through the normal polyadenylation sequences in the 30 LTR. Evidence suggests that increases in retroviral transcriptional readthrough also result in transductive recombination. Potentially, this readthrough transcript could be packaged into virions, although it is generally believed that transcripts longer than 150% of the genome would be accommodated poorly in the virus capsid. Alternatively, a rare deletion of the cellular DNA or aberrant splicing could provide a truncated provirus or mutant transcripts that may encompass a much greater portion of the cellular transcripts. Third, hybrid oncogene transcripts may be packaged along with normal viral transcripts into

virions. Retroviruses have a diploid genome that includes cis-acting sequences (usually near the 50 end of the viral genome) necessary for packaging into the viral capsid (designated the psi sequence). Thus, hybrid transcripts including the psi sequence would be preferentially packaged with normal retroviral sequences to give an RNA heterodimer instead of the normal RNA homodimer. However, normal cellular RNAs can be packaged into retroviral particles at low frequency, and co-packaging would allow copying by reverse transcriptase, which is not template specific. Fourth, the hybrid oncogene transcript and the wild-type transcript both will be used as templates for reverse transcriptase, which also is incorporated into viral particles. In some cases, incorporation of the proto-oncogene and expression at high levels from the retroviral promoter appears

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to be transforming. Nevertheless, most transduced oncogenes have multiple genetic alterations. Reverse transcriptase has several properties that favor the types of genomic changes observed in transforming retroviruses. These properties include template switching, deletion formation, and the introduction of point mutations. If the resulting recombinants retain all of the cis-acting sequences needed for replication, the transforming virus will be capable of propagation in the presence of replication-competent retroviruses. Supporting the importance of readthrough transcripts for this process, transforming retroviral genomes have been observed that carry poly(A) stretches typical of mRNAs at the junction between the host and 30 viral sequences. Furthermore, the incorporated oncogenes lack introns. Lessons from Retroviral Transduction of Oncogenes Cancer is believed to be a multistep process, where several genetic events contribute to the generation of tumor cells. Some avian retroviruses are known to have transduced two oncogenes, including avian erythroblastosis virus-ES4 and avian erythroblastosis virus-R (AEV-ES4 and AEV-R) (erbB and erbA), Mill-Hill 2 (MH2) avian myelocytoma virus (mil (aka raf) and myc), and avian myeloblastosis virus (AMVE26) (ets and myb). Some viruses may have transduced more than one cellular proto-oncogene and been selected for increased transforming capacity, a process that may be similar to the acquisition of multiple genetic changes in cancer cells during tumor progression. For example, evidence suggests that erbA expression is necessary for the full transforming activity of the erbB oncogene in AEV. Also, the MH2 retrovirus requires expression of both mil/raf and myc to transform neuroretinal cells from 7-day-old chicken embryos, an event that myc-expressing retroviruses cannot induce. However, AMV-E26 contains two oncogenes, but only one of them has been shown to be necessary for full transforming ability. Deletion of the ets oncogene does not diminish the transforming ability of AMV-E26 relative to wild-type virus when injected in newborn chickens. Furthermore, many of the

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transduced oncogenes contain deletions or point mutations that reveal regulatory regions of the encoded gene products, leading to a greater understanding of their normal functions in cellular growth control. Retroviruses, including simple retroviruses that lack regulatory genes as well as lentiviruses, have become common vectors for therapeutic gene delivery. These viruses have been used to deliver genes for treatment of a variety of illnesses, including cancers and genetic disorders. Lentiviral vectors offer the advantage of being able to infect and replicate in non-dividing cell types. However, simple retroviruses may be advantageous for preferential infection of dividing tumor cells and delivery of the therapeutic gene relative to adjacent normal cells. In both cases, viral structural genes in the vector are replaced with a therapeutic gene of interest. Expression of the transduced genes can be controlled by nonviral promoters, internal ribosome entry sites, or splicing. Although these vectors have shown promise as therapeutic agents, the safety of such vectors has been the overriding concern, i.e., preventing the formation of replication-competent viruses and avoiding the ill effects of integration. Limitations to replication and the avoidance of multiple insertions within target cells should minimize insertional activation of oncogenes. Improvements to the retroviral vector system have resulted from removal of the 30 U3 region, thus creating a self-inactivating (SIN) virus after reverse transcription. Another possible safety concern associated with ▶ gene therapy is the generation of new transforming viruses, especially in human patients. These events should be extremely rare because of several precautions included during the construction of these vectors, including the removal of U3 sequences as well as viral structural genes, thus confining these retroviruses to a single round of replication. Furthermore, the absence of homologous endogenous lentiviruses in humans should reduce recombination events that lead to generation of transforming retroviruses. In the early part of this century, a major milestone in human gene therapy was achieved. Early treatments of 20 children with X-linked severe combined immune deficiency (SCID-X1) using a

Transforming Growth Factor-Beta

wild-type Moloney murine leukemia virus-based vector allowed normal expression of the defective gene and corrected the immune defect. Unfortunately, in five of the patients, this vector inserted in close proximity to the LMO2 or CCND2 protooncogene, leading to dysregulation of expression and development of leukemia. A current gene therapy clinical trial suggests that removal of the 3’ LTR enhancer in the gammaretrovirus vector (SIN vector) prevents or delays such adverse events. Additional improvements continue to be made, including prevention of transcriptional readthrough or inclusion of insulator elements to block the formation of hybrid viral-cellular transcripts that may lead to oncogene transduction.

Cross-References ▶ Gene Therapy ▶ Oncogene ▶ Signal Transduction

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Transfer of T Cells ▶ Adoptive T-Cell Transfer

Transforming Gene ▶ Oncogene

Transforming Growth Factor-Beta Jorma Keski-Oja1 and Katri Koli2 1 Departments of Pathology and of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland 2 Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland

References

Definition

An W, Telesnitsky A (2004) Human immunodeficiency virus type 1 transductive recombination can occur frequently and in proportion to polyadenylation signal readthrough. J Virol 78:3419–3428 Hacein-Bey-Abina S et al. (2014) A modified gammaretrovirus vector for X-linked severe combined immunodeficiency. New England J Med 371:1407–1417 Nevins JR (2001) Cell transformation by viruses. In: Knipe DM, Howley PM (eds) Fundamental virology, 4th edn. Lippincott William & Wilkins, Philadelphia, pp 245–283 Swain A, Coffin JM (1992) Mechanism of transduction by retroviruses. Science 255:841–855 Swanstrom R, Parker RC, Varmus HE et al (1983) Transduction of a cellular oncogene: the genesis of Rous Sarcoma Virus. Proc Natl Acad Sci U S A 80:2519–2523

Transforming growth factors were identified on the basis of their ability to induce soft agar growth and morphological changes in nonmalignant cells. The original observation was of an activity, which was named sarcoma growth factor. Soon afterward the term transforming growth factor, TGF, was adopted. Sarcoma growth factor was subsequently found to be composed of an epidermal growth factor like protein, which was named transforming growth factor alpha, TGF-a, and of TGF-b. TGF-a is a member of the epidermal growth factor family and is unrelated to TGF-b. Targeting of TGF-b activity by LTBPs (binding proteins) and different mechanisms of activation from the latent forms is a crucial feature in its biology.

See Also (2012) Endogenous Retrovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1232. doi:10.1007/978-3-642-16483-5_1879 (2012) Retrovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3296–3297. doi:10.1007/978-3-642-16483-5_5084 (2012) Reverse Transcriptase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3297. doi:10.1007/978-3-642-16483-5_5086

Characteristics TGF-bs are multifunctional polypeptide growth factors involved in the regulation of cellular growth and differentiation and immune functions. The number of known members of the TGF-b

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superfamily is close to 40 different growthmodulating proteins. Besides TGFbs, these include bone morphogenetic proteins (BMPs), growth and differentiation factors (GDF), nodal, inhibins, and activins. TGF-bs are in many senses unique among growth factors in their potent and widespread actions. Three different mammalian gene products, TGF-bs 1–3, have been molecularly cloned. Almost all types of cells in the body make some form of TGF-b, and nearly all cells have cell surface receptors for it. One of their major effects is inhibition of cell proliferation, a property needed in developmental processes, for instance. In addition, TGF-bs regulate differentiation and cellular plasticity during development and in adult tissue repair processes. TGF-bs have important roles in the control of the pericellular proteolytic balance and in the regulation of the production and structure of the components of the connective tissues and extracellular matrices. TGF-b stimulates the transcription and synthesis of various components of the extracellular matrix like collagens, ▶ fibronectin, vitronectin, tenascin, and proteoglycans. TGF-bs are potent chemotactic factors for many cell types like fibroblasts, eosinophils, and various inflammatory cells at very low concentrations. They also suppress matrix degradation by decreasing the expression of proteinases (▶ serine proteases (type II) spanning the plasma membrane), such as plasminogen activators (▶ plasminogenactivating system) and numerous metalloproteinases, and by inducing proteinase inhibitors, such as plasminogen activator inhibitor-1 and ▶ tissue inhibitors of metalloproteinases (TIMPs). In addition, TGF-bs regulate cellular functions by modulating the expression of matrix receptors, the integrins. For these reasons the activities of TGF-bs must be tightly regulated. TGF-b Receptors and Signaling Mechanisms Members of the TGF-b superfamily have diverse functions in cell-cell signaling. TGF-bs play different roles in tissue homeostasis and at various stages of development. The mechanisms of regulation of TGF-b activity are multifaceted and complex. Three different TGF-b isoforms and the types, affinity, and signaling functions of its

Transforming Growth Factor-Beta

receptors also add complexity to the regulation of their effects. The effects of TGF-bs and the other family members are mediated from the cell membrane to nucleus through distinct combinations of type I and type II serine/threonine kinase receptors and downstream effectors, the Smad proteins. TGF-b signals are mediated by TGFbRII and two different type I receptors, activin-like kinase (ALK) receptors ALK5 and ALK1. Endoglin and betaglycan, also called TGF-b type III receptors, act as coreceptors for the signaling complex. The receptor-regulated Smads become phosphorylated by activated type I receptors, and they form heteromeric complexes with a common partner, Smad4, which gets translocated into the nucleus for gene transcription control. In addition to the signal-transducing Smads, inhibitory Smads also play a role in the outcome of the signaling. They downregulate the activation of receptor-regulated Smads. TGF-b can also elicit non-Smad signaling responses in target cells, such as activation of mitogen-activated protein kinase (MAPK) cascades, PI3K/AKT/mTOR pathway, and RhoA. These play an important role in the tumorpromoting activities of TGF-b. Latency TGF-b TGF-bs are produced by the majority of cells in latent complexes unable to associate with TGF-b signaling receptors. Some primary cells and established cell lines secrete active TGF-b. TGF-bs are secreted from cells as latent dimeric complexes containing the mature C-terminal TGF-b and its N-terminal pro-domain, LAP, the TGF-b latency-associated peptide. The two polypeptide chains of pro-TGF-b associate to form a disulfide-bonded dimer. TGF-b is cleaved from its propeptide by furin-like endoproteinase during secretion at RRXR sequence. The LAP propeptide dimer remains associated with the TGF-b dimer by non-covalent interactions. This complex is referred to as small latent TGF-b. TGF-bs are secreted in most cultured cell lines as large latent complexes, consisting of small latent TGF-b covalently bound to one of the three latent TGF-b binding proteins (LTBP-1, LTBP-3, or LTBP-4) covalently linked to LAP (Fig. 1). The expression and secretion of LTBPs

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Large latent TGF-β complex

LAP Hinge region (Plasmin and MMP cleavage sites) LTBP

Proteinase sensitive region

TGF-β

ECM binding site Latent TGF-β binding site

8-Cys repeat ECM binding sites

Hybrid domain Ca2+ binding EGF-like repeat

Extracellular matrix fibers

Non-Ca2+ binding EGF-like repeat

Transforming Growth Factor-Beta, Fig. 1 Large latent TGF-b complex. The small latent complex contains the C-terminal mature TGF-b and its N-terminal pro-domain, LAP (TGF-b latency-associated peptide). This complex

forms a disulfide-bonded complex with the third 8-Cys repeat of LTBP. LTBP associates with the ECM mainly via the 8-Cys domains and some adjoining regions

and TGF-bs is, in general, coordinately regulated. Interestingly, LTBP-2 is unable to form complexes with any of the small latent TGF-bs and has other functions as a microfibril-associated protein. In addition, LTBP-4 binds only to TGF-b1 and with much lower affinity than LTBP-1 and LTBP-3. LTBPs have a central role in the processing, secretion, and matrix targeting of TGF-bs, but they also have other roles as regulators of the structure and function of the extracellular matrix.

complexes can be a two-step process, the release of the large latent complex from ECM by proteolytic truncation and subsequent activation, which can be achieved by different mechanisms. It appears that integrins can activate TGF-b complexes directly without proteolytic release. Matrix stiffness plays an important role in integrinmediated activation of TGF-b. Since TGF-b regulates the cellular production of ECM components as well as the proteolytic balance, the matrix association and activation of TGF-b complexes form a finely tuned control network for the maintenance of the organization of extracellular structures. Cancer cells produce frequently aberrant amounts of both the matrix components and TGF-b. Malignant cells do also frequently fail to deposit TGF-b complexes to the extracellular matrix, probably due to their perturbed deposition of fibronectin-collagen matrix, as well as altered LTBP production.

Matrix Association and Release of TGF-b LTBPs have a central role in the targeting of TGF-b to extracellular matrix structures. LTBPs are produced in excess to TGF-b, and since TGF-b secretion is very inefficient in the absence of LTBP, most secreted cellular TGF-b is in the large latent complexes (Fig. 1). The release of active TGF-b from matrix-associated latent

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Transforming Growth Factor-Beta

Association of LTBPs with different TGF-β isoforms TGF-β1

TGF-β2

TGF-β3 RGD

RGD

LTBP-4

Latent complexes of TGF-b in the ECM may provide tissues with a readily available storage form of this growth factor. The release and activation of stored growth factors by proteases or migrating cells can generate rapid and highly localized signals like in wound healing or during radiotherapy. Cell movement causes traction of the latent matrix-associated complexes and induces activation. Rapid activation of extracellular signaling mechanisms could be important in the healing of tissues after damage, in the control of cells of the immune system during acute infections, and in the initial stages of angiogenesis. It is unclear how soluble growth factors could form gradients in highly cellular tissues. Matrix-bound growth factors might generate this kind of an immobilized activity gradient. LTBPs: Expression and Functions Data of the functions of LTBPs is accumulating rapidly. Structurally they resemble fibrillins, which are components of the extracellular microfibrils. LTBPs have a typical structure consisting of four eight-cysteine (8-Cys) repeats and several EGF-like repeats. The association of latent TGF-b with the matrix is mediated by LTBPs (Fig. 1). Not only the N-terminal domains but also a region of the C-terminus of LTBP is important in this association. The N-terminus contains transglutaminase

LTBP-1

LTBP-3

LTBP-2

substrate motifs, and transglutaminase is required for the covalent ECM association. In addition, TGF-b2 and TGF-b3 become associated with LTBPs. It is thus likely that LTBPs mediate and target the binding of all three TGF-b isoforms to various extracellular matrices. The TGF-b1 binding region in LTBPs is located close to their C-terminus in the third 8Cys repeat. The association between LTBP-1 and the propeptide part, LAP, is mediated by disulfide bonding. The respective 8-Cys repeats of LTBP-3 and LTBP-4 also bind small latent TGF-bs. Of the numerous known 8-Cys repeats of the LTBPs and fibrillins, only three have been found to have the capacity to associate in a covalent manner with the small latent TGF-bs (modeled in Fig. 2). LTBPs bind fibrillins, which form the extracellular microfibrils. This binding is important also for non-TGF-b-related functions of LTBPs. LTBP-4 is an important regulator of elastogenesis through association with fibulins and elastin. Activation of Soluble and Extracellular Matrix Forms of Latent TGF-b TGF-b can be activated in vitro by multiple mechanisms, including proteolysis, enzymatic deglycosylation, and extremes of pH. Activation of latent TGF-b involves proteolytic disruption of the non-covalent interaction between the

Transforming Growth Factor-Beta

propeptide LAP and TGF-b, which releases biologically active TGF-b capable of binding to its signaling receptors. LAP may also undergo conformational changes in such a manner that TGF-b is released or exposed to its receptors. The existence of different TGF-b isoforms and latent complexes, as well as the number of different LTBPs, suggests that there are variable mechanisms for the activation of TGF-bs. The electrostatic interaction between LAP and TGF-b can be dissociated in vitro by extremes of pH, chaotropic agents, and heat treatment. From the physiological point of view, the acidic environment in the bone (osteoclasts) or during wound healing could induce this kind of TGF-b activation. In vivo analyses of tumor-bearing mice indicated that irradiation causes rapid activation of TGF-b in the tumors. This effect appears to result from the activation of existing, most probably of matrix-bound latent TGF-b. Irradiation produces reactive oxygen species leading to redoxmediated activation of latent TGF-b complexes. Redox-mediated TGF-b activation may be involved in chronic tissue processes, where oxidative stress is implicated, such as carcinogenesis. The processing of pericellular matrixassociated LTBPs and activation of TGF-b are constant events in the ▶ apoptosis or ▶ anoikis of endothelial and epithelial cells, pinpointing the importance of pericellular latent complexes as a physiological source of TGF-b. Thrombospondin-1 (TSP-1), a platelet a-granule and ECM protein, plays a role in the activation of latent TGF-b complexes via a mechanism that does not involve cell surfaces or proteases. Using purified plasma TSP-1 or the recombinant protein, it was found that it is able to activate both small and large latent TGF-b complexes. The activation mechanism is not fully understood but seems to involve the N-terminal end of LAP and the type I repeats of TSP-1, possibly by inducing a change in the conformation of LAP and thus releasing the active TGF-b. TSP-1 interacts with LAP as a part of a biologically active complex, and this may prevent the reassociation of the inactive complex of LAP with TGF-b. The expression of TSP is induced

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during wound healing. TGF-b may thus get focally activated at sites of injury by enhanced TSP synthesis. Accordingly, TSP-deficient mice display many phenotypic features, similar to those detected in TGF-b1-deficient mice. The abnormalities in some tissues of the TSP null animals were even reverted by TSP-derived TGF-b activating peptides, further emphasizing the role for TSP in TGF-b activation. The LAP part of TGF-b contains an RGD-motif, which is recognized by many integrins (▶ Integrin Signaling). Integrin avb6 is able to activate TGF-b. This activation model is particularly interesting, because avb6 integrin is expressed solely on epithelial cells, which are very sensitive to TGF-b-mediated growth inhibition, and also because the overlap of the phenotypes of TGF-b1 and integrin b6 chain-deficient mice. b6 integrin-deficient mice show increased inflammation and decreased fibrosis, processes that are regulated by TGF-b. In addition, other av-containing integrins have been suggested to activate TGF-b directly through a mechanism requiring proteolytic activity or a change in the conformation of the latent complex. Hormonal effectors can also affect TGF-b activation. Originally it was found that antiestrogens could induce the production and secretion of active TGF-b in cultured breast cancer cells. Activation of TGF-b has subsequently been observed in a number of cell culture models using estrogens and antiestrogens, retinoids, and vitamin D derivatives. Steroid hormone superfamily members are efficient regulators of the expression of TGF-b isoforms, and TGF-bs are likely to act as local mediators of the diverse actions of steroids. Estrogens and antiestrogens regulate TGF-b1 formation in different cells and tissues like in mammary carcinoma cells and in fetal fibroblasts. TGF-b functions, for instance, as an autocrine negative growth regulator in breast carcinoma cells. TGF-b and LTBP Knockout Mice The importance of the three different TGF-bs is elucidated in the gene knockout studies. Knockout of Tgf-b1 results in multifocal inflammatory disease leading to the death of the animal. Tgf-b2

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knockout is embryonally lethal. The mice develop severe cardiac, lung, and craniofacial defects. The inner ear and eye are also affected. Tgf-b3 null mice develop cleft palate. Accordingly, null mice unable to produce LTBPs develop serious physiological defects. Ltbp-3 knockout mice develop multiple defects such as growth retardation, emphysema, bone malformations, and abnormalities of thymus and spleen. Their lifespan is, however, normal, and they are able to reproduce. Hypomorphic Ltbp-4 / mice develop early emphysema and colorectal tumors. The mice have also severe defects in elastogenesis. The short and long splice forms of LTBPs have different functions. Ltbp-1L null mice exhibit a cardiac phenotype, which reveals a crucial role for Ltbp-1L and matrix as extracellular regulators of Tgf-b activity in heart organogenesis. Ltbp2 / mice developed lens luxation caused by compromised ciliary zonule formation. Perspective Growth factors of the TGF-b family are important autocrine and paracrine regulators of cell proliferation and differentiation. The regulation levels of their activities include the expression of TGF-b receptors, availability of TGF-bs, their activities, and modulation of the cellular response. Most cells secrete TGF-b in a large latent complex, which associates with the extracellular matrix and is unable to bind to the TGF-b signaling receptors. LTBPs have a central role in TGF-b secretion, extracellular matrix deposition, and activation. In addition, LTBPs have structural and other functions not directly related to TGF-b signaling. Structural diversity in LTBP proteins is tremendous, and the possible functions of the different forms include, among others, the modulation of cell adhesion and the functions of integrins. Focal activation of latent TGF-b in the matrix by physicochemical means offers a rapid way to induce TGF-b signaling. In addition to plasminmediated TGF-b activation, novel mechanisms have been found including other proteases, reactive oxygen species, and thrombospondin- and integrin-mediated activation. The modulation of pericellular proteolytic activity by TGF-b supports a general cascade of

Transforming Growth Factor-Beta

events, where proteinases and latent matrixbound growth factors are components of extracellular signal transduction machinery. This directs tissue construction and remodeling and probably also regulates the activity of infiltrating immune cells. Disturbances in these control systems could participate in the pathogenesis of a variety of disease states like atherosclerosis, cancer, various fibrotic diseases, and chronic inflammation.

Cross-References ▶ Anoikis ▶ Apoptosis ▶ Fibronectin ▶ Integrin Signaling ▶ Plasminogen-Activating System ▶ Serine Proteases (Type II) Spanning the Plasma Membrane ▶ Smad Proteins in TGF-Beta Signaling ▶ Tissue Inhibitors of Metalloproteinases

References Koli K, Saharinen J, Hyytiäinen M, Penttinen, Keski-Oja J (2001) Latency, activation and binding proteins of TGF-b. Microsc Res Tech 52:354–362 Moustakas A, Heldin CH (2009) The regulation of TGF-b signal transduction. Development 136:3699–3714 Munger JS, Sheppard D (2011) Cross talk among TGF-b signaling pathways, integrins, and the extracellular matrix. Cold Spring Harb Perspect Biol 3:a005017 Saharinen J, Keski-Oja J (2000) Specific sequence motif of 8-Cys repeats of TGF-b binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-b. Mol Biol Cell 11:2691–2704 Sporn MB (2006) The early history of TGF-b, and a brief glimpse of its future. Cytokine Growth Factor Rev 17:3–7 Todorovic V, Rifkin DB (2012) LTBPs, more than just an escort service. J Cell Biochem 113:410–418

See Also (2012) Latent TGF-β Binding Protein. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1986. doi:10.1007/978-3-642-164835_3288 (2012) TGF-β Activation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3662. doi:10.1007/978-3-642-16483-5_5754

Transgenic Mouse

4631

Characteristics

Transfusion of T Cells ▶ Adoptive T-Cell Transfer

Transgenic Mice ▶ Mouse Models

Transgenic Mouse Aparna Gupta Life Science Research Associate, Department of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA

Definition A transgenic mouse contains additional, artificially introduced genetic material/DNA in every cell. The foreign DNA that has been purposely inserted into the mouse genome can be a foreign gene (not usually present in mice), a gene from the mouse, or a gene with a mutation (mutation: a change in the DNA code that alters the protein properties). The foreign gene is carefully constructed using recombinant DNA technology. In addition to the target gene itself, the DNA also includes other sequences that ensure: • Incorporation into the genomic DNA of the mouse • Proper expression of the gene by the cells In order to incorporate the foreign DNA into every cell of the mouse, it has to be introduced into very early mouse embryo so that the germ cells (sperm and ovum) also receive the gene and pass it on to the next generation.

Methodology There are two major methods that can be used to generate a transgenic mouse: • Pronuclear microinjection: Foreign DNA is introduced directly into the mouse egg just after fertilization. Using a fine needle under the microscope, the DNA is injected into the large male pronucleus, which is derived from the sperm. The fertilized egg divides actively, and the DNA integrates at a random position in the genome. There can be a single copy of foreign DNA or multiple tandem copies of DNA integrated into the genome. The integration occurs after a few cell divisions; therefore, the resulting mouse is only partially transgenic (some cells lack the transgene, referred to a mosaic). If the transgenic cells contribute to the germ line, then some transgenic eggs or sperm will be produced, and the next generation of mice will be fully transgenic (Fig. 1). • Embryonic stem cell (ES) injection: In this method, DNA is introduced into embryonic stem cells (ES cells). ES cells are derived from the very early mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo. Foreign DNA is introduced into ES cells growing on plates; DNA may integrate randomly, as in the case of pronuclear microinjection. The manipulated ES cells are then selected for cells that express the transgene. These are injected into the inner cell mass (ICM) of a blastocyst (blastocyst is an early stage of embryo formation where the inner layer of cells called ICM gives rise to the embryo and the outer layer, called trophoblast, gives rise to the placenta.) The injected blastocysts are implanted into the uterus of a pseudopregnant (by mating a female mouse with a vasectomized male) female mouse. This triggers changes in the female that facilitate embryo implantation. If the procedure is successful, the implanted embryos will give rise to healthy pups (Fig. 1).

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Transgenic Mouse

Transgenic Mouse, Fig. 1 Methodology for generating a transgenic mouse

Pups are screened for the transgene, only a small percentage of them will be positive. The ones positive for the transgene are bred to each other to generate transgenic animals. Random Versus Targeted Gene Insertion Random insertion of DNA results in multiple copies of the transgene in the genome. It can also insert itself in a coding sequence or could disrupt some regulatory sequences in the genome. Thus, targeted gene insertion is now the preferred method as it ensures that the foreign DNA does not disrupt other genomic sequences. The targeted DNA sequence has (as shown in Fig. 2): • The gene of interest. • Neomycin-resistant gene that inactivates the antibiotic neomycin and allows the cells to grow in the presence of the drug, by which positive cells can be selected. • A gene that encodes thymidine kinase (TK), an enzyme that phosphorylates ganciclovir. Nonfunctional phosphorylated ganciclovir inserts into freshly replicating DNA and kills the cells. So ganciclovir in the growth medium

kills cells that contain the TK gene and have random insertion of the transgene. The foreign DNA with all the right components is given to the cells, and the cells are cultured in the presence of neomycin and ganciclovir in the medium: • The cells that failed to take up the foreign DNA are killed by neomycin. • The cells in which the DNA is inserted randomly are killed by ganciclovir (because they insert nonfunctional nucleotide due to presence of tk gene). • The surviving cells are the ones transformed by homologous recombination where the DNA integrates in the desired genomic site (Fig. 2). These cells are injected into blastocysts, implanted into pseudopregnant females, and pups are analyzed. Knockout Mice As the name suggests, these mice have a complete loss or knockout of the gene of interest. This is achieved by using a recombinant DNA without any sequence for gene G (the target gene to be

Transgenic Mouse

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Transgenic Mouse, Fig. 2 Random and targeted gene insertion

Transgenic Mouse, Fig. 3 Conventional gene knockout

deleted). The foreign DNA will recombine with the genomic locus of gene G and result in replacement of G with neomycin resistance gene (Fig. 3). This is the simplest method to generate knockout mice. Figure 3 shows a knockout strategy that results in gene loss in every cell of the body. Knockout of a critical gene can result in dead mice not allowing further study of the gene of interest. If the scientific question addresses to study the function of a gene in a

particular tissue or at a particular stage of development, then tissue-specific knockout mice will be generated. Tissue-Specific Knockout Mice (The Cre/loxP System) Cre recombinase, abbreviated Cre, is an enzyme from bacteriophage P1 that can recognize a specific DNA sequence (called loxP site) and excise and recombine them. This system involves two mice lines (Fig. 4):

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Transglutaminase Type-2

Transgenic Mouse, Fig. 4 Tissue-specific and conditional gene knockout

• Mice expressing the Cre enzyme: (a) The expression of Cre can be driven by a tissue-specific promoter that allows the gene loss only in a desired tissue/ cell type. (b) The promoter can also be designed such that the Cre is expressed only after a certain drug is given to the mice. This allows the loss of gene at certain desired age and not from birth. • Mice carrying the conditional gene (the target gene between two loxP sites)

Kos CH (2004) Cre/loxP system for generating tissuespecific knockout mouse models. Nutr Rev 62(6 Pt 1):243–246 Kuhn R, Torres RM (2002) Cre/loxP recombination system and gene targeting. Methods Mol Biol 180:175–204

The two transgenic mice are bred, and the pups are analyzed for mice carrying both the Cre and loxP conditional gene. These mice have a loss of the target gene in the tissue/ cell targeted by the promoter used for the Cre-expressing mice.

Transglutaminase-2

References Cameron ER (1997) Recent advances in transgenic technology. Mol Biotechnol 7(3):253–265 Cameron ER, Harvey MJ, Onions DE (1994) Transgenic science. Br Vet J 150(1):9–24

Transglutaminase Type-2 ▶ Transglutaminase-2

Kapil Mehta The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Cytosolic transglutaminase; Endothelial transglutaminase; Liver transglutaminase; TG2; TGc; Tissue transglutaminase; Transglutaminase type-2; tTGase

Transglutaminase-2 Integrin 28 kDa fragment) 139 aa1

4635 Catalytic triad

β-sandwich

N

81 Fn 140

520 358 GTP 460

277 335 358

Catalytic core C

H D

EGSEEERE 430 Ca2+ 453

β-barrel1 αIβ-adrenergic receptor

687

β-barrel2

C

LHMGLKLV 657 677 PLCδ1

Transglutaminase-2, Fig. 1 Schematic representation of various functional domains of the TG2 protein. In addition to catalyzing calcium-dependent protein cross-linking function, TG2 can catalyze calcium-independent GTPase, ATPase, protein kinase, and protein disulfide isomerase activities. TG2 can modulate the functions of other proteins by directly interacting or associating with them; examples include phospholipase-d1, members of the

b-integrin family, focal adhesion kinase, fibronectin, osteonectin, RhoA, multilineage kinases, and ▶ retinoblastoma protein. Through these activities, TG2 plays a role in biological processes such as ▶ apoptosis, wound healing, and cataract formation. Recent work suggests that TG2 can also serve as a signaling molecule and promote cell growth, drug resistance, and metastatic functions in tumor cells

Definition

types. An important feature of TG2 is its high binding affinity for ▶ fibronectin; in cancers, membrane-associated TG2 can promote a stable interaction between cell surface integrins and fibronectin and promote cell growth and survival (Fig. 1).

Tissue transglutaminase (TG2; EC 2.3.2.13) is a ubiquitous and most diverse member of the transglutaminase family of enzymes. TG2 catalyzes calcium-dependent posttranslational modification of proteins by inserting highly stable isopeptide bonds between polypeptide chains or by conjugating ▶ polyamines to proteins. In addition, TG2 exhibits ▶ GTPase activity and can serve as a ▶ signal-transduction G protein. Less studied functions of TG2 include its protein disulfide isomerase and ▶ protein kinase activities.

Characteristics TG2 is a multifunctional protein whose expression in some cell types (e.g., endothelial and smooth muscle cells) is constitutively high. In other cell types, TG2s expression is upregulated via discrete signaling pathways, such as those induced by certain stress factors, inflammatory stimuli, differentiation agents, and growth factors. Although predominantly a cytosolic protein, TG2 can translocate to the nucleus by “piggyback riding” other proteins, such as importin-alpha-3, or translocate to membranes in association with integrins. TG2 can also be secreted outside the cell (by an as-yet-unknown mechanism), where it can cross-link extracellular matrix (ECM) proteins and promote ▶ adhesion of several cell

TG2 and Apoptosis A role for TG2 in apoptosis was initially suggested by Dr. Laszlo Fesus and his coworkers in 1987 based on the observation that leadinduced hypertrophy of the liver in rats was associated with cellular expression of increased TG2. Since then, many reports have supported the role of TG2 in apoptosis. In general, the expression of TG2 is markedly increased in cells undergoing apoptosis. Forcibly increasing the expression of TG2 in several cell types results in apoptosis or makes them susceptible to death-inducing stimuli. Conversely, reducing TG2 levels by antisense RNA renders the cells more resistant to apoptosis. It is believed that TG2 promotes apoptosis by cross-linking intracellular proteins, preventing their leakage from cells and induction of an inflammatory response. These observations suggest that cells generally do not tolerate the increased expression of TG2 and that TG2 overexpression leads to apoptotic death. However, some reports have provided paradoxical evidence and suggest that TG2 expression and apoptosis do not always go hand in hand. For example, TG2/ knockout mice (mice lacking

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4636

all TG2 expression) did not show any genetic alterations that are suggestive of perturbed apoptosis. The possibility that some other proteins compensate for the loss of TG2 in these mice cannot be ruled out. Furthermore, various other studies have provided data suggesting that increased expression of TG2 can prolong cell survival by preventing apoptosis. TG2 in Drug Resistance and Metastasis Evidence is accumulating that cancer cells that are resistant to chemotherapeutic drugs or that are isolated from metastatic sites express elevated levels of TG2. Also, there is evidence that drugresistant and metastatic cancer cells share some common pathways. For example, cells from advanced-stage cancers accumulate a large number of genetic alterations that can render them resistant to apoptosis. Resistance to apoptosis can enable cancer cells not only to grow and survive in the stressful environment of distant tissues (i.e., to metastasize) but also to withstand the toxic effects of drugs. Moreover, cell lines selected in vitro for resistance against chemotherapeutic drugs are more metastatic in vivo, while cancer cells isolated from metastatic sites, in general, exhibit higher resistance to chemotherapeutic drugs. Based on these observations we hypothesized that aberrant expression of TG2 in drugresistant and metastatic cancer cells may dysregulate some intrinsic apoptotic pathways in order to protect cells from apoptosis. Indeed, downregulation of endogenous TG2 by antisense RNA, TG2-specific ribozyme, or small interfering RNA (▶ siRNA) could reverse drug resistance in ▶ lung cancer, ▶ ovarian cancer and ▶ breast cancer cells. Similarly, inhibition of TG2 by siRNA in breast cancer and malignant ▶ melanoma cells augmented their response to chemotherapeutic drugs and reduced their invasiveness in laboratory experiments. In pancreatic cancer cells, inhibition of TG2 by siRNA resulted in massive accumulation of lysophagosomes and onset of ▶ autophagy (type II apoptosis). These properties suggest that TG2 expression in cancer cells contributes to the development of drug resistance and ▶ metastasis. Studies to elucidate the mechanisms involved in the development of TG2-mediated drug

Transglutaminase-2

resistance in cancer cells revealed that TG2 expression augments cell survival signaling by promoting a stable interaction between cell surface integrins and the ECM proteins. Depending on the cell type, 20–30% of total TG2 can exist in complex with b-integrins (e.g., b1, b4, and b5). The association of TG2 with integrins occurs primarily at their extracellular domains and promotes their interaction with ECM ligands such as fibronectin, collagen, and vitronectin. Downregulation of TG2 in glioblastoma cells resulted in decreased assembly of fibronectin in the ECM and cell death. Importantly, treatment of mice that had orthotopic glioblastomas with the TG2 inhibitor KIP1 sensitized the tumors to chemotherapy, induced apoptosis of cancer cells, and prolonged survival of the animals. Further, the interaction between TG2 and integrins is independent of the cross-linking activity of TG2 and results in increased cell adhesion, ▶ migration, and activation of downstream survival signaling pathways such as ▶ focal adhesion kinase (FAK). Interestingly, TG2 can also interact directly with focal adhesion kinase and result in its autophosphorylation (pY397) and consequent activation of the downstream PI3K and Akt signaling. Activation of the ▶ nuclear factor-kB (NF-kB), which plays an important role in regulating cell growth, apoptosis, and metastasis, has also been associated with increased TG2 expression in cancer cells. Tumor cells that overexpressed TG2 exhibited increased levels of constitutively active NF-kB. Activation of TG2 led to activation of NF-kB, and conversely, inhibition of TG2 activity inhibited the activation of NF-kB. Similarly, ectopic expression of TG2 caused activation of NF-kB, and inhibition of TG2 expression by siRNA abolished the NF-kB activation and rendered drug-resistant breast cancer cells sensitive to doxorubicin-induced cytotoxicity. Notably, immunohistochemical analysis of pancreatic ductal adenocarcinoma tumor samples further supported a strong correlation between TG2 expression and NF-kB activation. These observations suggest that TG2 induces constitutive activation of NF-kB in tumor cells via a novel pathway. Therefore, TG2 may be an

Transitional Cell Carcinoma

attractive target for inhibiting constitutive NF-kB activation and rendering cancer cells sensitive to anticancer therapies. Clinical Relevance Drug resistance and metastasis are major impediments to the successful treatment of cancer. More than 90% of cancer-related deaths can be attributed to the failure of chemotherapy. On the basis of published results that drug-resistant and metastatic tumors and tumor cell lines express high levels of TG2, that TG2 expression promotes cell survival and invasion, and that downregulation of TG2 results in increased sensitivity of cancer cells to chemotherapeutic drugs and to undergo programmed cell death (apoptosis or autophagy), TG2 may offer an attractive target for treating drug-resistant and metastatic tumors.

4637

Transitional Cell Carcinoma Jun Hyuk Hong1, Seong Jin Kim2 and Isaac Yi Kim1 1 Division of Urologic Oncology, The Cancer Institute of NJ, Robert Wood Johnson Medical School, New Brunswick, NJ, USA 2 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, USA

Synonyms TCC; Transitional cell carcinoma of bladder; Transitional cell carcinoma of renal pelvis; Transitional cell carcinoma of ureter; Urothelial carcinoma; Urothelial tumor

References

Definition

Eckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GV, Mehta K (2014) Transglutaminase regulation of cell function. Physiol Rev 94:383–417 Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140–156 Mehta K, Eckert R (eds) (2005) Transglutaminases: family of enzymes with diverse functions. Prog Exp Tumor Res 38:125–138 Mehta K, Fok J, Miller FR et al (2004) Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer. Clin Cancer Res 10:8068–8076 Mehta K (2005) Mammalian transglutaminases: a family portrait. In: Transglutaminases: family of enzymes with diverse functions. Karger Publ pp 1–18 Mehta K, Fok JY, Mangala LS (2006) Tissue transglutaminase: from biological glue to cell survival cues. Front Biosci 11:173–185 Verma A, Wang H, Manavathi B et al (2006) Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis. Cancer Res 66:10525–10533

Transitional cell carcinoma (TCC) arises in the urothelium that covers the lining of the renal calyx, renal pelvis, ureter, bladder, and part of the urethra. Although the WHO/ISUP consensus conference has determined that the term urothelial cancer is preferable to the term transitional cell cancer, the latter remains in widespread use. “Urothelial cancer” may also be confusing because cancers of other histological types, such as squamous cancers and adenocarcinoma, also arise in the urothelium.

Transin-1 ▶ Stromelysin-1

Characteristics Epidemiology It was estimated that in 2007, 67,160 new cases of bladder cancer would be diagnosed and 13,750 patients would die of invasive bladder cancer in the United States. ▶ Bladder cancer is nearly three times more common in men than in women, and more than 90% of bladder cancers are TCCs. The median ages at diagnosis for TCC are 69 years in males and 71 years in females.

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Upper urinary tract urothelial tumors involving the renal pelvis or ureter are relatively uncommon, accounting for about 5–7% of all renal tumors and about 5% of all urothelial tumors. Etiology One of the genetic changes that must occur for malignant transformation is the induction of oncogene. Oncogenes associated with TCC include those of the RAS gene family, including ▶ P21 RAS oncogene, and up to 50% of TCCs have been claimed to have RAS mutations. Another important molecular mechanism in the process of carcinogenesis is the inactivation of tumor suppressor genes. These include that of P53, the most frequently altered gene in human cancers, the retinoblastoma (RB) gene (▶ Retinoblastoma Protein, Biological and Clinical Functions), and genes on chromosome 9. Overexpression of normal genes including those for EGF receptor (ERBB1) and ERBB2 (epidermal growth factor receptor ligands) occurs in most TCCs. Cigarette smokers have a fourfold higher incidence of TCC than do people who have never smoked (▶ tobacco-related cancers). Ex-smokers have a reduced incidence of TCC, but the reduction of this risk down to baseline takes nearly 20 years. Nitrosamines, 2-naphthylamine, and 4aminobiphenyl are suggested as being responsible for TCC in cigarette smoke. Women treated with radiation for carcinoma of the uterine cervix or ovary have a two- to fourfold increased risk of developing bladder cancer. Patients treated with cyclophosphamide have up to a ninefold increased risk of developing bladder cancer. Signs and Symptoms Microscopic or gross hematuria is the most common presenting symptom. Patients with gross hematuria have reported rates of bladder cancer of 13–35%. With microscopic hematuria, the rates decreased to 0.5–10.5%. So, if a patient has unexplained hematuria, either microscopic or gross, cystoscopic examination is usually warranted, especially in individuals older than age 60 or younger people with a smoking history. The second most common presentation is the constellation of lower urinary tract irritative

Transitional Cell Carcinoma

symptoms such as urinary frequency, urgency, and dysuria. These irritative symptoms usually occur with hematuria. In fact, the risk of TCC may be doubled in patients with irritative voiding symptoms that coexist with hematuria. Evaluations In all patients with signs and symptoms suggestive of bladder cancer, excretory urography (IVU) is indicated. It is useful in examining the upper urinary tracts for associated urothelial tumors. Large bladder tumors may appear as filling defects in the bladder, but small ones may not be detected. Computed tomography (CT) has replaced IVU in the evaluation of hematuria. After imaging studies, all patients suspected of having bladder cancer should have cystoscopy. Retrograde pyelography should be done if the upper tracts are not visualized on IVU or CT. CT can help to assess the extent of the primary tumor and provides information about the presence of pelvic and para-aortic lymphadenopathy and visceral metastases. But CT fails to detect nodal metastases in up to 40–70% of patients who have them. MRI is not much more helpful than CT. Pelvic lymphadenectomy, which can be done with cystectomy, is the most accurate means of determining regional node involvement. The primary regions of lymphatic drainage of the bladder are the perivesical, hypogastric, obturator, external iliac, and presacral nodes. As some patients with limited nodal metastases can be benefited by lymphadenectomy, bilateral node dissection should be done. The usually recommended metastatic evaluation for invasive bladder cancer includes a chest radiograph, abdominal-pelvic CT, bone scan, and liver function tests. A flexible cystoscope is often used for the initial diagnosis and follow-up of patients with bladder tumors. It has much less discomfort than a rigid cystoscope. Bugbee electrode devices can be inserted through a flexible cystoscope to allow destruction of small, noninvasive papillary tumors. Malignant urothelial cells have large nuclei with irregular, coarsely textured chromatin and can be observed on microscopic examination of

Transitional Cell Carcinoma

the urinary sediment. This microscopic cytology is more sensitive in patients with high-grade tumors or carcinoma in situ (CIS). The specificity and positive predictive value of cytology are quite high. Staging (1997 AJCC-UICC, TNM Staging) As tumor stage forms the foundation for determining therapy, accurate staging is critical. The first treatment decision based on tumor stage is the presence or absence of muscle invasion. Because metastases are very rare with a superficial (non-muscle-invasive) tumor, treatment strategy can be grouped into superficial (Ta, T1, and Tis), muscle-invasive, and metastatic tumors. About 70% of bladder tumors are superficial at presentation. Of these, 70% present as stage Ta, 20% as T1, and 10% as CIS. Ta Tis T1 T2a T2b T3a T3b T4a T4b N0 N1 N2 N3 M0 M1

Papillary, epithelium confined Flat carcinoma in situ Lamina propria invasion Superficial muscularis propria invasion Deep muscularis propria invasion Microscopic extension into perivesical fat Macroscopic extension into perivesical fat Cancer invading pelvic viscera Extension to pelvic sidewalls, abdominal walls, or bony pelvis No histological pelvic node metastasis Single positive node 2 cm in diameter, below common iliacs Single positive node 2–5 cm in greatest diameter or multiple positive nodes Positive nodes >5 cm in diameter No distant metastases Distant metastases documented

Treatment of Superficial Bladder Cancer Local resection of a bladder tumor usually enables complete removal of the tumor and provides diagnostic information about the depth and the grade of the tumor. For this, first the bulk of the tumor and then the deep portion with some underlying bladder muscle should be resected. To detect dysplasia or CIS elsewhere in the bladder, selected site mucosal biopsies from areas adjacent to the tumor, bladder dome, trigone, and prostatic urethra have been recommended.

4639

If 5-aminolevulinic acid (ALA) is administered into the bladder in conjunction with fluorescent cystoscopy, lesions invisible with normal cystoscopy can be detected. The most important issue in tumor biology of superficial tumors is recurrence and progression to higher stages. Low-grade Ta tumors recur at a rate of 50–70% and progress in about 5%. High-grade T1 lesions recur in 80% and progress in 50% of patients. The most important risk factor for progression in superficial bladder tumors is grade, not stage. Prognosis also correlates with the presence of CIS, tumor size, multiplicity, and lymphovascular invasion and the configuration of the tumor (papillary vs. sessile). Of patients with CIS, 40–83% will develop muscle invasion if untreated. For T1 tumors, the depth of lamina propria invasion determined by the muscularis mucosa invasion or the extent of invasion below the urothelial surface has been known to be correlated with prognosis. CT and MRI appear to be inaccurate in determining the microscopic muscle infiltration and the minimal extravesical spread, which can also be aggravated by post-tumor recurrence (TUR) changes. To prevent recurrence and progression of bladder tumors, intravesical immunotherapy using BCG (Bacillus Calmette-Guerin) has been used. Treatments are generally begun 2–4 weeks after TUR and a 6-week course is usually administered. With BCG, tumor recurrence was reduced by 20–65% and progression was reduced by 23–27%. Intravesical chemotherapy using ▶ mitomycin C, doxorubicin, thiotepa, epirubicin, and ▶ gemcitabine also has been administered. Treatment of Invasive Bladder Cancer The standard surgical approaches to muscleinvasive bladder cancer are radical cystoprostatectomy in the male patient and anterior exenteration in the female patient, with bilateral pelvic lymphadenectomy. Anterior exenteration in the female requires removal of the uterus, fallopian tubes, ovaries, bladder, urethra, and a segment of the anterior vaginal wall. A nervesparing modification has been proposed in the

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male patient and results in improved postoperative return of erectile function. The more prevalent the orthotopic reconstruction becomes, the stricter indications for urethrectomy have been applied. The most significant factor for the anterior urethral recurrence and local/distant failure in the male patient has been identified as prostatic urethral involvement. The estimated 5-year probability of urethral recurrence is 5% without any prostate involvement and 12–18% with prostate involvement. CIS of the bladder neck and trigone was also significantly associated with prostatic urethral involvement. In the female patient, overt cancer at the bladder neck and urethra, diffuse CIS, or positive margin at surgery should be treated by en bloc urethrectomy as a part of the radical cystectomy. The mortality rate for radical cystectomy is 1–2% and the overall complication rate is about 25%. After urinary tract diversion, bowel obstruction rate is 4–10%. Stricture of anastomosis between ureter and bowel is found in less than 3%. Depending on the type of neobladder, metabolic disorders, vitamin deficiency, and urinary tract infection can occur. As for the neoadjuvant chemotherapy, results suggested improvement in overall survival of 5–6% among patients with locally advanced disease (stages T3–T4a). Some reports suggest that for patients with locally advanced disease and lymph node involvement, adjuvant chemotherapy may also provide a survival advantage. Due to the small numbers of patients, these results are as yet insufficient for the routine use of adjuvant therapy. Treatment of Metastatic Bladder Cancer These patients are routinely treated with systemic chemotherapy. The most commonly used agents are methotrexate, vinblastine, doxorubicin, and ▶ cisplatin (MVAC). MVAC chemotherapy produces a complete response in about 20% of patients, although long-term disease-free survival is rare. The combination of cisplatin and a newer agent, ▶ gemcitabine (GC), has produced similar survival outcomes with less toxicity compared with MVAC. ▶ Paclitaxel and ▶ docetaxel have also been used in clinical trials and demonstrate response rates of 25–83%.

Transitional Cell Carcinoma of Bladder

Cross-References ▶ Urothelial Carcinoma, Clinical Oncology

References Herr HW, Dotan Z, Donat SM et al (2007) Defining optimal therapy for muscle invasive bladder cancer. J Urol 177:437–443 Messing EM (2007) Urothelial tumors of the bladder. In: Wein AJ, Kavoussi LR, Novick AC, Partin AW, Peters CA (eds) Campbell-Walsh urology, 9th edn. SaundersElsevier, Philadelphia, pp 2407–2446 Sengupta S, Blute ML (2006) The management of superficial transitional cell carcinoma of the bladder. Urology 67:48–54 Sternberg CN, Donat SM, Bellmunt J et al (2007) Chemotherapy for bladder cancer: treatment guidelines for neoadjuvant chemotherapy, bladder preservation, adjuvant chemotherapy, and metastatic cancer. Urology 69:62–79

Transitional Cell Carcinoma of Bladder ▶ Transitional Cell Carcinoma

Transitional Cell Carcinoma of Renal Pelvis ▶ Transitional Cell Carcinoma

Transitional Cell Carcinoma of the Urinary Bladder ▶ Urothelial Carcinoma

Transitional Cell Carcinoma of Ureter ▶ Transitional Cell Carcinoma

Translesion DNA Polymerases

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Pol K

Translesion DNA Polymerases

Pol I

W. Glenn McGregor University of Louisville, Louisville, KY, USA

REV1 3⬘

6 18

TT

REV7 Pol ␨ REV3

Definition Translesion DNA synthesis (TLS) is a highly conserved mechanism for the completion of replication of damaged genomes. Analogous pathways exist in bacteria, and homologs with remarkable similarity exist in all eukaryotic cells, including postmitotic organisms such as D. melanogaster. Advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements and can insert bases across from adducted templates, but with potentially mutagenic consequences.

Characteristics Most mutations induced by genotoxic carcinogens occur when a DNA template that contains residual (unrepaired) damage is replicated during S-phase of the cell cycle. Presumably, the replication complex is blocked by bulky adducts in the DNA such as those induced by ultraviolet light (UV) or a variety of chemical carcinogens (▶ Adducts to DNA). As diagrammed in Fig. 1, advances indicate that error-prone translesion synthesis (TLS) is responsible for the majority of base substitutions induced in the DNA. TLS is defined as the incorporation of a nucleotide across from DNA damage followed by extension of the potentially mispaired primer-template. This process is undertaken by at least five accessory DNA polymerases, several of which have been purified and studied in vitro (▶ DNA damage responses). The properties of these polymerases have been extensively reviewed. Based on structural homology, these

Pol ␩ XPV/hRAD30A 5⬘

Pol ␦ TT GA

Translesion DNA Polymerases, Fig. 1 Model for translesion replication. The replicative polymerase complex stalls at sites of helical distortion induced by DNA damage, such as UV-induced photoproducts. The presumed ubiquitin ligase RAD18 targets a ubiquitinconjugating enzyme, RAD6, to the site of damage. There are two closely related homologs of RAD6 in higher eukaryotic cells, termed RAD6A and RAD6B. One of the targets of ubiquitination appears to be PCNA, which signals accessory polymerases in ways that are not fully understood, although at least one TLS polymerase, pol Z, has a higher affinity for monoubiquitinated PCNA. Current thinking is that one of the Y-family polymerases (pol Z, pol ι, or pol k) may insert a base directly across from the lesion, but pol z is required to extend the resulting primer such that the pol d can continue processive DNA replication. REV1 is required for mutagenesis, but this role is probably separate from its dCMP transferase activity (data indicate that REV1 may tether pol z to the other accessory polymerases)

polymerases fit into one of two families: the Y-family (REV1, pol Z, ι, and k) or the B-family (pol z). The cellular roles of this universe of polymerases are not known. In particular, the extent to which each of these polymerases participates in TLS most likely depends on the structure of a particular adduct and on the sequence context. As shown in Fig. 1, it has been suggested that pol Z, ι, and/or k inserts a base directly across from a lesion and that pol z extends the mispair to form a template-primer that can be extended by pol d. Although REV1 is a DNA polymerase, its role in mutagenesis is thought to be structural rather than catalytic. The unrestrained activity of error-prone polymerases would lead to widespread mutagenesis and genomic instability, so there are signaling mechanisms that tightly control polymerase

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Translesion DNA Polymerases

PCNA

MMS2Ubc13; Rad5

Rad6 Rad18

K-63 Damage avoidance

K-164-Ubc Translesion synthesis

PCNA

PCNA Ubc9 siz1

K-164-SUMO PCNA

RAD52dependent recombination

Translesion DNA Polymerases, Fig. 2 Regulation of lesion bypass in the budding yeast Saccharomyces cerevisiae is thought to be signaled by modification of PCNA at lysine 164 (K-164). The presence of a stalled DNA replication fork recruits Rad18, which is a presumed ubiquitin ligase, and Rad6, a ubiquitin conjugase, to the site of the replication-blocking lesion. Monoubiquitination at K-164 leads to recruitment of TLS polymerases with potentially mutagenic consequences. Polyubiquitination at lysine 63 (K-63) of ubiquitin by MMS2-Ubc13 leads to damage avoidance. A proposed mechanism for damage

avoidance is uncoupling of the replication fork, such that the undamaged strand is replicated for some distance beyond the blocked replication complex. The nascent strand, which has the same sequence as the damaged strand, then acts as a template for replication. The damage is thereby avoided in an error-free manner. A competing reaction is sumoylation at K-164 by the SUMO (small ubiquitin modifier)-specific ligase Siz1 and conjugase Ubc9. This reaction is thought to suppress Rad52dependent recombination and damage-induced genomic instability

switching events. Although not fully understood, the mechanisms used by cells to accomplish polymerase switching events at blocked primer termini have been studied most intensively in the budding yeast, Saccharomyces cerevisiae. In this organism, replication-blocking lesions in the template strand can be bypassed by proteins in the Rad6dependent DNA damage tolerance pathway. This process prevents the collapse of stalled replication forks, and replication of the damaged template is completed by TLS with potentially mutagenic consequences or by damage avoidance mechanisms mediated by recombination that are largely error-free. As diagrammed in Fig. 2, the ubiquitinconjugating enzyme encoded by Rad6 and the presumed ubiquitin ligase encoded by Rad18 are central to this process, since mutants cannot bypass replication-blocking lesions in the template and are sensitive to many DNA-damaging

agents. Insights into the biochemical function of this complex were gained when the Rad18/Rad6 complex was found to be responsible for the monoubiquitination of PCNA at K-164. PCNA modified in this fashion is thought to signal translesion synthesis and further ubiquitination is thought to signal damage avoidance (Fig. 2). Although the molecular details of the signaling pathways downstream of monoubiquitination are unknown, at least one TLS polymerase, pol Z, has been shown to have enhanced affinity for monoubiquitinated PCNA. The strategies used by yeast cells to complete the replication of damaged genomes appear to have been conserved in higher eukaryotes, but with additional layers of complexity. For example, higher eukaryotic cells have at least two Y-family polymerases that are not found in yeast and one of these (pol k) appears to be independent of RAD18. Human RAD18 was cloned and the

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protein was purified. It is a 56 kDa protein that shares 26% identity and 59% similarity with its yeast counterpart. The protein interacts with the two RAD6 homologs found in higher eukaryotes (RAD6A and RAD6B) with equal affinity and is ubiquitously expressed in all human tissues. Among the conserved regions are a RING finger motif found in the N-terminus that is required for the interaction with RAD6A/B and a zinc finger that is presumably required for interaction with DNA. In principle, the accessory DNA polymerases and associated proteins described herein represent potential targets for antimutagenesis strategies. However, deficiency of individual polymerases may result in enhanced carcinogenesis. The most well-studied example of this is the human syndrome xeroderma pigmentosum variant, which is a skin cancer-prone condition that results from an inherited deficiency of DNA polymerase Z. This enzyme is posited to be specialized for the error-free bypass of cyclobutane dimers between adjacent thymidine bases. In its absence, data indicate that polymerase iota assumes its function and is error-prone when doing so. Unexpectedly, however, when both polymerases are deficient in mouse models, UV-induced skin cancer is accelerated despite reduced UV-induced mutant frequencies in the double knockout. These data support a role for polymerase iota as a tumor suppressor separate from its role in TLS.

References Dumstorf CA, Clark AB, Lin Q et al (2006) Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer. Proc Natl Acad Sci U S A 103:18083–18088 Friedberg EC, Lehmann AR, Fuchs RP (2005) Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 18:499–505 Wang Y, Woodgate R, McManus TP et al (2007) Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. Cancer Res 67: 3018–3026

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Transmembrane 4 Superfamily Protein ▶ Metastasis Suppressor KAI1/CD82

Transmembrane Protease Serine 1–13: Hepsin (TMPRSS 1) ▶ Serine Proteases (Type II) Spanning the Plasma Membrane

Transplacental Carcinogenesis Mark Steven Miller Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC, USA

Synonyms Fetal exposure to carcinogenic agents; In utero exposure to carcinogenic agents

Definition Transplacental carcinogenesis is a subfield of cancer research that looks at the effects of exposure of the fetus to chemical and physical agents that may cause cancer. The best known example of a chemical administered during pregnancy that resulted in tumors in the children of the treated mothers is diethylstilbestrol (DES). Young women of mothers who were treated during pregnancy with DES exhibited an increased incidence of vaginal cancer (Miyagawa et al. 2011). The best known example of a physical agent that resulted in cancer in the offspring of exposed mothers is radiation. Studies from the survivors of the atomic bomb blasts in Japan showed that exposure of the fetus to radiation caused increased incidences of cancer at several organ sites (Pierce and Preston 2000).

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Characteristics Types of Transplacental Carcinogens Studies conducted in the field of transplacental carcinogenesis use a variety of experimental techniques to demonstrate the effects of different agents on the developing fetus. These include statistical analysis of incidences of cancer following exposure of pregnant women to chemical or physical carcinogens (such as the DES and atomic bomb studies discussed in the previous section), studies in animal models, and cell culture studies using embryonic cells. Transplacentally active chemical carcinogens can come from a number of different sources. These can include: 1. Pharmaceutical agents such as DES, which are administered for medical reasons but have unintended side effects. Because of concerns over the potential for new pharmaceutical agents to cause harm to the fetus, the Food and Drug Administration requires specific testing in animal models before the agency will allow any testing in people. Since this is often very expensive, many companies will not test new pharmaceutical candidates in younger age-groups, and many drugs are thus not approved for use during pregnancy in the United States. 2. Environmental contaminants. Many chemicals found in the environment are the result of industrial processes. Some of these are chemicals that have been manufactured for a specific use and later determined to be a threat to human health, such as pesticides. Other chemicals are industrial by-products that have leaked into the environment and can be found in water, air, or in the ground. Agents such as pesticides, cigarette smoke, and benzene, as well as a variety of other chemicals, have been linked to increased incidences of both childhood- and adult-onset cancers (Anderson et al. 2000; Alexander et al. 2001). 3. Diet. Many chemicals can be found in the diet as a result of exposure of fish, chicken, cattle, and food animals to environmentally

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prevalent chemicals. While it is unlikely that one can consume sufficient quantities of transplacental carcinogens from a normal diet to affect their developing fetus, the high levels of mercury found in certain fish would be an example where dietary moderation would be advisable. In addition, the safety of a variety of food supplements and nutriceuticals for consumption during pregnancy is not known. Mechanisms of Cancer Causation It is now known that many chemicals that the pregnant mother is exposed to have the potential to cross through the placental circulation into the developing fetus. A number of studies using carefully controlled animal experiments have demonstrated that the developing embryo and fetus exhibit very different sensitivity to many environmental chemical and physical agents than does an adult. Exposure during fetal development can thus pose significant risk to the developing organism at doses that would not affect an adult, including the pregnant mother. For example, treatment of pregnant mice with polycyclic aromatic hydrocarbons (chemicals that are found in cigarette smoke and diesel exhaust) causes a high incidence of lung tumors in the offspring at doses of the chemical that do not affect the pregnant mother (Miller 2004). On the other hand, with some environmental agents, the fetus may be less sensitive than the adult organism to the toxic effects of these agents (Rice 1979). A number of physiological mechanisms have been proposed to account for the differential sensitivity of the fetus to chemical and physical agents. It is likely that this differential sensitivity to cancer induction is the result of combinations of a number of different biological factors. The developing fetus is not a “little adult” and exhibits significant differences in the way they respond to chemical, physical, pharmaceutical, and dietary agents. In addition, the developing fetus is a remarkably adaptable organism, presenting a constantly changing picture of gene expression as a variety of gene systems are turned on and off at different stages of development. As such, the ability of the fetus to mount a defensive response to

Transplacental Carcinogenesis

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Transplacental Carcinogenesis, Fig. 1 Conversion of nonpolar benzene to either water soluble, sugar conjugated, inactive polar metabolites that are readily excreted in the urine and feces (Eqs. 1 and 3); or DNA-bound benzene adduct that can cause gene mutations and disease (Eqs. 1 and 2)

environmental insults can be markedly different at different stages of fetal development (Miller 2004; Shorey et al. 2011). Most living organisms respond to potentially toxic environmental chemicals by producing enzymes that can eliminate the environmental toxin. Many environmental chemicals are actually fairly inert. When they enter the body, these compounds tend to be poorly soluble in water (hence they are referred to as “nonpolar” compounds) and prefer to remain in fattytissue deposits. In order to get rid of these agents, the cells in our bodies increase the production of drug-metabolizing enzymes. Phase I enzymes are the first step in this process. These enzymes catalyze the conversion of the parent nonpolar compound to slightly more watersoluble compounds by adding oxygen to the parent compound. The presence of the oxygen molecule makes the parent compound more reactive, thus creating a target site for Phase II enzymes. These enzymes can then add very water-soluble molecules, such as sugar molecules, to the parent compound. This converts the nonpolar compounds to water-soluble compounds (also called polar compounds) that can be readily excreted in either the urine or feces, as shown in Eq. 1 and schematically in Fig. 1 using benzene metabolism as an example.

Equation 1 Phase I metabolism, the addition of oxygen to a nonpolar compound (R) RH þ O2 ! ROH þ H2 O The initial conversion step carried out by Phase I enzymes can also result in converting the original parent compound into a highly reactive molecule that can bind to DNA, cause gene mutations, and thus initiate the carcinogenic process. Equation 2 Binding of reactive molecules to DNA ROH þ DNA ! R  DNA þ H2 O ! genetic damage Phase II enzymes will bind to these toxic metabolites and aid in their elimination from the body before they can do any harm. Equation 3 Phase II metabolism, inactivation of toxic compounds ROH þ sugar ! R  sugar þ H2 O In the fetus, the levels of both Phase I and Phase II enzymes can differ drastically from

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Trask (Transmembrane and Associated with Src Kinases)

those found in the adult. The fetus lacks many of the enzymes that are normally expressed in adult tissues and thus contain only a subset of these enzymes. In addition, the levels at which these enzymes are expressed can differ by the gestational age of the fetus. In general, both Phase I and Phase II enzymes are poorly expressed during the first trimester, making the fetus at early stages of development less susceptible to chemicals that require enzymatic conversion to become toxic. However, once these enzyme systems start to become prevalent in fetal tissues, the Phase I enzymes become more responsive to chemical toxins than do the Phase II enzymes. This can result in a greater production of toxic metabolites and an enhanced sensitivity of the fetus to certain chemicals relative to the adult organism, which has a greater ability to carry out the second phase of metabolism and eliminate these chemicals (Miller 2004). In addition to drug metabolic enzymes, living organisms have other defensive mechanisms to prevent genetic damage. These include DNA repair enzymes which can fix the damage caused by chemical binding to DNA as shown in Eq. 2 and Fig. 1. Because the fetus is constantly growing and developing, fetal tissue cells exhibit a high rate of cell proliferation. As a result, when genetic damage occurs as a result of exposure to chemical or physical carcinogenic agents, the cells may synthesize new DNA before the cell has time to repair the damage. In this case, the bound chemical will cause gene mutations that are then genetically inherited and passed on to new cells. Although the levels of DNA repair enzymes differ between fetal and adult organisms, their role in the relative protection of fetal DNA is still uncertain. Scientists are continuing to identify new compounds that can cause cancer following in utero exposures, the mechanisms by which transplacental carcinogens exert their effects, and are also looking for preventive measures that can be taken to protect the fetus from known environmental exposures. In addition to regulating environmental release of

potentially harmful chemical and physical transplacental carcinogens, one promising area is chemoprevention. This would allow a pregnant woman to take a relatively harmless drug that would protect her fetus from environmental toxins. As our understanding of transplacental carcinogens increases, new strategies will be developed to protect this vulnerable population.

References Alexander FE, Patheal SL, Biondi A, Brandalise S, Cabrera ME, Chan LC, Chen Z, Cimino G, Cordoba JC, Gu LJ, Hussein H, Ishii E, Kamel AM, Labra S, Magalhaes IQ, Mizutani S, Petridou E, de Oliveira MP, Yuen P, Wiemels JL, Greaves MF (2001) Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion. Cancer Res 61:2542–2546 Anderson LM, Diwan BA, Fear NT, Roman E (2000) Critical windows of exposure for children’s health: cancer in human epidemiological studies and neoplasms in experimental animal models. Environ Health Perspect 108(Suppl 3):573–594 Miller MS (2004) Transplacental lung carcinogenesis: molecular mechanisms and pathogenesis. Toxicol Appl Pharmacol 198:95–110 Miyagawa S, Sato M, Iguchi T (2011) Molecular mechanisms of induction of persistent changes by estrogenic chemicals on female reproductive tracts and external genitalia. J Steroid Biochem Mol Biol 127:51–57 Pierce DA, Preston DL (2000) Radiation-related cancer risks at low doses among atomic bomb survivors. Radiat Res 154:178–186 Rice JM (1979) Perinatal period and pregnancy: intervals of high risk for chemical carcinogens. Environ Health Perspect 29:23–27 Shorey LE, Castro DJ, Baird WM, Siddens LK, Lohr CV, Matzke MM, Waters KM, Corley RA, Williams DE (2011) Transplacental carcinogenesis with dibenzo [def,p]chrysene (DBC): timing of maternal exposures determines target tissue response in offspring. Cancer Lett

Trask (Transmembrane and Associated with Src Kinases) ▶ CDCP1

Trastuzumab

Trastuzumab Wen Jin Wu and Milos Dokmanovic Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA

Synonyms Anti-c-erB-2; Anti-c-erbB2 monoclonal antibody; Anti-ERB-2; Anti-erbB-2; Anti-erbB2 monoclonal antibody; Anti-HER2/c-erbB2 monoclonal antibody; Anti-p185-HER2; MOAB HER2; Monoclonal antibody c-erb-2; Monoclonal antibody HER2; rhuMabHER2

Definition Trastuzumab (US brand name: Herceptin ®) is a humanized monoclonal antibody IgG1 directed against the human epidermal growth factor receptor 2 (HER2) and is approved for the treatment of HER2-positive breast cancer and HER2-positive metastatic adenocarcinomas of the stomach or gastroesophageal junction (GEJ).

Characteristics Antibody Antibodies, also known as immunoglobulins, are large protein molecules produced by the body’s immune system in response to antigenic stimuli that are either infectious agents, such as a bacteria, fungi, viruses, or parasites, or other molecular components recognized by the body’s immune system as foreign. Antibodies consist of two

The entry “Trastuzumab” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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identical pairs of polypeptide chains, comprised of a heavy chain and a light chain. The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, or IgM). These polypeptide chains are arranged in a large Y-shaped protein. Both heavy and light chains have constant and variable regions. Variable regions are contained within the amino (NH2) terminus of the polypeptide chain where complementarity determining regions (CDRs) are found. CDRs serve to recognize and bind specifically to antigen. Monoclonal antibodies are antibody molecules with identical amino acid sequences expressed by a single clone of cells. Molecular Target of Trastuzumab HER2 (also known as neu and ErbB2) is encoded by ERBB2/neu gene and is a member of the HER family of receptor tyrosine kinases. HER family receptors are composed of four type I receptors: EGFR/HER1/ErbB1, HER2/ErbB2, HER3/ ErbB3, and HER4/ErbB4. All receptors share a similar structure composed of an extracellular ligand-binding region, a single transmembrane lipophilic segment, and a cytoplasmic tyrosine kinase-containing domain. The extracellular ligand-binding region of HER family receptors is composed of four domains (I–IV). Domains I and III are important for ligand binding. Domain II mediates receptor dimerization. Domain IV forms intramolecular interactions with domain II and thus blocks dimerization. Ligand binding to the extracellular domain of HER family members disrupts the autoinhibition conformation. This results in receptor homo- or heterodimerization and transphosphorylation followed by the activation of the downstream signaling pathways. A ligand for HER2 has not been identified yet. However, the HER2 extracellular domain adopts a fixed conformation that resembles a ligand-activated state that permits it to form a dimer in the absence of a ligand. This conformation of HER2 probably explains why HER2 is the preferred dimerization partner for the other HER family members. Moreover, although none of the ligands for the HER family receptors directly binds to HER2, activation of EGFR, HER3, or HER4 by their ligands

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can facilitate transactivation of HER2 through ligand-induced heterodimerization. Overexpression of HER2 as a result of amplification of the HER2 gene (ERBB2/neu) appears to mediate the initiation, progression, and metastasis of many types of human cancer, including breast and gastric cancers. HER2 is overexpressed in approximately 20–25% of breast cancers and is associated with poor disease-free survival and poor response to chemotherapy. Gene amplification is the most common mechanism resulting in HER2 overexpression in breast cancer. HER2positive expression was also observed in 22.1% metastatic gastric or gastroesophageal junction (GEJ) patients. Murine Anti-HER2 Monoclonal Antibody (mAb) 4D5 The murine anti-HER2 monoclonal antibody, 4D5, was shown to recognize human HER2 and to suppress the growth of HER2-overexpressing tumor cells, as well as to enhance the sensitivity of tumor cell killing by the host immune system. Further studies demonstrated that radiolabeled 4D5 localized to HER2-overexpressing tumors in patients. In order to make a therapeutic monoclonal antibody that would maintain the half-life and effector functions of human antibodies and not be recognized as foreign by a patient’s immune system, 4D5 was humanized by engineering the 4D5 CDR heavy and light chain sequences into the framework of a consensus human monoclonal antibody IgG1 isotype. The humanized version of 4D5 (also known as rhuMabHER2; later named trastuzumab) alone or in combination with other chemotherapy agents showed significant inhibitory effects in HER2-overexpressing breast cancer cells and in mouse xenograft models of HER2-overexpressing breast cancer. Clinical Indications For the past 20 years, the development of monoclonal antibodies targeting HER family receptor tyrosine kinases has been intensely pursued as an important cancer therapeutic strategy. Based on results from a phase III investigational clinical trial of trastuzumab, which showed that trastuzumab in combination with chemotherapy increased time to

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disease progression and response rates compared to chemotherapy alone, trastuzumab received FDA approval in September 1998 for use in women with HER2-overexpressing metastatic breast cancer. Trastuzumab is indicated for treatment of patients both as first-line therapy in combination with paclitaxel chemotherapy and as a single agent for those who have received one or more chemotherapy regimens. Trastuzumab was the first HER2 targeted treatment for metastatic breast cancer. Dako’s HercepTest™, which is a semi-quantitative immunohistochemical (IHC) assay for determination of HER2 protein overexpression in breast cancer tissues, was approved simultaneously to aid in the identification of patients eligible for trastuzumab treatment. On November 16, 2006, the FDA granted approval to trastuzumab as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of women with early-stage HER2-positive and node-positive breast cancer. This approval was based on evidence of a significant prolongation in disease-free survival in women receiving trastuzumab and chemotherapy compared to those receiving chemotherapy alone. On October 20, 2010, the FDA granted approval for trastuzumab in combination with cisplatin and a fluoropyrimidine (either capecitabine or 5-fluorouracil) for the treatment of patients with HER2-overexpressing metastatic gastric or GEJ adenocarcinoma who have not received prior treatment for metastatic disease. This approval is based on results of a single international multicenter openlabel randomized clinical trial BO18255 (ToGA trial), which enrolled 594 patients with locally advanced or metastatic HER2-overexpressing adenocarcinoma of the stomach or GEJ. HER2 Status Trastuzumab is used for the treatment of breast or gastric/GEJ cancers where HER2 is overexpressed. Two testing methodologies can be used clinically to determine the HER2 status of tumor samples: immunocytochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC detects the level of HER2 protein in cancer samples, whereas FISH detects the level of HER2 gene amplification. The American Society of

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Clinical Oncology/College of American Pathologists guideline recommends the use of both IHC and FISH testing to determine the HER2 status of human breast cancer. According to this guideline, a positive HER2 result is the IHC staining of 3+ and a FISH result of more than six HER2 gene copies per nucleus or a FISH ratio [HER2/chromosome 17 (CEP17)] of more than 2.2. In the ToGA trial, patients were eligible for trastuzumab treatment if their tumor samples were scored as 3+ on ICH or if they were FISH positive (HER2:CEP17 ratio 2). HER2 heterogeneity in gastric/GEJ tissue is greater than in breast cancer tissue, which may cause discordance between FISH and IHC. Mechanisms of Action of Trastuzumab Trastuzumab directly binds to the extracellular domain IV of HER2 to mediate an inhibitory effect on cancer cells. While the mechanisms by which trastuzumab induces regression of HER2positive breast cancers are still being investigated, it is currently believed that the binding of trastuzumab to HER2 contributes to its therapeutic effect either by direct modulation of proliferative and pro-survival signaling downstream of HER2 or by its effect on angiogenesis and immune cell recruitment. Binding of trastuzumab to the extracellular domain of HER2 directly inhibits HER2 signaling by (a) prevention of the cleavage of HER2 extracellular domain by metalloproteinase ADAM10, (b) inhibition of either HER2 homodimerization or heterodimerization. and (c) induction of HER2 endocytosis followed by receptor degradation. Taken together, binding of trastuzumab to HER2 leads to the inhibition of pro-survival and proliferative pathways, such as the phosphatidylinositol 3kinase (PI3K) pathway, mitogen-activated protein kinase (MAPK) pathway, and cell cycle progression (Fig. 1). Trastuzumab has also been shown to inhibit tumor angiogenesis, resulting in the decreased microvessel density of tumor in vivo and reduced endothelial cell migration in vitro. Trastuzumab has been demonstrated to kill tumor cells not only by its direct action on tumor cell signaling but also through antibodydependent cell-mediated cytotoxicity (ADCC). More specifically, trastuzumab is an IgG1 isotype

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and its constant region is capable of binding to Fc receptors presented on certain immune cells, which activates antibody effector functions. These immune cells release enzymes and factors that kill the tumor cells. Studies have demonstrated that when trastuzumab is present, immune cells preferentially target HER2-overexpressing cancer cells compared to cancer cells that do not overexpress HER2. Mechanisms of Trastuzumab Resistance Treatment with trastuzumab has significantly improved the outcome in women with HER2positive breast cancer. However, tumor resistance to trastuzumab poses a significant hurdle in breast cancer therapy. Clinical data has shown that approximately two thirds of HER2-positive metastatic breast cancer patients demonstrated primary resistance to single-agent trastuzumab and that the majority of patients with HER2-positive breast cancer who achieve an initial response to trastuzumab acquire resistance within 1 year. While the mechanisms of trastuzumab resistance are still being investigated, the following are proposed mechanisms based on clinical and preclinical studies: (1) overexpression of membraneassociated glycoprotein mucin-4 (MUC4), which may mask the epitope of HER2 recognized by trastuzumab; (2) formation of homodimers and heterodimers among EGFR, HER2, and HER3 due to overexpression of HER family ligands, which interferes with trastuzumab-mediated growth inhibition; (3) upregulation of Rac1, a member of Rho family small GTPases, which may impair trastuzumab-induced HER2 endocytic downregulation; (4) increased heterodimerization between HER2 and IGF-1R, which may interfere with trastuzumab-mediated induction of the cell cycle progression inhibitor, cyclindependent kinase p27 (kip1); (5) activation of PI3K either by constitutive activation, such as PIK3CA mutant, or by loss of PTEN which results in activation of PI3K signaling; (6) cyclin E amplification/overexpression; (7) increased cleavage of HER2 extracellular domain, which results in the formation p95HER2, a hyperactive membrane anchored fragment that drives breast cancer progression in vivo; and (8) loss of HER2

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Trastuzumab, Fig. 1 Signal transduction by the HER family and potential mechanisms of action of trastuzumab (Reproduced from Hudis (2007)). As shown in Panel A, the four members of the HER family are HER1, HER2, HER3, and HER4. There are receptor-specific ligands for HER1, HER3, and HER4. An intracellular tyrosine kinase domain exists for HER1, HER2, and HER4. Phosphorylation of the tyrosine kinase domain by means of

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homodimerization or heterodimerization induces both cell proliferation and survival signaling. HER2 is the preferred dimerization partner for the other HER family members. The phosphorylated (activated) tyrosine residues on the intracellular domain of HER2 activate the lipid kinase phosphoinositide 3-kinase (PI3-K), which phosphorylates a phosphatidylinositol that in turn binds and phosphorylates the enzyme Akt, driving cell survival. In parallel,

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expression in HER2-overexpressing breast cancer cells. Trastuzumab-Induced Cardiotoxicity HER2 signaling in the heart is essential for cardiac development and function, as well as for the prevention of dilated cardiomyopathy. Deletion of HER2 gene in a mouse model has been shown to result in early death of the animal. In the adult heart, HER2 may continue to have an important function in modifying the cardiac response to stress. It is possible that trastuzumab treatment results in a loss of HER2-mediated signaling in cardiomyocytes and that this interferes with the heart’s ability to respond to stress. Molecular mechanisms by which trastuzumab induces cardiac dysfunction still remain elusive. Trastuzumab cardiotoxicity in the metastatic clinical trial: Cardiotoxicity was initially reported in a phase III trial, which tested the efficacy of combining chemotherapy with trastuzumab versus chemotherapy alone in metastatic breast cancer disease. This study found that the combination of anthracyclines and cyclophosphamide (AC) alone was associated with a rate of overall cardiac dysfunction (CD) of 8% and a New York Heart Association (NYHA) class III/IV rate of 4%. However, when trastuzumab was added to AC, the overall CD rate was 27% with a rate of NYHA class III/IVCHF of 16%. Paclitaxel alone was associated with a CD rate of 1% and a NYHA class III/IV rate of 1%. The addition of trastuzumab to paclitaxel resulted in a CD rate of 13% with a NYHA class III/IV rate of 2%. Trastuzumab associated CD is manifested as severe congestive heart failure

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(CHF) and a significant decrease in left ventricular ejection fraction (LVEF). Trastuzumab cardiotoxicity in the adjuvant trials: Adjuvant trastuzumab prolonged survival, including both overall survival (OS) and diseasefree survival (DFS), among women with HER2positive early breast cancer, but the benefits were accompanied by the risks of cardiac toxicity. Based on the assessment of several major adjuvant trastuzumab clinical trials, including the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31, North Central Cancer Treatment Group N9831, Herceptin Adjuvant, Breast Cancer International Research Group 006, and Finland Herceptin trials, up to 4% of patients experienced severe CHF during treatment. However, a large number of patients on these trials experienced some form of cardiotoxicity that ultimately required discontinuation of trastuzumab. Approximately 14% of patients in the NSABP B-31 trial were reported to discontinue trastuzumab treatment due to asymptomatic decrease in LVEF. A systematic review of eight clinical trials, which involved 11,991 women with HER2-positive operable breast cancer who were treated with trastuzumab or the standard therapy (with no trastuzumab), found that breast cancer mortality was reduced by one third, but the risk of cardiac toxicity (i.e., CHF and LVEF) was five times more likely for women receiving trastuzumab than women receiving standard therapy alone. The incidence of cardiac toxicities may be associated with duration of trastuzumab administration, such that the longer treatment (1 year) may involve a greater

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ä Trastuzumab, Fig. 1 (continued) a guanine nucleotide exchange factor, the mammalian homologue of the son of sevenless (SOS), activates the rat sarcoma (RAS) enzyme that, in turn, activates receptor activation factor (RAF) and then the mitogen-activated protein kinase (MAPK) and mitogen extracellular signal kinase (MEK). MEK phosphorylates, among others, the MAPK, driving cellular proliferation. One of many other downstream effects of HER2 signaling is the production of vascular endothelial growth factor (VEGF) supporting angiogenesis. The most well-documented potential mechanisms of action are shown in Panels C through F. Cleavage of the extracellular domain of HER2 leaves a membrane-bound

phosphorylated p95, which can activate signal transduction pathways (Panel C). Binding of trastuzumab to a juxtamembrane domain of HER2 reduces shedding of the extracellular domain, thereby reducing p95 (Panel C). Trastuzumab may reduce HER2 signaling by physically inhibiting either homodimerization, as shown, or heterodimerization (Panel D). Trastuzumab may recruit Fc-competent immune effector cells and the other components of antibody-dependent cell-mediated cytotoxicity, leading to tumor cell death (Panel E). Additional mechanisms such as receptor downregulation through endocytosis have been postulated (Panel F)

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risk of severe heart toxicities than shorter treatment (6 months or less). Trastuzumab-associated cardiotoxicity is believed to be reversible upon stopping the treatment. However, the concept that trastuzumabrelated cardiotoxicity is reversible has been challenged due to the lack of sufficient long-term follow-up cardiac data. Additionally, in NSABP B-31trial among those diagnosed with a cardiac event, two thirds of patients continued to receive cardiac medications and 71% had a decrease in LVEF relative to baseline on follow-up, suggesting persistent cardiac dysfunction. Disclaimer The information presented in this entry represents publically available information. Any opinions expressed reflect the views of the authors and do not represent the policy of the US Food and Drug Administration.

Cross-References ▶ Drug Design ▶ Herceptin

Treatment-Refractory Germ Cell Tumors

Trefoil Factors Christian Gespach Laboratory of Molecular and Clinical Oncology of Solid tumors, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, France INSERM U. 673, Paris, France

Synonyms Spasmolytic polypeptide SP/TFF2; Trefoil peptides pS2/TFF1

Keywords Angiogenesis; Epithelial tumors; Gastric ulcers; Inflammation; Intestinal trefoil factor (ITF/TFF3); Invasion; Metastasis; Migration; Mucosal protection and regeneration

Definition References Dokmanovic M, Wu WJ (2011) Trastuzumab: resistance and breast cancer. In: Gunduz M, Gunduz E (eds) Breast cancer: carcinogenesis, cell growth and signaling pathways. InTech, Rijeka, pp 171–204 Hudis CA (2007) Trastuzumab: mechanism of action and use in clinical practice. N Engl J Med 357:39–51 http://www.cancer.gov/cancertopics/druginfo/trastuzumab Moja L, Tagliabue L, Balduzzi S, Parmelli E, Pistotti V, Guarneri V, D’Amico R (2012) Trastuzumab containing regimens for early breast cancer. Cochrane Database Syst Rev 4:CD006243 Telli ML, Hunt SA, Carlson RW, Guardino AE (2007) Trastuzumab-related cardiotoxicity: calling into question the concept of reversibility. J Clin Oncol 25: 3525–3533

Treatment-Refractory Germ Cell Tumors ▶ Platinum-Refractory Testicular Germ Cell Tumors

Trefoil factors (TFF) belong to a family of heat, acid, and protease-resistant regulatory peptides ubiquitously expressed in the brain, blood, and peripheral organs. In inflammatory conditions and generation of cancer lesions, they are induced, lost, or modified by gene silencing and somatic mutations. Thus, TFF overexpression or invalidation is either the consequence or the causal origin of human solid tumors and their ▶ progression to metastatic situations. While the TFF receptors or recognition systems are not still clearly identified, TFF are involved in mucosal and epithelial cell cytoprotection, wound healing, cancer cell survival and ▶ invasion, and ▶ angiogenesis, through several oncogenic pathways involved in neoplasia. Finally, TFF are now considered as multifaceted factors with beneficial and pejorative functions on inflammatory and cancer diseases, according to their dual and divergent impacts at early and late stages of these pathological states.

Trefoil Factors

Characteristics TFF Discovery and Expression Since the discovery and molecular annotation of the trefoil factor pS2 (TFF1) in human breast cancer, much attention has been devoted on TFF1 and its structurally related protease and acid-resistant factors spasmolytic polypeptide (SP-TFF2) and intestinal trefoil factor (ITF-TFF3). These TFF contain either one (TFF1 and TFF3) or two (TFF2) trefoil domains delimited by three disulfide bridges. TFF are involved in the stabilization of the mucus layers secreted by mucosal epithelial cells. The three human trefoil genes are located in a cluster region of 55 kb on chromosome 21q22.3. A novel two trefoil domains Bm-TFF2 protein-activating platelet aggregation has been purified from the frog Bombina maxima skin secretions. TFF are widely expressed in the brain, the urogenital system (breast, kidney, prostate), the lymphoid tissue, the respiratory and the digestive tract (esophagus, stomach, intestine, exocrine and endocrine pancreas, and liver), and in conjunctival goblet cells and pterygium. TFF1 and TFF2 are predominantly detected in the normal stomach, whereas TFF3 is found mainly in the small and large intestine. TFF are regulated via genetic, ▶ epigenetic, and tissue-specific mechanism including amplification of the chromosomal region 21q22 harboring TFF family genes in ▶ cholangiocarcinoma, promoter methylation, chromatin modification, histone H3 acetylation, and transcription factor downstream signaling pathways involved in cellular ▶ stress responses, ▶ inflammation, and cancer. These pathways include gastrin and bFGF growth factors, the ▶ interleukin 6 family cytokine receptor gp130/ STAT1–3 and SHP-2/ERK cascades, ras, the ▶ hypoxia-induced HIF-1a transcription factor, allergens in the lungs, nuclear ▶ estrogen receptors and GEFs, NF-kB, peroxisome proliferatoractivated receptor gamma (PPAR-g), hepatocyte nuclear factor 3 (HNF3), the homeodomain transcription factor CDX2, and the activator protein ▶ AP-1 via the negative control of COBRA1, the cofactor of BRCA1 (breast cancer-associated protein 1) involved in DNA damage repair.

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TFF are secreted by the gastrointestinal mucosa in a mucus, inflammation, and ulcerassociated cell lineage (UACL)-dependent manner. Chronic inflammation and ulceration in the gastrointestinal tract is associated with the development of the reparative UACL from mucosal stem cells. UACL was originally described as pyloric metaplasia in the ileum, reflux esophagitis associated with Barrett esophagus, peptic ulcer in the stomach, and chronic cholecystitis. TFF display multifaceted roles in mucosal repair and during cancer progression. TFF in Mucosal Repair and Protection It is now well accepted that TFF are involved in maintenance and repair of the mucosal barrier, wound healing, and cytoprotection during hypoxia and transient inflammatory situations in experimental ulcerative colitis. Local administration of recombinant TFF and ectopic expression of TFF3 in cellular models and transgenic animals supported this general idea of a cytoprotective role for TFF in mucosal repair. Both epithelial and stromal cells contribute to wound healing and mucosal repair. Consistent with a signaling role of TFF in mucosal protection, the tetraspanin family member Vangl1 is involved in the migratory response to TFF3 through Ser/Thr phosphorylation in intestinal epithelial cells. In addition TFF3 improved intestinal crypt stem cells survival following combined radiation and ▶ chemotherapy in both wild-type and TFF3/ knockout transgenic mice. TFF in Chronic Inflammation and Cancer Progression Although some studies argue for a therapeutic potential of TFF in mucosal injury and wound healing, advances in the field support their adverse effects during chronic inflammation and cancer progression. Persistent inflammatory situations initiate several genetic, molecular, and cellular dysfunctions associated with tumor promotion and cancer progression. Notably, selfinduction and cross-talk between TFF at their regulatory sequences have been described as a molecular signature of chronic inflammatory situations and neoplasia.

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Trefoil Factors Adenoma - edenocarcinoma transitions: hypoxia and angiogenesis

Cin-loh tumors Normal mucosa

Premalignant ACF APC, Ki-ras, c-myc, src

Colon crypt

Msi tumors

MUC-5AC APC, COX-2 PAR-1, IGF p21, p16, cyclin D1

ADENOMA (FAP) Wnt-2, DCC SMAD-2/-4

TGFβ-RII, IGF-RII, ß-catenin/ TCF4 PTEN,E2F, BAX c-kit, EGF-R, Axin

ADK: Local Invasion, Metastasis

Invasion Angiogenesis Survival

TFF-1

Duke’s stages TP53 - MET - src A B C D

Target organs

hMSH2, hMLH1, hMSH6, hPMS1, hPMS2 Mutations, Hypo-hypermethylations (HNPCC)

Trefoil Factors, Fig. 1 Genetic and molecular alterations linked to the multistep progression of familial and sporadic human colorectal cancers

For example, TFF1 exerts divergent functions in the digestive mucosa. In the stomach, gastric TFF1-deficient mice develop antropyloric adenomas and carcinomas, suggesting that TFF1 is a candidate gastric-specific ▶ tumor suppressor gene to protect the mucosa against repetitive injury from digestive secretions, ulceration, and chronic inflammation induced by acid, proteases, and pathogens, such as ▶ Helicobacter pylori. Somatic mutations and loss of heterozygosity (LOH) of the TFF1 gene is observed in human gastric cancers, in association with TFF3 overexpression. In coherence with these observations, ▶ cyclooxygenase (COX)-2 was strongly induced in pyloric adenomas induced by genetic ablation of the TFF1 gene in mice. Similarly, COX-derived products are reported to exert beneficial roles in mucosal protection and wound healing, but deleterious functions during chronic inflammation and neoplasia. Conversely, in the normal human colon, TFF1 is absent but is induced at high levels in Crohn disease, colitis, and colorectal cancers. It is therefore likely that TFF1 is a cancer progression factor in the human colon, according to its aberrant expression and

transforming functions at the adenoma and carcinoma transitions (Fig. 1). Thus, TFF exert opposing functions, one counteracting transient inflammatory situations and the other linked to pejorative functions, in cooperation with other dominant genetic and molecular alterations during the neoplastic progression in the human colon. These include the cancer predisposition pathways controlled by Wnt/APC/b-catenin, TGF-b/ SMAD-4, ras, ▶ src, and deleted in colon cancer (DCC). Validation of this model can be explored in transgenic animals harboring selectively these oncogenic alterations, in cooperation with forced expression of TFF1 in intestinal stem cells, through intestinal promoters that are functional in this cellular compartment, such as the villin promoter (pVIL) and the carcinoembryonic antigen (pCEA) regulatory regions. The emergence of colorectal adenocarcinomas (ADK) is a complex multistep process linked to genomic and ▶ chromosomal instability (CIN) and LOH, ▶ microsatellite instability (MSI), DNA ▶ aneuploidy, and generalized deregulation of gene expression and signal transduction pathways. The first mechanism, which accounts for

Trefoil Factors

80% of sporadic cases, is connected with CIN and LOH targeting the tumor suppressors APC (5q), ▶ TP53 (17p), DCC (18q), and TGFb pathway signaling elements SMAD-2 and SMAD-4 (SMAD-4/DPC4) at 18q. Sporadic MSI tumors are frequently mucinous, predominantly localized in the right colon, and generally diploid. In MSI patients, alterations in TGFb-RII, IGF receptors IGF-RII, b-catenin, TCF-4, and E2F transcription factors, as well as loss of the PTEN tumor suppressor, and the ▶ apoptosis regulator BAX are frequently reported. Sporadic cancers are also driven by epigenetic mechanisms, hypo- and hypermethylation of promoter genes encoding cancer markers and/or effectors, such as TFF, MUC-5 AC, COX-2, protease-activated receptors PAR-1, IGF, and ▶ p21/p16/cyclin D1. Dominant activation of proto-oncogenes by point mutations or constitutive activation by other oncogenic pathways is also frequently observed at early stages (ACF, polyps: src, ras, c-myc) and late stages concomitant with cellular invasion, angiogenesis, and ▶ metastasis (c-Kit, EGF-R, VEGF, the hepatocyte growth factor receptor MET, src, and many others). Familial adenomatous polyposis (FAP) is induced by mutations of the APC gene, a defect that contributes to CIN and appearance of more than 100 colorectal adenomas. Molecular alterations in other elements of the Wnt pathways are also concerned in sporadic colon cancers, including Wnt-2, axin, b-catenin, and TCF4 transcription factor. Nonpolyposis form of the hereditary colon cancer (HNPCC) is more frequent than FAP and is caused by germ cell mutations that invalidate the DNA repair systems. DNA mismatch repair is deficient in 90% of the HNPCC patients. The mutations concern mostly the hMSH2 and hMLH1 DNA repair enzymes, less frequently hPMS1 and hPMS2. Such genetic and molecular changes lead to the formation of aberrant crypt foci (ACF), which precede the appearance of premalignant adenomas anchored in the colon mucosal wall. The next stage is the evolution of the adenoma toward more aggressive lesions (ADK) and irreversible acquisition of dominant and anarchic functions, chronic inflammation, oxidative ▶ DNA damage,

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autocrine and paracrine regulatory loops linked to IGF-R, VEGF-R, EGF-R ligands, induction of thrombin protease-activated receptors PAR-1, trefoil factor pS2 (TFF-1), and the immediate response gene COX-2. Aberrant Expression of TFF as Clinical Markers of the Neoplasia TFF are involved in the neoplastic progression in human epithelial tumors according to their ability to confer several transforming functions including resistance to apoptosis, induction of cellular scattering, anchorage-independent growth in soft agar, and proinvasive and proangiogenic activities in vitro and in vivo. Both TFF1 and TFF3 reduced apoptosis induced by serum privation and loss of cellular adhesion (▶ Anoikis), a major response linked to cancer cell transformation, survival, and dissemination. Accordingly TFF are connected with several oncogenic and tumor suppressor elements such as ▶ E-cadherins, EGF-R, the RhoAROCK axis, PI3-kinase (PI3K), phospholipase C, COX-2, nitric oxide synthase 2, NF-kB, STAT3, and Cdc25. The nuclear phosphatases Cdc25A and B are associated with hypergrowth activity and control of the G2/M checkpoint in response to DNA damage and repair. This is supported by clinical investigations on aberrant expression of TFF in human solid tumors of the prostate (TFF3), premalignant changes and neuroendocrine differentiation in human prostate cancer (TFF1), human hepatocellular carcinomas (promoter hypomethylation of the TFF3 gene), hepatolithiasis, and cholangiocarcinomas (TFF1–MUC5AC), primary mucinous carcinomas of the skin (TFF1–TFF3), and ulcerating Barrett esophagus, a precancerous lesion considered as a gastric-type metaplasia. Barrett esophagus is characterized by the specific expression of the gastric-type markers TFF1 and MUC5AC with high levels and strong colocalization in the surface epithelium. In contrast, TFF3, MUC6, and MUC5B were found in the deeper glandular structures. Similarly, gastric metaplasia of the duodenum (GMD) is characterized by replacement of the intestinal epithelium with gastric-type mucus cells, villus damage, and atrophy and is frequently found in association with inflammation and

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gastritis induced by H. p. GMD expressed TFF1, TFF2, and the colon cancer -associated ▶ mucin MUC5AC, a marker of ACF now considered as precancerous lesions in the large bowel. Progressive loss of TFF1 and TFF2 associated with reciprocal induction of TFF3 is likely to be involved in the early stages of gastric ▶ carcinogenesis. In the normal gastric mucosa, TFF2 is expressed in surface mucus neck cells. Decreased TFF2 expression in chronic atrophic gastritis possibly attributes to the decrease in the number of surface epithelial cells expressing TFF2. Reexpression of TFF2 in gastric epithelial dysplasia implies that TFF2 possibly contributes to the progression of gastric carcinoma. It has been reported that TFF3 induction in gastric tumors correlated with an aggressive phenotype with advanced stages, infiltrative growth pattern, and positive lymph nodes. TFF3 is now considered as a marker of poor prognosis in human gastric carcinomas and is associated with aggressive behavior and lethality of colon cancer cells in rats. Ectopic expression of TFF3 promoted the invasive phenotype in Rat-2 fibroblast cells associated with upregulation of b-catenin, MMP-9 matrix metalloproteinase, and downregulation of the tumor suppressor gene product ▶ E-cadherin involved in b-catenin-associated ▶ adherens junctions. In fact, several reports suggest that TFF participate to morphogenesis and differentiation programs for epithelial cells in breast, gastrointestinal tract, and lungs. Conversely, depletion of TFF3 in the human gastric cancer cell line SNU-1 that expresses TFF3 resulted in decreased ability to form colonies in soft agar and in a marked increase in apoptosis and chemosensitivity to anticancer agents. The situation is probably more complicated since both TFF2 and TFF3 are induced in advanced gastric carcinomas and linked with neoangiogenesis, thus having a negative impact on patient survival, and are an independent predictor of disease recurrence. Both TFF1 and TFF2 are strongly expressed in diffuse-type gastric cancers, suggesting that the academic definition of TFF1 as a gastric-specific tumor suppressor gene should be applied in relation with the corresponding status of TFF2/TFF3, and the clinicopathologic context and oncogenic

Trefoil Factors

status of human gastric tumors. For example, TFF1 can be considered as a bona fide gastric tumor suppressor acting against ulcerative and procarcinogenic inflammarory situations. Exocrine gastric secretions and digestive functions as well as stress conditions are known to induce predisposition to the neoplasia. In this scenario, TFF1 mediates gastric mucosal protection and epithelial cell reconstruction of the gastric gland units. However, when the TFF1 gene is silenced by loss of heterozygosity in man, experimental knockdown in transgenic mice, or methylated at the TFF1 promoter region (epigenetic silencing), the non-expressing TFF1 gastric mucosa is now subjected to the agressive secretions of acid and pepsinogen by the stomach. In the Tff1-KO transgenic mice, a direct link between TFF1 loss and bcatenin signaling to its target genes c-Myc and Ccnd1 during gastric oncogenesis. This experimental data was validated in clinical samples from human gastric tumors showing increased nuclear localisation of b-catenin linked to TFF1 deficiency. In contrast, TFFs are found overexpressed during the progression of several human epithelial tumors, including colorectal and breast cancers. Thus, TFFs are now usually considered as critical growth, migratory and invasion-promoting factors in epithelial cancers through direct and indirect mechanisms inducing tumor angiogenesis and metastasis. Several reports indicate that TFF2 is a gastric marker of tumor metastasis frequently upregulated in diffuse gastric cancers in correlation with decreased survival. TFF2-expressing cells are upregulated in the stomach of Helicobacter-infected mice and seem to give rise to invasive cancerous lesions. Consequently, both TFF2 and COX-2 are overexpressed in patients with H. pylori-induced chronic fundic gastritis in association with dysplasia. In the established H. felis/C57BL/6 mouse model of gastric cancer induced by chronic infection with Helicobacter felis, bone marrow-derived mesenchymal progenitor cells, but not hematopoietic stem cells, are recruited to the site of gastric mucosa injury and inflammation. This proliferative zone gives rise to spasmolytic-expressing metaplasia (SPEM) and differentiation toward an epithelial phenotype, evidenced by positive

Trefoil Factors

staining for TFF2 and the epithelial cell cytokeratin KRT1–19 in deep antral and fundic glands. Thus, experimental Helicobacter infection can give rise to a new mucosal microenvironment in the infected gastric mucosa following upregulation of the stem cell factor SCF-1 (the ligand of the c-kit tyrosine kinase) and the ▶ chemokine SDF-1 binding the ▶ G-protein-coupled receptor CXCR4, two key factors involved in the mobilization of bone marrow progenitors and cancer metastasis. It remains to be elucidated whether subpopulations of human gastric cancers may originate from the neoplastic transformation of bone marrow progenitors with gastric mucosal cell gene expression pattern. In addition, SPEM was suppressed by invalidation of the TNF-a gene in Tnf / K19-C2mE transgenic animals expressing simultaneously COX-1/-2 in gastric mucosa via the cytokeratin 19 gene promoter. Finally, we cannot exclude the possibility that illegitimate and constitutive expression of TFF2 by mesenchymal bone marrow stem cells may also target the gastric progenitor niche for metaplasia and dysplasia. Notably, SPEM is associated with gastric H. pylori infection, aberrant expression of the mucin 6 (MUC-6) gene, and progression of human gastric adenocarcinoma. Therefore, the combined loss of the gastric-specific tumor suppressor gene TFF1 with induction of TFF2 and TFF3 provides insights into the complex mechanisms underlying the biological significance and versatility of TFF in gastric cancer progression and neoplasia. In breast cancer, TFF1 and TFF3 but not TFF2 were identified as informative markers for the detection of ▶ micrometastases in the axillary lymph nodes and blood. Significance analysis of microarrays identified a positive correlation between TFF1 overexpression and breast cancer metastasis to the bone in a cohort of 107 patients with primary breast tumors who were all lymph node negative at the time of diagnosis. The involvement of the FGF signaling pathway was also incriminated in preference of tumor cells that relapse to the bone. The fact that TFF1 may contribute to tumor relapse to the bone is underscored by its abundant presence in breast cancer micrometastases.

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In addition, morphogenetic effects have been attributed to TFF2 in human breast cancer cells following the induction of highly complex branched ductular structures typical of migratory, invasive, and survival functions, in a TFF1dependent manner. TFF3 was also expressed in breast ductal and lobular breast carcinomas in situ and invasive lobular carcinomas. While TFF1 is a surrogate indicator for the response to antihormonal therapy and favorable outcome in estrogen receptor-a (ERa)-positive and welldifferentiated breast cancers, its deregulated expression is now considered to contribute to the progression of both ER-a-positive and -negative human breast cancers. Of note, plasma levels of TFF were found elevated in patients with advanced prostate cancer. Conclusions TFF are now considered as valuable therapeutic tools for the treatment of injured mucosal epithelial cells and protection against mucosal and tissular damages following transient injury and other damages caused by radiation therapy and chemotherapy. Selective loss, induction, and overexpression of TFF observed during inflammatory processes and neoplasia deregulate TFF signaling cross-talks and signals and compensatory functions linked to TFF. The molecular complexity of TFF is further illustrated by their ability to form covalent disulfide-linked dimers in vitro and in vivo. The possibility that TFF could form heterodimers adds further complexity for their relevance in receptor and signal transduction. Despite increasing interest on TFF in molecular research and clinical applications, TFF are still orphan signaling peptides facing unknown receptors in the classical definition of receptormediated signal transduction pathways from the plasma membrane, cytoplasmic and nuclear domains, and vice versa. Advances in the field pointed the discovery of new TFF-binding proteins apparently linked to mucosal protection, such as MUC-5 AC and the gastrokine-1-like peptide blottin. It is conceivable that TFF are not released via normal secretory pathways in inflammatory situations and neoplasia. The epithelial cell polarity and its normal microenvironment

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with stromal and vascular cells, immune cells, and extracellular matrix components and their receptors are lost during cancer progression. In this case, it is tempting to assume that TFF are in the abnormal situation interacting with key signal transducers and cancer-associated stromal cell lineages during cancer progression, via illegitimate, pejorative, and persistent mechanisms. In this scenario, TFF were shown to signal through distinct transduction pathways following external addition of the peptides versus ectopic expression. Future attempts and new strategies to identify the sensu stricto TFF receptors and direct transducers are therefore expected in order to exploit the positive facets of TFF for therapeutic purposes and to fight against their deleterious functions in pathological states.

Trefoil Peptides pS2/TFF1

TRF1-Interacting, Ankyrin-Related ADP-Ribose Polymerases ▶ Tankyrases

TR-FRET ▶ Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

Trident ▶ Forkhead Box M1

References Emami S, Rodrigues S, Rodrigue CM et al (2004) Trefoil factors (TFFs) and cancer progression. Peptides 25:885–898 (Review) Lefebvre O, Chenard MP, Masson R et al (1996) Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 274:259–262 Rodrigues S, Rodrigue C, Attoub S et al (2006) Induction of the adenoma-adenocarcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells. Oncogene 25:6628–6636 Soutto M, Peng D, Katsha A et al. (2015) Activation of βcatenin signalling by TFF1 loss promotes cell proliferation and gastric tumorigenesis. Gut 64(7):1028–39 Taupin D, Podolsky DK (2003) Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 4:721–732 Thim L, May FE (2005) Structure of mammalian trefoil factors and functional insights. Cell Mol Life Sci 62(24):2956–2973 (Review)

Trefoil Peptides pS2/TFF1 ▶ Trefoil Factors

40 ,5,7-Trihydroxyisoflavone ▶ Genistein

3,40 ,5-Trihydroxystilbene ▶ Resveratrol

Triple-Negative Breast Cancer Benoit Paquette Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada

Synonyms

Treg ▶ Regulatory T Cells

Estrogen receptor, progesterone receptor, and HER-2 negative; TNBC

Triple-Negative Breast Cancer

Definition Triple-negative breast cancer (TNBC) is a subgroup that accounts for 10–20% of all breast carcinomas. TNBC is characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2).

Characteristics Breast cancer is a heterogeneous disease, encompassing a number of distinct biological entities associated with specific morphological and clinical behavior. Technological developments have allowed to sequence the complete genome of breast cancer and compared a large number of tumor samples. From these, studies have emerged five subtypes: luminal A, luminal B, HER2-enriched, basal-like, and claudin-low. TNBC is sometime referred to as basal-like tumors, as a majority of basal-like cancers are TNBC and approximately 80% of TNBC are also basal-like breast cancers. TNBC is also associated with younger patients and high-grade tumors. Whereas hormone receptor-positive and HER2-positive breast cancers have had favorable outcomes with chemotherapy and treatment targeting the ER or HER-2, TNBC on the contrary has got mixed result and still lacking a targeted therapy. For some TNBC patients, the prognosis is poor when compared to other subtypes of breast cancer because distant recurrence appears between the first and third years after treatment. However, 5 years after diagnosis, in TNBC patients whose cancer didn’t reappear early, the probability to develop metastasis is not significantly different compared to non-TNBC patients. This suggests that TNBC patients can be divided in two groups, the good responders to treatment and the poor responders where an early recurrence is observed. However, there is presently no biomarker to identify them. Breast Cancer Treatment by Radiotherapy Microfoci of cancer cells are often dispersed throughout the breast and may be present at few

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cm from the edge of the primary tumor. Conservative surgery removes the primary tumor, while microfoci of cancer cells are targeted by radiotherapy. The treated area frequently covers the whole breast and the axillary and subclavian lymph nodes. Unfortunately, the radiation dose used in clinic does not always eradicate all cancer cells scattered in the breast because it is rather aimed at optimizing long-term results with minimal adverse effects. Many cancer cells are effectively killed since radiotherapy leads to a measurable reduction of local recurrence and distant metastases. A moderate hypofractionated schedule is frequently used in postoperative breast irradiation (2.66 Gy/day, 16 fractions, boost of 2.5 Gy/day, 4 fractions in the tumor bed, 52.6 Gy). More aggressive hypofractionated treatments are under study. For example, whole-breast irradiation of 3 Gy/day in 13 fractions for 39 Gy, followed by a tumor bed boost of 9 Gy in 3 fractions, and even more aggressively with 5 weekly fractions of 6.0 Gy for a total of 30 Gy. The aim is to further improve the local recurrence and overall survival, with acceptable side effects to normal tissues. Radiation and Metastases Development Radiotherapy induces an inflammatory response in all patients treated. The level of inflammation induces by the radiotherapy can vary greatly from one patient to another. Dry skin after breast cancer radiation treatment is fairly common. In some women, a significant inflammation of the skin can occur that looks like a sunburn in treated area (dermatitis). Rarely, this dermatitis is accompanied by severe pain that will require to stop the radiation treatment. Accumulating studies suggest that the burst of inflammatory cytokines induced by radiation may stimulate the development of new metastases. Supporting the observations made in clinic, it has been shown in a mouse model that pre-irradiation of mammary gland before implantation of TNBC D2A1 cells stimulated the migration of cancer cells in the mammary gland, increased the number of circulating tumor cell, and favored the development of

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lung metastases. A similar stimulation of metastases development was also observed after irradiating a TNBC D2A1 tumor already implanted in the mammary gland of the mouse. The inflammatory pathways involved still need to be investigated in details. Nevertheless, these adverse effects of radiation were associated with the cytokine interleukin-6 (IL-6) and the inflammatory enzyme cyclooxygenase-2 (COX-2). These observations raise some concerns about the hypofractionated radiation protocols for breast cancer. On one hand, a higher radiation dose per fraction may be a promising alternative to standard radiotherapy since a greater number of cancer cells would be eliminated by fraction. However, a more important inflammation could be induced. Therefore, the balance between the removal of a large number of cancer cells and the deleterious effects caused by significant inflammation must be determined. The hypofractionated protocols may also not be appropriate for patients who are at higher risk of early recurrence.

Biomarkers of TNBC Poor Responders Radiation oncologists are aware that the risk of recurrence is non-negligible in TNBC patients. As recurrence may appear shortly after radiotherapy, it is possible that inflammatory cytokines induced by radiation might stimulate the development of metastases. On the other hand, physicians also know that TNBC good responders will benefit from radiotherapy. Identification of the TNBC poor responders before treatment is therefore crucial. Some commonly applied approaches for biomarker discovery, such as gene expression profiling, have not yet succeeded in identifying highly sensitive and specific markers related to disease progression of TNBC. A protein signature has been reported to identify the TNBC poor prognosis (recurrence within 5 years). However, their analysis was restricted to patients who did not receive adjuvant therapy. Studies are therefore required to determine

Tripterine

whether TNBC poor responders can be identified based on a specific pattern of gene expression in the tumors or by an inflammatory response characterized by an exaggerated intensity or involving specific cytokines.

Cross-References ▶ Breast Cancer Immunotherapy

References Bouchard G, Bouvette G, Therriault H, Bujold R, Saucier C, Paquette B (2013) Pre-irradiation of mouse mammary gland stimulates breast cancer cell migration and development of lung metastases. Br J Cancer 109:1829–1838 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:4429–4434 Holland R, Veling SH, Mravunac M, Hendriks JH (1985) Histologic multifocality of Tis, T1-2 breast carcinomas. Implications for clinical trials of breastconserving surgery. Cancer 56:979–990 Liu NQ, Stingl C, Look MP, Smid M, Braakman RBH, De Marchi T, Sieuwerts AM, Span PN, Sweep FCGJ, Linderholm BK et al (2013) Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative breast cancer. J Natl Cancer Inst 106:djt376 Pogoda K, Niwinska A, Murawska M, Pienkowski T (2013) Analysis of pattern, time and risk factors influencing recurrence in triple-negative breast cancer patients. Med Oncol 30:388

Tripterine ▶ Celastrol

TROP-1 ▶ EpCAM

Tuberous Sclerosis Complex

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Trophectoderm

TSC

Isabelle Gross INSERM U1113, Université de Strasbourg, Strasbourg, France

▶ Tuberous Sclerosis Complex

TSC1 Definition The fertilized egg of mammals cleaves several times to generate a 16-cell morula consisting of small inner cells enclosed within larger outer cells. Most of the outer cells are then epithelialized (▶ epithelium) and form the trophectoderm, whereas the inner cells go on to generate the inner cell mass in blastocysts. Cell-fate analyses revealed that the inner cell mass gives rise to all of the embryonic cells and the extraembryonic endoderm, whereas the trophectoderm forms the embryonic portion of the placenta and represents the first differentiated cell lineage of mammalian embryogenesis.

Cross-References ▶ Epithelium

▶ Hamartin

tTGase ▶ Transglutaminase-2

Tuberous Sclerosis Complex Andrew R. Tee1, Julian R. Sampson1, Jeremy P. Cheadle1 and David Mark Davies2 1 Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK 2 Department of Oncology, South West Wales Cancer Centre, Swansea, UK

Synonyms TSC

Trp63 ▶ p53 Family

Trp73

Definition Tuberous sclerosis complex (TSC): An autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 genes and characterized by the development of benign tumors in multiple organs, seizures, and neurocognitive impairment.

▶ p53 Family

Characteristics

Trx ▶ Thioredoxin System

Clinical Aspects Tuberous sclerosis complex (TSC) is a genetic, multisystem disorder caused by mutations in

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Tuberous Sclerosis Complex

Tuberous Sclerosis Complex, Fig. 1 Tuberous sclerosis complex is characterized by the development of hamartomas in a variety of tissues and organs. (a) A facial angiofibroma, (b) an abdominal MRI scan showing a renal angiomyolipoma (arrowed) (c) a cranial MRI scan showing a partly intraventricular subependymal giant cell astrocytoma (arrowed), and, (d) a renal ultrasound scan showing a renal cell carcinoma (within a cyst) (arrowed)

either of the TSC1 or TSC2 genes. TSC occurs in up to 1 in 6,000 live births. The condition has a variable phenotype, both in terms of type and severity of clinical features. Neurological problems are often the most disabling feature of TSC. Approximately, 75–90% of people with TSC have epilepsy. The seizures usually begin in the 1st year of life, although they can also occur for the first time in later childhood or in adulthood. There are correlations between the early onset and severity of seizures and neurodevelopmental and cognitive problems. A wide variety of seizure types occur. Infantile spasms are particularly associated with an increased risk of neurodevelopmental impairments and occur in about one third of infants with TSC. Rapid recognition and treatment is imperative. Infantile spasms are typically characterized by clustered repetitive flexion of the limbs. However, infantile spasms can also present with rapid extension of the limbs, a mixture of flexion and extension, or more subtle signs such as head nods, upward eye deviation, or shoulder

elevation. Onset peaks at 4–6 months. There is characteristic chaotic interictal electroencephalography (EEG) pattern, which, when typical, is termed hypsarrhythmia. TSC is associated with several types of brain lesions. Cortical tubers are focal developmental abnormalities of the cerebral cortex and occur in 80–90% of patients. Subependymal giant cell astrocytomas (SEGAs) are low grade tumors occurring in approximately 10% of patients with TSC (Fig. 1). Growth, typically occurring in childhood or adolescence, may lead to hydrocephalus and present with a deterioration in epilepsy and/or behavior as well as with symptoms such as headache, vomiting, and visual problems due to raised intracranial pressure. SEGAs are thought to arise from subependymal nodules, asymptomatic hamartomas found on the walls of the lateral and third ventricles in most patients with TSC. A minority of individuals with TSC are profoundly intellectually impaired; many more have a slight reduction in IQ, while others haven normal intellectual ability. Specific cognitive deficits

Tuberous Sclerosis Complex

in areas such as memory, attention, and executive function are very common. Developmental disorders such as autistic spectrum disorder and attention deficit hyperactivity disorder are also common in TSC as are depression and anxiety. TSC is associated with a range of behavioral disorders such as aggressive outbursts, sleep problems, and poor social interaction. Whilst behavioral problems are more often seen in patients with intellectual impairment, they also occur in those with normal intelligence. Renal angiomyolipomas (AMLs) are benign tumors found in approximately 80% of patients (Fig. 1). They often develop during later childhood and adolescence. They are usually multiple, bilateral, and asymptomatic but can cause flank pain, impair renal function, and bleed causing hematuria and occasionally major hemorrhage. Renal cysts are also common in TSC and are usually asymptomatic, but patients with a contiguous deletion of the TSC2 and adjacent PKD1 genes develop polycystic kidney disease that often progresses to renal failure in early adult life. The incidence of renal cancer in TSC is similar to the general population, but it tends to occur in younger patients and may be bilateral (Fig. 1). Lymphangioleiomyomatosis (LAM) is a disorder of the lungs and lymphatics, which can occur sporadically or in association with TSC. LAM occurs almost exclusively in females, and radiographic studies suggest 40% of female patients with TSC are affected, although many are asymptomatic. LAM is characterized by proliferation of abnormal “LAM” cells and cystic changes within the lung parenchyma that can lead to respiratory failure. LAM usually presents with dyspnea or recurrent pneumothorax. The sporadic form is caused by acquired mutations in TSC2. It is thought that “LAM” cells arise from renal angiomyolipomas or other remote sites and migrate to the lung in a process that has been termed “benign metastasis.” The skin is affected in approximately 90% of individuals with TSC. The main skin manifestations are facial angiofibromas, shagreen patches, periungual fibromas, and hypopigmented macules. Hypomelanotic macules are often present at birth or develop during infancy and are best seen

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under ultraviolet light using a Wood’s lamp. They are, however, one of the least reliable diagnostic signs. Facial angiofibromas are red/reddish brown papules, typically occur over the nose, cheeks, and chin, often first developing at around 3–5 years of age (Fig. 1). Shagreen patches are elevated irregular light brown or flesh colored plaques often present in the lumbar sacral area that often develop during childhood. Ungual fibromas are fleshy nodules that occur on the finger- or toe-nail beds. A linear depression in the nail can suggest the presence of a subungual fibroma. Fibrous plaques can develop on the forehead, and some individuals with TSC develop “confetti-like” hypopigmentation on the limbs. Cardiac rhabdomyomas are an early manifestation of TSC, appearing at 22–28 weeks of gestation. They usually regress during childhood and are normally asymptomatic but can be associated with obstructive heart failure or arrhythmias. They are often detected on antenatal ultrasound and be sporadic but are associated with a substantial risk of tuberous sclerosis, especially if the lesions are multiple. The Genetic Basis of TSC

TSC is caused by mutation in either TSC1, located on chromosome 9 or TSC2, located on chromosome 16, adjacent to PKD1, one of the genes responsible for autosomal dominant polycystic kidney disease. TSC is an autosomal dominant condition, but about two thirds of cases result from a new mutation with neither parent being affected. Each child of a person with TSC has a 50% chance of inheriting the mutation. All those who inherit the mutation develop TSC (i.e., the disorder shows full penetrance). TSC1 mutations are associated with less severe disease manifestations than TSC2 mutations, but it is not possible currently to predict severity at an individual level on the basis of genotype. Parents of a child with TSC who are apparently unaffected should be evaluated for subtle signs of the disease. If neither parent has any features of TSC nor a TSC-causing mutation in DNA is extracted from leucocytes, the risk of each future child having TSC is 1–2% because of germline mosaicism

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where two or more genetically distinct cell lines are present in the testis or ovary. In addition to an inherited TSC1 or TSC2 mutation an acquired “second hit” mutation in the second TSC1 or TSC2 allele in a cell appears to be required for the formation of most lesions in TSC, consistent with the two-hit tumor suppressor gene model of Knudson. However, mutation of just one allele may contribute to some of the manifestations of TSC, such a cognitive impairment, through a gene dosage effect.

Tuberous Sclerosis Complex 1

angiomyolipomas have been successful, leading to regulatory approval in many countries. Sirolimus has been demonstrated to slow the deterioration in lung function in patients affected by sporadic or TSC associated LAM.

References Kwiatkowski DJ, Whittemore VH, Thiele EA (eds) (2010) Tuberous sclerosis complex: genes, clinical features and therapeutics. Willey Blackwell, Weinheim

The Molecular Pathology of TSC

The TSC1 and TSC2 tumor suppressor proteins together with TBC1D7 form a complex, which regulates multiple cellular processes and functions to inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1, in turn, regulates many processes such as protein synthesis and the biosynthesis of ribosomes, lipid and glucose metabolism, nucleotide synthesis, mitochondrial biogenesis, and autophagy. Rheb (Ras homolog enriched in brain) is a GTPase complex that functionally links TSC1/ TSC2 to mTORC1. The TSC1-TSC2-TBC1D7 complex (termed the TSC-TBC complex) has GTPase-activating protein activity converting Rheb from an active GTP-bound state, which in turn potently activates mTORC1, to an inactive GDP-bound state which turns off mTORC1. Functional TSC1-TSC2-TBC1D7 allows the integration of multiple stimuli such as the levels of cellular oxygen, energy, and nutrients as well as growth factor signaling into the nuanced control of the processes regulated by mTORC1. Loss-offunction mutations in either TSC1 or TSC2 cause aberrant signal transduction through mTORC1 leading to many of the pathological features of tuberous sclerosis.

Tuberous Sclerosis Complex 1 ▶ Hamartin

Tubulin-Interacting Proteins ▶ Microtubule-Associated Proteins

Tumor Antigens Roberto Bei Department of Clinical Sciences and Translational Medicine, Faculty of Medicine, University of Rome “Tor Vergata”, Rome, Italy

Definition Tumor antigens (TAs) are antigens recognized by specific immune effector cells and/or antibodies and differentially expressed (qualitatively and/or quantitatively) in the tumor tissue as compared to the normal counterpart tissue.

Targeted Therapy for TSC

The direct link between deregulated mTORC1 signaling and the pathogenesis of TSC provided a strong rational for the use of mTORC1 inhibitors, such as everolimus and sirolimus, as a targeted therapy. Clinical trials of everolimus for the treatment of TSC-related subependymal giant cell astrocytoma (SEGA) and renal

Characteristics Tumor Antigens Classification, Expression, and Immune Response TAs can be divided into tumor-specific antigens (TSAs) that are unique antigens expressed by

Tumor Antigens

tumor cells and not by normal cells and tumorassociated antigens (TAAs), which are also expressed by normal cells although at lower levels than in tumor cells (see also Yang and Yang 2005). TAs include mutated antigens, oncofetal antigens (OFs), cancer–testis antigens (CTs), aberrantly glycosylated and expressed antigens, tissue-lineage antigens, overexpressed antigens, and virally encoded antigens. Classification and examples of tumor antigens are shown in Table 1 (see also Khodadoust and Alizadeh 2014). Mutated antigens are antigens which display structural mutations, including point mutations and chromosomal translocations, the latter leading to the generation of chimeric fusion proteins (see also Khodadoust and Alizadeh 2014). Mutations in genes which encode for proteins involved in normal regulation of growth (proto-oncogenes) induce their activation to oncogenes, whose products contribute to cellular transformation. The prototype of a tumor antigen carrying a point mutation is the product of the RAS protooncogene, which encodes for a protein (p21) which induces growth factor-mediated signal transduction and whose point mutation makes it always active. Missense p21 mutations were detected in several types of cancer. Antibody responses to wild-type and mutated p21 ras were detected in a high portion of patients with colon cancer. In addition, in vitro stimulation of human lymphocytes from cancer patients with mutant ras peptides induced the expansion of CD4+ and CD8+ T-cell precursors. Mutations associated with the development of papillary thyroid cancer involve the B-type Raf kinase (BRAF). Mutations of the BRAF gene are the most common genetic alterations in melanoma. Mutated B-Raf/B-Raf (V599E)-specific antibodies and CD8+ T cells were found in melanoma patients. The t(9;22) chromosomal translocation (Philadelphia chromosome, Ph) results in the formation of the Bcr-abl fusion protein which has deregulated protein kinase activity as compared to the normal kinase abl and contributes to the pathogenesis of chronic myelogenous leukemia (CML). The Bcr-abl chimeric protein represents

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a target for therapy in CML, in Ph+ acute lymphoblastic leukemia (ALL), and in some acute myelogenous leukemias (AMLs). Bcr-ablspecific T cells were detected in CML patients. Autoantibodies to p210 Bcr–Abl were found in both Ph+ and Ph- leukemias. A mutated cyclin-dependent kinase 4 (CDK4) and mutated b-catenin were found in melanoma and shown to induce specific CD8+ T cells generated from tumor-infiltrating lymphocytes. Tumor suppressor genes, i.e., those genes that negatively regulate cell growth, are also modified by mutations that make them inactive during tumor development. The prototype of a tumor suppressor gene that is mutated in the majority of tumors is the p53 protein which is capable of blocking the cell cycle and induces apoptosis in cells with DNA damage. An altered activity of p53 leads to accumulation of DNA damage and contributes to neoplastic transformation. Antibodies to p53 were shown to be elicited in patients with tumor mutated p53, and human cytotoxic T lymphocytes (CTLs) that preferentially recognize tumor cells bearing a conformational p53 mutant could also be isolated. Wilms’ tumor 1 (WT1) is a transcription factor expressed in embryonic kidney cells and hematopoietic stem cells. WT1 is mutated in patient with Wilms’ tumor and in most AMLs and CMLs. Patients with AML show antibodies reactive with full-length or NH2-terminal WT1 protein. Direct recognition and lysis of leukemia cells by WT1-specific T lymphocytes were also reported. OF antigens are expressed in fetal tissues, partially repressed in adult tissues and expressed at high levels by cancer cells (see also Reuschenbach et al. 2009). OF antigens include carcinoembryonic antigen (CEA), alphafetoprotein (AFP), oncofetal antigen/immature or precursor laminin receptor protein (OFA/iLRP), oncofetal antigen 5T4, glypican-3 (GPC-3), insulin-like growth factor II mRNA-binding protein (IMP) 3 (IMP-3) and the identical KOC (KH domain-containing protein overexpressed in cancer), human chorionic gonadotropin-b (hCGb), and pancreatic oncofetal antigen (POA) (see also Canevari et al. 1996).

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Tumor Antigens

Tumor Antigens, Table 1 Classification and examples of tumor antigens Antigen group Mutated

Name RAS BRAF BCR/ABL b-catenin CDK4 p53

Oncofetal

WT1 CEA

AFP

OFA/iLRP 5T4 oncofetal antigen GPC-3 IMP-3 hCGb

Cancer testis

Aberrantly glycosylated and expressed

MAGE-A1, MAGE-A3, MAGE-A10, NY-ESO-1, SSX-2, GAGE1, KP-OVA-52, XAGE-1b MUC1

Thomsen–Friedenreich (TF or T) antigen LeY

Tissue lineage

Stage-specific embryonic antigen-1 (SSEA-1) (LeX) Gangliosides (GM1, GM2, GD1, GD2, GD3) FucGM1 Melanoma differentiation antigens (tyrosinase, TYRP1/gp75, Melan-A/ MART-1) Prostate-specific antigen (PSA) Idiotype

Site/type of the tumor Colon, lung, pancreas, prostate, leukemias, bladder, etc. Thyroid, melanoma CML, ALL, AML Melanoma Melanoma Breast, colon, lung, colon rectum, ovary, thyroid, bladder, pancreas, B-cell lymphoma, etc. AML, CML, Wilms’ tumor Colorectal, stomach, pancreas, lung, breast, gallbladder, ovary, endometrium Testicular, liver, pancreas, lung, embryonal cell carcinoma, genitourinary tract, yolk sac (ovary) Hematological, breast, mesenchymal tissue, kidney Breast, kidney, colorectal, prostate, ovary Liver, melanoma, yolk sac, stomach Pancreas, lung, stomach, esophagus, colon, kidney, soft tissue Colon, lung, pancreas, esophagus, breast, bladder, cervix, stomach, prostate, trophoblast, testis Bladder, brain, colon, head and neck, lung, liver, esophagus, melanoma, myeloma, neuroblastoma, prostate, thyroid, ovary, NHL, lung Breast, pancreas, ovary, endometrium, lung, prostate, bladder, gastrointestinal tract, multiple myeloma, T-cell and some B-cell lymphomas Colon, breast, bladder, prostate, liver, ovary, stomach Ovary, prostate, colon, breast, pancreas, lung, embryonal tissues, yolk sac, testis Colon, stomach, breast, ovary, kidney, bladder Neuroblastoma, melanoma, lung

Spontaneous immune response in cancer patients Humoral and cell mediated Humoral and cell mediated Humoral and cell mediated Cell mediated Cell mediated Humoral and cell mediated

Humoral and cell mediated Humoral and cell mediated

Humoral and cell mediated

Humoral and cell mediated Cell mediated Cell mediated Cell mediated Cell mediated

Humoral and cell mediated

Humoral and cell mediated

Humoral Humoral

Humoral Humoral

Lung Melanoma

Humoral Cell mediated and humoral (TYRP1/gp75)

Prostate

Humoral and cell mediated

B-cell malignancies

Humoral (continued)

Tumor Antigens

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Tumor Antigens, Table 1 (continued) Antigen group Overexpressed

Name ErbB receptors (EGFR, ErbB2, ErbB3, ErbB4) Ribosomal P0 protein Ribosomal protein S6 Ribosomal protein L19 HSPs MYC

OPN MDM2 Mesothelin Survivin

Virally encoded

HPV (E6, E7) EBV (EBV-encoded nuclear antigens (EBNAs), BZLF1 protein)

Site/type of the tumor Breast, colon, head and neck, lung, pancreas, prostate, bladder Colon, liver, head and neck, breast NHL, breast, colon, kidney Lung Breast, ovary, lung, pancreas, colon, prostate, urinary tract, AML Neuroblastoma, small cell lung cancer, alveolar rhabdomyosarcoma, retinoblastoma Prostate CLL Mesothelium, pancreas, ovary Breast, ovary, lung, pancreas, colon, liver, prostate, glioma, esophagus meningioma, urinary tract Cervix, oral cavity B-cell malignancies, nasopharyngeal carcinoma

CEA is present in normal epithelial cells of several tissues. CEA expression in these organs normally begins during the early fetal period (week 9–14) and appears to continue throughout life. High levels of CEA are found on a wide range of human carcinomas. The presence of anti-CEA antibodies was observed in the serum of patients with gastrointestinal malignancies, and CEA-specific CTLs could be generated using CEA peptide-pulsed dendritic cells. AFP is synthesized in the fetal yolk sac at 9week gestation and later in the fetal liver and gastrointestinal tract. In adult life, AFP is reexpressed in multiple tumors of endodermal and mixed mesodermal/endodermal origin. There are different AFP isoforms and lectin glycan-associated forms demonstrable by electrophoretic and chromatographic procedures. Autoantibodies and T-cell immune responses to AFP have been reported to occur in patients with hepatocellular carcinoma (HCC) or with liver diseases. OFA/iLRP is widely expressed in many types of human tumors, while it is absent in normal adult differentiated tissues. Gained expression of the OFA/iLRP facilitates cancer cells to penetrate tissue and vessel barriers expressing laminin.

Spontaneous immune response in cancer patients Humoral and cell mediated Humoral Humoral Cell mediated Humoral Humoral

Humoral Cell mediated Humoral and cell mediated Humoral and cell mediated

Humoral and cell mediated Humoral and cell mediated

A spontaneous tumor-specific humoral immune response against OFA/iLRP was detected in a significant proportion of chronic lymphocytic leukemia (CLL) patients, while several OFA/iLRPspecific T-cell clones were established in breast cancer patients. The oncofetal antigen 5T4 is highly expressed in several carcinomas but has limited expression in normal tissues. This antigen has been isolated from the term placenta. 5T4 was found to be expressed in tumor-initiating cells and associated with worse clinical outcome in non-small cell lung cancer. The presence of a CD8+ T-cell repertoire specific for 5T4 was demonstrated in an apparently healthy donor. In addition, CTLs specific for a 5T4 epitope were induced in colorectal cancer patients following vaccination with a recombinant modified vaccinia Ankara (MVA) virus expressing 5T4. GPC-3 is a membrane-anchored heparin sulfate proteoglycan which is normally expressed in fetal liver and placenta, but not in normal adult liver. GPC-3 is overexpressed in HCC and melanoma. In HCC patients, GPC-3 peptide-reactive CTLs could be established from PBMCs by in vitro stimulation with these peptides.

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IMP-3 is produced in developing epithelia, muscle, and placenta during early stages of human and mouse embryogenesis. The expression of IMP-3/KOC is also observed in malignant tumors, but it is weakly or not detectable in adjacent benign tissues. The existence of specific T-cell responses to HLA-A24-restricted IMP-3 epitopes in esophageal squamous cell carcinoma patients was established. hCG is glycoprotein hormone secreted by trophoblastic cells during normal gestation. The b-subunit of hCG (hCGb) was found to be overexpressed in several tumors. Peripheral blood mononuclear cells (PBMCs) from patients with hCGb-productive bladder and testis tumors displayed an hCGb-specific proliferative response. CT antigens are aberrantly expressed by tumors of different histological origins and normal adult reproductive tissues such as testis and placenta. CT antigens comprise the melanomaassociated antigen family (MAGE-A1, MAGEA2, MAGE-A3, etc.), the GAGE/PAGE/XAGE superfamily, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), synovial sarcoma X (SSX), synaptonemal complex protein 1 (SCP-1) and BAGE, etc (see also Van den Eynde and van der Bruggen 1997). Melanomareactive CTLs could be induced from the peripheral blood lymphocytes (PBLs) of melanoma patients or normal donors by stimulation with the HLA-Al-binding epitope of MAGE-1 or MAGE-3. Spontaneous MAGE-A10- and/or SSX-2-specific CD8+ T cells were detected in HCC patients (see also Yang and Yang 2005). In addition, humoral immune responses to NY-ESO-1 were detected in patients with ovarian cancer, and a CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro was identified from PBMCs of healthy donors. GAGE1 was found to elicit autoantibodies in 6% of patients with thyroid cancer but not with benign nodules. A novel cancer/testis antigen KP-OVA-52 was discovered by SEREX in ovarian cancer and was found to be regulated by DNA methylation. 11.3% of non-Hodgkin lymphoma (NHL) samples were found to express at least 1 CT antigen including MAGE-A family (6.6%), GAGE (5.7%), and NY-ESO-1 (4.7%). Humoral and

Tumor Antigens

T-cell responses against XAGE-1b (GAGED2a) were observed in non-small cell lung carcinoma (NSCLC) and in adenocarcinoma lung patients (see also Van den Eynde and van der Bruggen 1997). Altered glycosylations frequently occurring in tumors lead to the creation of aberrantly glycosylated and expressed antigens. Glycosyltransferases are the key enzymes for the biosynthesis of carbohydrate chains. In cancer cells, glycosylation often is similar to that performed in fetal or immature cells. Changes in patterns of glycosylation may involve incomplete synthesis and variation of normally existing carbohydrates or changes in the backbone or in the inner core structures of the carbohydrates (see also Heimburg-Molinaro et al. 2011). Mucins are high molecular weight glycoproteins composed of 20 amino acid residues repeated in tandem, always heavily glycosylated with N-acetylgalactosamine O-linked to serine and threonine residues. Cancer tissues show increased levels of mucin mRNA and an aberrant glycosylation as compared to normal tissues, which lead to unusual expression of the core of the protein. Mucins exist as transmembrane (MUC1, MUC3, MUC4, MUC10-18) or soluble (gel forming) (MUC2, MUC5AC, MUC5B, MUC6-9, and MUC19) glycoproteins. MUC1 is present on normal ductal epithelial cells as a heavily glycosylated protein. However, it was found that cancer cells have reduced or no activity of the b1–6GlcNAc transferase, while they have increased activity of a2-3sialyltransferase which competes for the same galactose substrate by the addition of sialic acid, thus precluding additional carbohydrates insertion. In cancer cells, this phenomenon leads to the uncovering of novel peptide antigenic determinants and the production of new oligosaccharide epitopes. MUC1 expression is upregulated in the majority of adenocarcinomas as well as in hematological malignancies. Autoantibodies to MUC1 were found in breast, ovarian, pancreas, and non-small cell lung cancers. In addition, MUC1 peptide-stimulated CTLs could be isolated from patients with different adenocarcinomas (see also Heimburg-Molinaro et al. 2011).

Tumor Antigens

Similarly, aberrant glycosylation in cancer cells often results in the exposure of tumorassociated carbohydrate structures and in enhanced expression of GalNAca1–Ser/Thr (Tn antigen), Neu5Aca2–6GalNAc (sialyl-Tn antigen), and Galb1–3GalNAca1–Ser/Thr (Thomsen–Friedenreich, TF or T, antigen). The TF or T antigen represents the core 1 structure of O-linked mucin-type glycans. The TF antigen is hidden in normal epithelium by sialic acids and sulfates or by the addition of other sugar chains to produce branched and complex O-glycans. However, unsubstituted Galb1–3GalNAc antigen frequently occurs in a high percentage of tumors. Immunoglobulins G to TF, Tn, and aGal were detected in patients with breast cancer. Blood group antigens related to the ABO and Lewis system are found in the peripheral structure of glycoproteins and glycolipids. The expression of Lewis Y antigen structure (LeY) appears to be low in normal tissues, while it was found high in several tumors. LeY is also expressed on granulocytes. LeY circulating immune complexes were found in serum of breast and gastric cancer patients. Stage-specific embryonic antigen-1 (SSEA-1) (LeX) was found to be expressed on normal epithelial cells, leukocytes, bone marrow, macrophages, spleen cells, and in some areas of the central nervous system. Increased SSEA-1 expression was found in several tumors. Antibodies to LeX were detected in patients with gastric cancer. Gangliosides (G) are complex glycosphingolipids mono- (M), di- (D), or tri-sialylated; the number (1, 2, 3) in their nomenclature represents the order of their distance of migration in thinlayer chromatography. Aberrant expression of gangliosides has been found in neural crestderived tumors. As a result of neoplastic transformation, normal melanocytes expressing GM3 begin to produce large amounts of GD3. GD3 and GM2 were also found in small cell lung cancers (SCLCs). Antibodies to GD1a/b and GM1 were found in patients with lung and gastric cancer, melanoma, and B-cell lymphoma. In addition, antibodies against GM1 gangliosides were associated with metastatic melanoma (see also Reuschenbach et al. 2009).

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Fucosyl GM1 (FucGM1) appears to be a particular ganglioside form expressed in SCLC and with a very low expression in normal tissues. Autoantibodies against FucGM1 were found at low titer in few SCLC, renal carcinoma patients, and in healthy controls. Tissue-lineage antigens are expressed in a tumor of a certain histotype and in the normal tissue from which the tumor is derived. This group of antigens contains melanoma differentiation antigens such as gp100, Melan-A, tyrosinase and tyrosinase-related protein 1 (TYRP1/gp75), prostate-specific antigen (PSA), and idiotypic antigens. Melanoma differentiation antigens are abundantly expressed in most primary and metastatic melanomas. The in vitro stimulation of peripheral PBMCs with peptides from Melan-A/ MART-1 (MART-127–35), gp100, tyrosinase, and TYRP1/TYRP2 was demonstrated to induce CTLs in melanoma patients. In addition, serum antibodies from a melanoma patient were able to immunoprecipitate TYRP1/gp75 (see also Yang and Yang 2005). PSA is a 33- to 34-kDa serine proteinase. The serum PSA test is still the most significant biomarker for the detection and follow-up of prostate cancer; the usefulness may be improved by determination of PSA isoforms in conjunction with free PSA. Circulating autoantibodies to PSA were found in the serum of benign prostatic hyperplasia and prostate cancer patients. In addition, recognition of PSA-derived peptide antigens by T cells from prostate cancer patients was demonstrated. An idiotype represents a unique and characteristic antigenic determinant of an immunoglobulin (Ig) or T-cell receptor. A unique Ig is expressed on the surface of a B lymphocyte. The variable regions of the heavy and light chains of the Ig contain unique determinants called idiotype. Accordingly, a B-cell lymphoma following clonal proliferation of a B cell will express an antigen which cannot only be regarded as a tissue-lineage antigen but has to be considered also as a tumorspecific antigen. It has been demonstrated that CD5-positive B-cell malignancies frequently express cross-reactive idiotypes associated with IgM autoantibodies.

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Overexpressed antigens represent antigens that even if present in normal tissues are present at higher levels in tumor tissues. According to this definition, several of the antigens listed above can be grouped into this category (see also Khodadoust and Alizadeh 2014). Certainly in this category can be included several receptors with tyrosine kinase activity (RTKs) and whose overexpression contributes to the development of the tumor. Among other RTKs, members of the epidermal growth factor receptor (EGFR) family, including EGFR, ErbB2, ErbB3, and ErbB4, have frequently been implicated in human neoplasia by overexpression in the presence or absence of gene amplification. Figure 1 shows overexpression of EGFR and ErbB2 in well-differentiated head and neck squamous cell carcinomas. Humoral response to all ErbB family receptors and T-cellmediated immunity to ErbB2 were demonstrated. Another example of molecules overexpressed by cancer cells is ribosomal proteins which were also demonstrated to induce immune responses in cancer patients. Among ribosomal proteins, the ribosomal P0 protein is overexpressed in colon cancer, hepatocarcinoma, head and neck cancer, and in breast cancer. Overexpression of the 22 amino acid C-terminal epitope of P0 in a well-differentiated head and neck squamous cell carcinoma is provided in Fig. 1. Humoral response to the 22 amino acid C-terminal peptide of P0 has been demonstrated in breast cancer and head and neck cancer patients. Ribosomal protein S6 was found to be overexpressed in non-Hodgkin lymphoma, breast, colon, and renal cell carcinomas. Autoantibodies to S6 were detected in patients with breast cancer. Similarly, ribosomal protein L19 (RPL19) was found to be overexpressed in NSCL cancer tissues and to induce CTL. Overexpression of heat shock proteins (Hsp27, Hsp70, Hsp90) has been found in several tumors. Several HSPs were shown to elicit antibodies in breast and ovarian cancer patients. Patients with AML showed significantly higher anti-Hsp70 antibody concentrations compared to the control group. The transcription factor MYC is involved in cell growth, transformation, angiogenesis, and

Tumor Antigens

cell-cycle control. MYCN amplification has been reported in several tumors. Humoral responses to MYC have been detected in serum of breast, lung, hepatocellular, and colorectal cancer patients. Osteopontin (OPN) expression is deregulated in prostate neoplastic lesions. The frequency of anti-OPN antibodies was found higher in prostate cancer (66%) patients as compared to benign prostate hyperplasia patients (33%) and healthy donors (10%). The human homolog of the murine doubleminute 2 oncoprotein (MDM2) is a putative TAA because it is overexpressed in several malignancies, including CLL cells. MDM2specific T cells were generated in 7/12 CLL patients. Mesothelin is a glycoprotein expressed on normal mesothelial cells and highly expressed on mesothelioma, ovarian, and pancreatic cancer. Autoantibodies to mesothelin were detected in serum of mesothelioma and ovarian cancer patients. In addition, specific mesothelin T cells were generated at high frequency from PBLs of pancreatic cancer patients. Survivin, a member of the inhibitor of apoptosis protein family, is frequently expressed in cancers. Anti-survivin antibodies were observed in patient with brain tumors. Survivin-specific T-cell responses in healthy donors and cancer patients were also demonstrated. Virus-induced tumors express virally encoded proteins. Examples of virally encoded proteins are those produced after human papillomavirus (HPV) and Epstein–Barr virus (EBV) infection. Infection with HPV is a risk factor for the development of cervical cancer. Oral HPV infection has been associated with some cases of oropharyngeal cancer. Lymphoproliferative responses to specific HPV16 E6 and E7 peptides and antibodies to E6 and E7 proteins were detected in HPV-infected individuals (see also Yang and Yang 2005). EBV is a gamma herpes with potent B-cell transforming activity associated with various B-cell malignant diseases and nasopharyngeal carcinoma (NPC). T lymphocytes can discriminate EBV-infected or EBV-transformed B cells.

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Tumor Antigens, Fig. 1 EGFR, ErbB2, and P0 overexpression in head and neck squamous cell carcinoma. Immunohistochemical detection. Immunoperoxidase counterstained with hematoxylin, original magnification 200

Elevated titers of IgA antibodies to EBV replicative antigens frequently precede the appearance of NPC.

Cross-References ▶ Alpha-Fetoprotein ▶ BCR-ABL1 ▶ Cancer ▶ Carcinoembryonic Antigen

▶ Cyclin-Dependent Kinases ▶ Epidermal Growth Factor Receptor ▶ Gangliosides ▶ MDM2 ▶ Mesothelin ▶ Mesothelioma ▶ Mucins ▶ MYC Oncogene ▶ Oncofetal Antigen ▶ Osteopontin ▶ Prostate-Specific Antigen

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▶ Retinoblastoma ▶ Survivin ▶ Wilms’ Tumor

References Canevari S, Pupa SM, Ménard S (1996) 1975–1995 revised anti-cancer serological response: biological significance and clinical implications. Ann Oncol 7:227–232 Heimburg-Molinaro J, Lum M, Vijay G, Jain M, Almogren A, Rittenhouse-Olson K (2011) Cancer vaccines and carbohydrate epitopes. Vaccine 29:8802–8826 Khodadoust MS, Alizadeh AA (2014) Tumor antigen discovery through translation of the cancer genome. Immunol Res 58:292–299 Reuschenbach M, von Knebel Doeberitz M, Wentzensen N (2009) A systematic review of humoral immune responses against tumor antigens. Cancer Immunol Immunother 58:1535–1544 Van den Eynde BJ, van der Bruggen P (1997) T cell defined tumor antigens. Curr Opin Immunol 9:684–693 Yang F, Yang XF (2005) New concepts in tumor antigens: their significance in future immunotherapies for tumors. Cell Mol Immunol 2:331–341

Tumor Cell Invasion ▶ Invasion

Tumor Cell-Induced Platelet Aggregation Mei-Chi Chang1 and Jiiang-Huei Jeng2 1 Biomedical Science Team, Chang Gung Institute of Technology, Taoyuan, Taiwan 2 Laboratory of Pharmacology and Toxicology, School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan

Synonyms Cancer cell-platelet microemboli; Pathological tumor cell-platelet interaction; Tumor cell-platelet aggregate; Tumor cell-platelet interaction; Tumor-induced platelet aggregation

Tumor Cell Invasion

Definition Tumor cell-induced platelet aggregation (TCIPA) is the ability of cancer cells to generate crucial molecules or surface receptor molecules that mediate platelet aggregation and accelerate the survival advantages of tumor cells in the vasculature, which is important for distant ▶ metastasis of cancer.

Characteristics Hematogenous metastasis of cancer cells to critical distant organs is one of the major reasons for death in most cancer patients. After ▶ invasion into blood vessels, circulating cancer cells may interact with various vascular cells such as leukocytes, platelets, and endothelial cells that may affect the survival and metastasis of tumor cells. Although normal platelet functions are important to control vascular hemostasis and thrombosis, cancer cells may express many factors which modulate the platelet activities. Clinically, the interactions between circulating cancer cells and platelets are crucial for tumor metastasis in patients with cancers. Cancer cells may induce the morphological changes and aggregation of platelets and subsequently trap the cancer cells in capillaries and enhance the adhesion of cancer cells to capillary endothelial walls. These events facilitate further tumor cell invasion and metastasis into distant organs. During these processes, the activated platelets release various factors (like serine proteases factor VIIa, factor Xa, ▶ plateletderived growth factor (PDGF), ▶ vascular endothelial growth factor (VEGF), fibrinogen, and thrombospondin), which increase vascular permeability and promote the growth, survival, motility, and the extravasation of metastatic cancer cells to neighboring organs. Expression of PDGFreceptor in breast cancer tissues has been reported to increase the risk of lymph node metastasis. Similarly, the expression of von Willebrand factor (vWF) in tumor tissues and serum may also affect the metastasis of ▶ osteosarcoma patients. A number of clinical studies further suggest the increased risk of thrombosis in patients with

Tumor Cell-Induced Platelet Aggregation

various cancers, indicating the roles of TCIPA and activation of the coagulation system in the progression of cancer. Mechanisms Induction of TCIPA may form platelet-cancer cell aggregates to prevent cancer cells from killing by human cellular and humoral immunities and from the damage by sheer force of blood flow. Platelet activation further facilitates ▶ angiogenesis, tumor cell adhesion to endothelium, and subsequent invasion into distant organs. Various cancer cells may produce differential amounts of critical factors such as ADP, thromboxane A (TXA), thrombin, cathepsins, ▶ matrix metalloproteinases (MMPs), membrane-type matrix metalloproteinases (MT-MMP), cancer procoagulant, mucin, tissue factors (TF), aggrus/ ▶ podoplanin, etc. These molecules may activate various receptors (P2Y12 purinergic receptor, thromboxane receptors, ▶ protease-activated receptors (PARs), integrin receptors, etc.) and downstream signaling cascades to initiate platelet aggregation and coagulation disorders. TF-bearing cancer cells can stimulate platelet aggregation via generation of thrombin and signaling by glycoprotein IIb/IIIa and VEGF release. The localized production of thrombin and fibrin may increase endothelial cell motility as well as platelet activation. Tumor cells have the capacity to convert fibrinogen into fibrin in vitro, and histologically the deposition of fibrin is popularly noted in the connective tissue surrounding tumor cells, an event being critical for tumor angiogenesis and metastasis. Generation of MMPs by tumor cells may activate platelet integrin receptors followed by induction of platelet aggregation. This event can be inhibited by anti-MMP antibodies and phenanthroline, an MMP inhibitor. MMPs generated by tumor cells and platelets are further shown to degrade the basement membrane and facilitate metastasis. Cancer cells may also activate platelets to release ADP, which binds further to platelet P2Y12 receptors, leading to full platelet aggregation. The ADP-mediated platelet aggregation by tumor cells can be attenuated through degradation of ADP by apyrase (APT102). Moreover, the

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interaction between platelets and tumor cells can be mediated by cell adhesion proteins, e.g., P-selectin in platelets and P-selectin glycoprotein ligand-1/CD162, heparin sulfate proteoglycan, and sialyl-Leqis A/X in cancer cells. This is followed by b3 integrin-mediated processes with concomitant activation of thrombospondin and fibrinogen. Using venous blood from cancer patients for testing, current techniques can detect the presence of circulating tumor cells in the bloodstream. Interestingly, podoplanin (Aggrus), a mucin-line glycoprotein, has been shown to induce platelet aggregation and affect the invasion/metastasis of cancer. The expression of podoplanin in astrocytic tumors has been linked to the malignancy of astrocytic tumors. This is possibly due to early interactions by surface mucins and other selectin ligands of tumor cells with the platelet P-selectin and L-selectin. Heparin as an antithrombotic agent is able to inhibit metastasis further by blocking the tumor cell mucin P-selectin ligands and thus also the platelet-tumor cell interaction. Clinical Aspects and Therapy Higher platelet numbers have been found in many kinds of cancers including lung, gastric, colorectal, and breast cancers. Clinically, cancer patients also have a higher risk for development of vascular thromboembolism. This can be attributed to abnormalities in platelet functions as well as in the thrombotic and hemostatic systems by tumor cellreleased factors. Understanding the mechanisms for TCIPA is helpful for the clinical design of therapeutic agents to control tumor metastasis. A number of clinical studies have found higher levels of thromboxane and lower levels of prostacyclin in plasma and tumor tissues from lung, bone, and breast. Moreover reports have also observed the overexpression of cyclooxygenase2 (COX-2) and thromboxane synthase in the tumors of colon, breast, prostate, brain, and endocrine. Some tumors may generate excessive amounts of TXA2 to enhance tumor growth and metastasis. An elevation of urinary 11-dehydroTXB2 (a major enzymatic metabolite of TXB2) in patients with colorectal cancers has been noted, suggesting the beneficial use of a low-dose

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COX-1 inhibitor to reduce platelet activity in some cancer patients. BM-567, a thromboxane synthase inhibitor and TXA2 receptor antagonist, can inhibit the MG-63 osteosarcoma cell-induced platelet aggregation. Administration of SQ29548, the other TXA2 receptor antagonist, also reduces chondrosarcoma tissue TXB2 levels, vascular permeability, and tumor size in experimental animals in vivo. Whether TXA2 receptor antagonists can be effectively used clinically to prevent tumor metastasis should be further addressed. ▶ Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen may reduce the risk of cancer in colon, esophagus, stomach, prostate, and lung as well as the metastasis of prostate cancer. Aspirin as a COX-1 inhibitor and antiplatelet agent is also tested for its anti-metastatic effects. In experimental animals, aspirin may reduce the metastasis of injected ▶ hepatocellular carcinoma cells to lung. However, a multicenter study shows little effect of aspirin in combination with chemotherapy to improve survival of 303 patients with small cell ▶ lung cancer. More studies on the dosage, duration, and timing of aspirin or other therapeutic administrations are needed. In experimental animals, heparin administration seems effective in the suppression of tumor metastasis, but not primary tumor growth. This can be due to its inhibition of blood coagulation, tumor cell-platelets, and ▶ tumor-endothelial cross talks. Accordingly, clinical administration of aspirin and heparin to humans may suppress platelet aggregation and therefore promote survival in cancer patients. In addition, heparin, warfarin (a vitamin K antagonist), and antiplatelet agents (prostacyclin and dipyridamole) are able to inhibit the metastasis of ▶ pancreatic cancer cells and melanoma cells to the liver and lung. Intriguingly, studies have found that low molecular weight heparin (LMWH) successfully prevents the progression of cancer and improves the survival of patients with advanced ▶ non-small cell lung cancer, colon, pancreatic, breast, and pelvic cancers. Since tumor cells may also express PARs, glycoprotein IIb/IIIa, and integrin avb3, inhibition of these receptors and activation and signaling by agents such as hirudin, PPACK,

Tumor Cell-Induced Platelet Aggregation

antithrombin III, and receptor neutralizing antibodies are potentially useful methods for inhibition of TCIPA and metastasis by some tumor cells. XV454, a glycoprotein IIb/IIIa antagonist, has been shown to suppress the metastasis of Lewis lung carcinoma cells in experimental mouse metastasis models. A combination of LMWH and XV454 is even more effective in reducing cancer-induced thrombosis in vitro. Currently, some new reagents are being tested for their inhibition of TCIPA. Administration of antiplatelet agents (such as antiplatelet antibody, dansylarginine N-(3-ethyl-1, 5-pentanediyl) amide) before inoculation of tumor cells has been shown to effectively reduce tumor metastasis to lung and bone tissue. Integrilin, a plateletspecific integrin inhibitor, was able to effectively suppress TCIPA, but showed only partial inhibitory effects on physiological platelet functions. An elevated expression of aggrus/podoplanin, a new platelet aggregation-inducing factor in tumor tissues, has been found and promotes pulmonary metastasis in experimental animals. Administration of anti-podoplanin antibody is also shown to inhibit the neuroglioblastoma cell-induced platelet aggregation. More clinical trials by inhibition of TCIPA using antiplatelet and anticlotting agents are now in progress. These results will be helpful in developing effective clinical strategy and regimens for antimetastatic therapy of cancer in the near future.

Cross-References ▶ Angiogenesis ▶ Cyclooxygenase ▶ Hepatocellular Carcinoma ▶ Invasion ▶ Lung Cancer ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Non-Small-Cell Lung Cancer ▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Osteosarcoma ▶ Pancreatic Cancer ▶ Platelet-Derived Growth Factor

Tumor Microenvironment

▶ Podoplanin ▶ Protease-Activated Receptors ▶ Tumor–Endothelial Cross-Talk ▶ Vascular Endothelial Growth Factor

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Tumor Grading ▶ Grading of Tumors

References Gupta GP, Massague J (2004) Platelets and metastasis revisited: a novel fatty link. J Clin Invest 114:1691–1693 Hejna M, Raderer M, Zielinski CC (1999) Inhibition of metastasis by anticoagulants. J Natl Cancer Inst 91:22–36 Jurasz P, Alonso-Escolano D, Radomski MW (2004) Platelet-cancer interactions: mechanisms and pharmacology of tumor cell-induced platelet aggregation. Br J Pharmacol 143:819–826 Medina C, Jurasz P, Santos-Martinez MJ et al (2006) Platelet aggregation-induced by caco-2 cells: regulation by matrix metalloproteinase-2 and adenosine diphosphate. J Pharmacol Exp Ther 317:739–745 Timar J, Tovari J, Raso E et al (2005) Platelet-mimicry of cancer cells: epiphenomenon with clinical significance. Oncology 69:185–201

See Also (2012) Cyclooxygenase-2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1035. doi:10.1007/978-3-642-16483-5_1435 (2012) Glycoprotein. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2451 (2012) Thromboxane. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3682. doi:10.1007/978-3-642-16483-5_5798

Tumor Markers ▶ Alpha-Fetoprotein ▶ Clinical Cancer Biomarkers

Tumor Metabolism ▶ Antiglycolytics and Cancer

Tumor Microenvironment Isaac P. Witz Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel

Synonyms Cancer (or tumor) stroma

Definition

Tumor Cell-Platelet Aggregate ▶ Tumor Cell-Induced Platelet Aggregation

The specific conditions existing in the tumor tissue, the nonmalignant cells and the molecules present in proximity to the tumor cells.

Tumor Cell-Platelet Interaction

Characteristics

▶ Tumor Cell-Induced Platelet Aggregation

It is now widely accepted that “Although abnormalities of cancer genes (▶ Oncogene; ▶ tumor suppressor genes) are essential contributors to cancer, most abnormalities in these genes occur relatively early in the disease process and none of them is known to be associated with the metastatic stage. It is this final stage – the seeding and growth

Tumor Glucose Metabolism ▶ Warburg Effect

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of satellite lesions in other organs – that is ultimately responsible for the great majority of neoplastic deaths.” It is the tumor microenvironment that determines and shapes the malignancy phenotype of cancer cells, in other words its metastatic behavior (▶ Metastasis). The tumor tissue can be viewed as an ecosystem composed of two compartments being intimately associated with each other. The first compartment constitutes the malignant cells. The second is the tumor microenvironment composed of resident cells such as fibroblasts, endothelial cells (▶ Tumor-Endothelial Cross-talk), and other nonmalignant cells; of infiltrating cells such as lymphocytes or macrophages (▶ Tumorassociated macrophages) and of numerous molecules released by the tumor cells as well as by the non malignant cells. These molecules may be in complex with other molecules, for example, in the extracellular matrix. Other molecules such as growth factors, cytokines, ▶ chemokines, antibodies, proteases, other types of enzymes, various metabolites, or drugs may be present in soluble form. The microenvironment of many solid tumors may be characterized by ▶ hypoxia (Hypoxia and Tumor Physiology; ▶ hypoxia inducible factor-1); low extracellular pH and by low glucose concentration. Cellular products released from necrotic tumor cells are also present. Although the term tumor microenvironment is used most often with respect to solid tumors, other types of malignancies have also their specific microenvironments. The bone marrow serving as a microenvironment for certain leukemias and for ▶ multiple myeloma is a case in point. Stephen Paget, over 100 years ago, is credited with being the first to postulate the important role played by the microenvironment in metastasis formation. The concept of his “Seed and Soil” theory, explaining site specific metastasis in ▶ breast cancer, has been supported and confirmed. However, numerous studies published in the last three decades demonstrate very clearly that the “soil” functions also, or even primarily, as an active “educational/inductive” venue in which cancer cells are directed, by interacting with microenvironmental factors, into one of

Tumor Microenvironment

several molecular evolution pathways. In other words, by exerting regulatory functions and selective pressures, the tumor microenvironment determines and shapes the malignancy phenotype of cancer cells. The tumor microenvironment is an interaction arena between microenvironmental components and tumor cells and between different microenvironmental components. This arena is characterized by four major hallmarks: complex regulatory circuits; a yin-yang (double edged sword) interplay; plethora of vicious cycles; abnormality of its “normal,” nonmalignant compartment. Complex Regulatory Circuits The major function of the tumor microenvironment is a regulatory one. Many genes in the tumor cells and in nontumor cells residing in or infiltrating to the tumor microenvironment are regulated by microenvironmental components. Several 100 proteins were identified in the microenvironment of breast cancer. An extremely large number of signaling cascades (▶ Signal transducers and activators of transcription in oncogenesis; ▶ signal transduction) would operate in this microenvironment even if only a small portion of these proteins would interact with tumor cells or with nontumor cells. It is safe to predict that a similar number of proteins will be detected in the microenvironment of other types of solid tumors. These signaling cascades take part in the regulation of genes in tumor and in nontumor cells thereby shaping the phenotype of cancer cells and drive their ▶ progression. The regulatory power of the microenvironment can be amplified by the agonistic or antagonistic cross-talk (▶ Receptor cross-talk) between different signaling cascades. Furthermore, several signaling cascades in cancer cells are aberrant. This may well increase the number of combinatorial signaling pathways, augment their complexity, and decrease the capacity of physiological feedback mechanisms to confront these malignancyassociated processes. Another factor contributing to the complexity of the interactions taking place in the tumor microenvironment is tumor

Tumor Microenvironment

heterogeneity. It is thus to be expected that different tumor variants, expressing different profiles of signaling receptors (▶ Receptor tyrosine kinases), would respond differentially to microenvironment-derived signals. The Yin-yang (Double Edged Sword) Interplay in the Tumor Microenvironment The cross-talk between tumor cells and microenvironmental factors may result in diametrically opposed effects which could either enhance or block tumor formation or progression. There are several examples of such a yin-yang interaction. The activity of ▶ transforming growth factorbeta (TGFb) is an example for a microenvironmental molecule manifesting a “love–hate relationship” with tumor cells. Whereas TGFb is a potent inhibitor of normal mammary epithelial cells, it enhances tumor cell ▶ invasion and metastasis of advanced breast cancer cells (Epithelial Tumors). Moreover, cancer cells may secrete TGFb which augments ▶ angiogenesis and is capable of suppressing antitumor immune responses of the host. On the other side of the coin, it was demonstrated that the progression of pancreatic and of intestinal tumors is enhanced by the inactivation of the TGF signaling cascade. Another prominent example for yin-yang interplay in the tumor microenvironment is ▶ inflammation versus protective tumor immunity. Cells and molecules of the immune system may, under certain circumstances, inhibit tumor growth and under different circumstances promote it. Vicious Cycles in the Tumor microenvironment A vicious cycle may be described as an input event that drives and amplifies other events which, in turn, promote tumor progression. Among such activities, the input event may also augment itself (positive feedback). A well studied vicious cycle in the tumor microenvironment is the cross-talk between osteoblasts, osteoclasts and other microenvironmental factors on the one hand and breast, prostate (▶ Prostate cancer clinical oncology) and lung (▶ Lung cancer) tumor cells on the other hand. This

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cross-talk promotes bone metastasis (▶ Bone tropism). Tumor-derived molecules such as cytokines cause either an osteoblastic or an osteolytic response. Such molecules feedback on the tumor and on various cells in the microenvironment causing the release of factors driving tumor progression. Another example of a vicious cycle in the tumor microenvironment is the cross-talk between chemokines (▶ Chemoattraction; ▶ chemokine) such as CCL2 and CCL5 secreted from mammary tumors of mice or from breast cancer cells of humans and cytokines such as TNFa secreted from macrophages infiltrating into these tumors. The tumor-derived chemokines attract monocytes to the microenvironment. These monocytes differentiate into macrophages which secrete TNFa. This cytokine up regulates the secretion of CCL2 and CCL5 from the tumor cells. CCL2 and CCL5, in turn, promote the secretion of TNFa from the tumor-associated macrophages. In this vicious cycle, the tumor cells and the macrophages promote each other’s ability to express and secrete pro malignancy factors. Hypoxia (Hypoxia and tumor physiology; hypoxia inducible factor-1) characterizes the microenvironment of solid tumors. Hypoxiainduced changes in the proteome (▶ Proteomics) may lead to either impairment of tumor growth and spread or, alternatively, to tumor propagation and progression. In the later case, a vicious cycle is created in which tumor cells surviving and propagating under hypoxia will aggravate the state of tumor hypoxia which in turn promotes genomic instability and further progression. The Nontumor Cells in the Tumor Microenvironment may Express a Different Phenotype than Their Counterparts at Distant Sites The conditions in the tumor microenvironment, for example, hypoxia, may induce or promote genetic instability and cause mutations and alterations in gene expression profiles of cancer cells (Genetic polymorphisms). It is not unlikely that such conditions may induce genetic alterations also in nontumor cells present in the microenvironment. The question, if the phenotype and

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functions of nontumor cells in the tumor microenvironment are similar or different from those of their counterparts in normal microenvironments, is by and large open. Several studies clearly demonstrate that at least some of the nontumor cells in the tumor microenvironment may not represent faithfully the characteristics of their counterparts in other sites of the body. Cancer-associated fibroblasts and endothelial cells are two prominent examples that illustrate the abnormality of tumor-associated nontumor cells. Cancer-associated fibroblasts have genetic changes both at the DNA level as well as at the expression level. For example, fibroblasts in human carcinomas have tumor suppressor gene mutations. DNA microarrays (▶ Microarray (cDNA) technology) identified over 100 genes differentially expressed by prostate carcinoma (Prostate cancer, clinical oncology)-derived fibroblasts and by systemically derived ones. These alterations may well manifest themselves by altered functions of such tumor-associated cells. Similar findings were also reported for endothelial cells. Cytogenetic abnormalities have been shown to occur in tumor endothelium and such cells may also express proteins that are not expressed by endothelial cells of the corresponding normal tissue. Site Specific Metastasis and the Metastatic Microenvironment Paget already realized that there is a predilection of tumors to metastasize to specific organ sites and that the metastatic capacity of a certain tumor is not restricted to a single organ site. Each tumor type has therefore several different potential metastatic microenvironments. Since the tumor and its microenvironment regulate and shape each other’s phenotype, it is to be expected that the metastases arising in one organ site be different from metastases derived from the very same tumor developing in a different organ site. It is also assumed that different reciprocal signaling cascades take place between metastases and nontumor microenvironmental cells in different metastatic microenvironments. These assumptions are indeed supported by experimental evidence.

Tumor Microenvironment

Dormant Micrometastasis and Microenvironmental Control Many organs including those of healthy people harbor solitary tumor cells or very small clusters of such cells. These cells do not proliferate either due to a balance between proliferation and apoptosis or due to cell cycle arrest. There is strong experimental support that such cells are precursors for metastasis and that microenvironmental control mechanisms keep these micrometastatic cells under check, i.e., in a state of dormancy. It is not unlikely that breakdown of these control mechanisms is responsible for the awakening of dormant micrometastases and their progression towards frank metastasis. The Tumor Microenvironment as Target in Cancer Therapy Tumor autonomous factors as well as the tumor microenvironment cooperate in the formation of primary tumors and of metastasis. It is therefore not surprising that cells and molecules that originate in the microenvironment and that drive tumor progression do serve as candidates for cancer therapy. Moreover the fact that nontumor cells in the tumor microenvironment express a different phenotype than that expressed by the corresponding cells in the normal microenvironment (see above) might also be exploited in cancer therapy modalities, targeting these differentially expressed molecules. Numerous clinical trials targeting various components of the tumor microenvironment are in progress.

Cross-References ▶ Angiogenesis ▶ Breast Cancer ▶ Chemoattraction ▶ Chemokines ▶ Desmoplasia ▶ Epithelial Tumorigenesis ▶ Hypoxia ▶ Hypoxia-Inducible Factor-1 ▶ Invasion ▶ Lung Cancer

Tumor Necrosis Factor

▶ Metastasis ▶ Microarray (cDNA) Technology ▶ Multiple Myeloma ▶ Oncogene ▶ Progression ▶ Proteomics ▶ Receptor Cross-Talk ▶ Receptor Tyrosine Kinases ▶ “Seed and Soil” Theory of Metastasis ▶ Signal Transducers and Activators of Transcription in Oncogenesis ▶ Signal Transduction ▶ Tissue Inhibitors of Metalloproteinases ▶ Transforming Growth Factor-Beta ▶ Tumor-Associated Macrophages ▶ Tumor–Endothelial Cross-Talk ▶ Tumor Suppressor Genes

References Baglole CJ, Ray DM, Bernstein SH et al (2006) More than structural cells, fibroblasts create and orchestrate the tumor microenvironment. Immunol Invest 35:297–325 Bierie B, Moses HL (2006) Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 6:506–520 Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12:89–96 Goss PE, Chambers AF (2010) Does tumour dormancy offer a therapeutic target? Nat Rev Cancer 10:871–877 Klein-Goldberg A, Maman S, Witz IP (2014). The role played by the microenvironment in site-specific metastasis. Cancer Lett 352:54–58 Maman S, Witz IP (2013) The metastatic microenvironment. In: Shurin MR et al (eds) The tumor microenvironment. Springer Science+Business Media, pp 15–38 Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799 Witz IP, Levy-Nissenbaum O (2006) The tumor microenvironment in the post-PAGET era. Cancer Lett 242:1–10 Witz IP (2008) Tumor-microenvironment interactions: dangerous liaisons. Adv Cancer Res 100:203–229 Witz IP (2008) The selectin-selectin ligand axis in tumor progression. Cancer Metastasis Rev 27:19–30 Witz IP (2008) Yin-yang activities and vicious cycles in the tumor microenvironment. Cancer Res 68:9–13 Witz IP (2009) The tumor microenvironment: the making of a paradigm. Cancer Microenviron Suppl 1:9–17

See Also (2012) Genetic Polymorphism. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1528. doi:10.1007/978-3-642-16483-5_2382

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Tumor Necrosis Factor Wen-Ming Chu Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI, USA

Synonyms Cachectin; Differentiation-inducing Endotoxin-induced factor in serum

factor;

Definition The tumor necrosis factor (TNF) family includes TNF alpha (TNFa) and TNF beta (TNFb), which are among the most important cytokines involved in systemic inflammation, tumor lysis, and initiation of the acute-phase reaction.

Characteristics Discovery More than 100 years ago, Dr. Williams B. Coley used crude bacterial extracts to treat tumor patients. He found that the bacterial extracts had an ability to induce tumor necrosis. While tumors were regressive, patients receiving bacterial extracts also suffered from a severe systematic inflammatory reaction. One of the major inflammatory stimulators now known to cause this reaction was identified in 1975, when a protein factor in the serum of endotoxin-treated animals was found to cause lysis in tumor cells and was therefore named “tumor necrosis factor.” In 1984, TNF gene was isolated and characterized. About the same time, another gene encoding a protein, which was purified from T lymphocytes and named “T lymphotoxin alpha (TLa)” in 1968, was also isolated and characterized. It was found that these two genes were in the same family. Thus, TNF was named “TNFa,” while TLa was called “TNFb.” In 1985, Nobel Prize winner Bruce Beutler and his colleagues purified a protein called cachectin from the supernatant of

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endotoxin-treated macrophages. This protein induced wasting (cachexia) and septic shock in murine recipients. Cachectin and TNF were later revealed to be the same protein. Sources TNFa is mainly produced by macrophages, whereas TNFb is mainly produced by T lymphocytes. Other cells can also express TNFa and TNFb at low levels. Genes and Proteins The human and murine TNFa genes are located on chromosome 6 and chromosome 17, respectively, and are preceded by the TNFb gene. Both genes are present in a single copy, are approximately 3 kb in size, and contain four exons. Several DNA-binding sites for the transcription factor nuclear factor kappa B (NF-kB) have been identified within the promoter region of the TNFa gene; therefore, it appears that the expression of TNFa is NF-kB dependent. A DNA-binding site for the high-mobility group 1 (HMG1) protein is located in the promoter region of the TNFb gene. Two forms of TNFa exist: the membranebound form (mTNFa) and the soluble form (sTNFa). Human mTNFa contains 157 amino acids (aa) and a 76 aa leader sequence, while mouse mTNFa contains 156 aa plus a 79 aa leader sequence. During synthesis, TNFa translocates to the cell membrane where the TNFa converting enzyme (TACE) sheds the mTNFa into sTNFa. In contrast to TNFa, TNFb only exists in the soluble form (sTNFb). TNF is conserved among species. For example, human TNF is 80% homologous to mouse TNF. The homology between TNFa and TNFb in both species is approximately 30%. Biological Functions TNF exerts many important physiological and pathological actions. TNF causes tumor cell necrosis (a process that involves cell swelling, organelle destruction, and finally cell lysis) and apoptosis (a process that involves cell shrinking, the formation of condensed bodies, and DNA fragmentation). Furthermore, studies in

Tumor Necrosis Factor

TNFa- or TNFR-deficient mice have revealed that TNF plays an important role in the regulation of embryo development and sleep-wake cycle and that TNF is vitally important for host defense against bacterial and viral infection. TNF has been shown to be an endogenous pyrogen that causes fever. Additionally, chronic exposure to a low dose of TNF may cause cachexia, wasting syndrome, and depression. It has been suggested that TNF might exhibit antitumor activities. The most phenomenal and critical biological function of TNF is its stimulation of systematic inflammatory reactions. TNF is a vital player in animal models of endotoxin-induced septic shock and in chemotherapy-induced septic shock in latestage lung cancer patients. TNF plays a central role in rheumatoid arthritis (RA) and inflammatory bowel diseases including Crohn disease and ulcerative colitis, as well as other autoimmune diseases. Anti-TNF Therapy Although TNF induces lysis of tumor cells, it has never been considered as an anticancer drug. Instead, due to its crucial role in the pathogenesis of immune disorders as mentioned above, antiTNF treatment has been developed as a therapy for RA and Crohn disease patients. The strategies developed for blockade of TNF either utilize anti-TNF antibodies (Remicade/infliximab, adalimumab/Humira, and golimumab) to neutralize sTNF or utilize Fc fragments of human immunoglobulin 1 fused with extracellular domains of TNFR1 or TNFR2 (certolizumab/Cimzia and etanercept/Enbrel) to block the interaction of TNF with TNFRs. Overall, anti-TNF therapy has been very successful in ameliorating symptoms of immune disorders, although some side effects have been identified. For example, anti-TNF therapy may enhance the risk of tuberculosis infection and the development of lymphoma. Molecular Mechanisms The molecular mechanisms behind TNF’s actions have been extensively investigated. Particularly, scientists have been keen to elucidate how TNF

Tumor Necrosis Factor

triggers activation of the IKK/NF-kB and the MAPK/AP-1 pathways, which are essential for the expression of pro-inflammatory cytokines, and to understand how TNF induces apoptosis. TNF binds to the TNFR1 and TNFR2 receptors, which can be either membrane bound or soluble. TNFR1 and TNFR2 each interact with both mTNFa and sTNFa. However, TNFR1 signaling can be strongly activated by both mTNFa and sTNFa, while TNFR2 signaling can only be efficiently activated by mTNFa. TNFR1 is ubiquitously expressed, while TNFR2 is mainly expressed on lymphocytes and endoepithelial cells. Upon ligation, both TNFR1 and TNFR2 form homotrimers, but interestingly they do not form a TNFR1/TNFR2 heterodimer. TNFR1 has a death domain, which allows it to interact with other death domain-containing adaptor proteins, whereas TNFR2 lacks a death domain. Activation of the IKK/NF-kB and the MAPK/AP-1 Pathways

When TNFR1 binds to TNF, its conformation is changed such that its death domain can interact with TNFR-associated factor containing death domains (TRADD), which in turn recruits TNFR-associated factors (TRAFs) including TRAF2 and TRAF5, as well as the cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/ 2) to form the TNF receptor signaling complex (TNF-RSC). Next, the linear ubiquitin assembly complex (LUBAC), comprised of HOIL-1, HOIP, and SHARPIN, is recruited to the TNF-RSC by TRADD, TRAF2/5, and c-IAP1/ 2. LUBAC is not only required for the stabilization of TNF-RSC, but it also adds linear ubiquitin chains [e.g., methionine 1 (M1)-linked ubiquitin chain] to both RIP1 and the regulatory subunit of the IkappaB (IkB) kinase (IKK), IKKg (also called NEMO), thus bringing both RIP1 and IKKg to TNF-RSC. This results in the formation of the IKKg/IKK complex and the TAK1 (TGFb-activated kinase 1)/TAB1/2 (TAK1-binding proteins 1 and 2) complex. Intriguingly, c-IAP1/2, but not TRAF2, are E3 ligases that catalyze lysine (K) 11-, 48-, or 63-linked polyubiquitination of receptorinteracting protein (RIP1) and c-IAP1/2

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themselves, and this ligase activity is required for the recruitment of LUBAC to c-IAP-generated ubiquitin chains. The polyubiquitinated RIP1 triggers activation of TAK1, which in turn activates the catalytic subunits of IKK, IKKa, and IKKb. Although both IKKa and IKKb phosphorylate IkBa, IKKb is the major kinase leading to ubiquitination and degradation of IkBa and subsequently leading to NF-kB translocation to the nucleus where it initiates transcription of more than 200 NF-kBdependent genes, including cell survival genes, pro-inflammatory cytokines, chemokines, growth factors, and TNFa itself. TAK1 also activates mitogen-activated protein kinases (MAPKs), including JNK and p38. JNK phosphorylates both c-Jun and ATF2, and p38 phosphorylates ATF2 leading to the formation of a c-Jun/ATF2 heterodimer called activating protein 1 (AP-1). AP-1 is another critical transcription factor and has similar functions to NF-kB. Induction of Apoptosis

TNFR1 also uses TRADD to recruit the Fas-associated protein-containing death domain (FADD) and deubiquitinated RIP1. Both FADD and RIP1 interact with pro-caspase 8, resulting in its cleavage and activation. Activated caspase 8 in turn mediates the cleavage of pro-apoptotic protein Bid generating a truncated form tBid, which translocates to the mitochondria and decreases mitochondrial membrane potential resulting in cytochrome c release. Cytochrome c, together with the apoptotic protease-activating factor 1 (Apaf1), binds to the initiator pro-caspase 9 forming an apoptosome complex, which activates other caspases including 3 and 7 resulting in cell apoptosis. c-IAPs are able to interact with caspases 3 and 7 and inhibit TNF-induced apoptosis (Fig. 1). Conclusion TNF is one of the most important proinflammatory cytokines and plays a pivotal role in the pathogenesis of immune disorders. Further understanding of TNF’s actions and the mechanisms underlying TNF pathology will allow for the development of a new generation of anti-TNF

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Tumor Necrosis Factor

Tumor Necrosis Factor, Fig. 1 A model of TNFa signaling in inflammation and apoptosis. TNF binds to TNFR1 and TNRF2. TNFR1 interacts with TRADD using its death domain (DD), which in turn recruits TRAF2, TRAF5, and c-AIP1/2 to form the TNF-RSC. RIP1 and LUBAC are then recruited to the TNF-RSC, leading to ubiquitination of RIP1 and IKKg and formation of the IKKg/IKK and TAK1/TAB1/TAB2 complexes. Polyubiquitinated RIP1 triggers TAK1 activation, which in turn activates IKKa/b, JNK, and p38 leading to NF-kB and AP-1 activation, respectively. AP-1 and NF-kB bind to DNA-binding sites located in the promoter regions of their target genes and initiate gene expression. TNFR1 also uses

TRADD to recruit FADD and deubiquitinated RIP1. Both FADD and RIP1 use their DD domains to interact with the DD domain in the initiator pre-caspase 8, leading to its cleavage and activation. Activated caspase 8 triggers cleavage of Bid into tBid, which then enters mitochondria leading to a decrease in mitochondrial membrane potential and subsequent cytochrome C release. Cytochrome C, together with Aparf1 and caspase 9, forms an apoptosome complex that triggers apoptosis. Interestingly, TNFR2 does not have a DD domain, but it is able to form a complex with TRAF2 and TRAF5, leading to activation of NF-kB and AP-1 upon stimulation with TNF

therapies that will cause fewer side effects, yet still maintain high efficacy in the treatment of immune disorders.

complex and is required for TNF-mediated gene induction. Mol Cell 36(5):831–844 Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, Dikic I (2011) SHARPIN forms a linear ubiquitin ligase complex regulating NF-kB activity and apoptosis. Nature 471(7340):637–641 Tokunaga F, Nakagawa T, Nakahara M, Saeki Y, Taniguchi M, Sakata S, Tanaka K, Nakano H, Iwai K (2011) SHARPIN is a component of the NF-kBactivating linear ubiquitin chain assembly complex. Nature 471(7340):633–636

References Haas TL, Emmerich CH, Gerlach B, Schmukle AC, Cordier SM, Rieser E, Feltham R, Vince J, Warnken U, Wenger T, Koschny R, Komander D, Silke J, Walczak H (2009) Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling

Tumor Suppression

Tumor Necrosis Factor-Alpha Converting Enzyme ▶ ADAM17

Tumor Pathology ▶ Pathology

Tumor Progenitors ▶ Stem-Like Cancer Cells

Tumor Rejection of Non-Irradiated Tumor Areas ▶ Abscopal Effects

Tumor Spread ▶ Metastasis

Tumor Staging ▶ Staging of Tumors

Tumor Suppression Eric Stanbridge Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA

Definition Tumor suppression is the consequence of the functional presence and activity of ▶ tumor

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suppressor genes (TSGs). TSGs are recessive genes whose protein products appear to directly or indirectly negatively regulate cell proliferation, promote ▶ apoptosis, and maintain in vivo homeostatic growth and differentiation potential.

Characteristics Three major classes of genes are involved in cancer causation and progression: • Dominantly acting oncogenes, whose proteins serve to stimulate cell growth and survival • Recessive genes involved in ▶ repair of DNA • Recessive TSGs The recessive TSGs and DNA repair genes are often included under the rubric of tumor suppressor genes. The importance of TSGs in the genesis of cancer became apparent when individuals predisposed to early onset cancer were found to contain a mutated allele of a certain TSG in their germline. This condition predisposes the individual to earlier onset cancer at a significantly higher probability than individuals who possess a sporadic cancer of the same histologic type. This is best illustrated by the prototypic TSG, RB1, that predisposes to childhood ▶ retinoblastoma. The incidence of sporadic retinoblastoma is 1:40,000 and is often unilateral, whereas the incidence of heritable retinoblastoma within an affected family is 40% and often involves both eyes (bilateral retinoblastoma). The affected proband inherits the mutant allele from one of the parents. Thus, the classic presentation of a heritable recessive TSG is one of dominant autosomal inheritance. In the classic two-hit model, the affected individual inherits one mutant RB1 allele, and the second allele is eliminated somatically in the retinoblastoma tumor. In the case of sporadic retinoblastoma, both alleles are eliminated somatically. Since the identification of the RB1 TSG, a large number of cancer-predisposing germline mutations in TSGs have been found (Table 1), and they include DNA repair genes. Many, but not all (e.g., BRCA1 and BRCA2), are commonly

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Tumor Suppression

Tumor Suppression, Table 1 Predisposing germline mutations in tumor suppressor genes Associated cancer syndrome Familial retinoblastoma

Tumor suppressor gene RB1

Human chromosomal location 13q14

Wilms’ tumor Li-Fraumeni syndrome

Wt1 p53

11p13 17q11

Von Recklinghausen disease Neurofibromatosis type 2

NF1

17q11

NF2

22q12

Von Hippel-Lindau disease

VHL

3p25

Familial adenomatous polyposis Familial melanoma

APC

5q21

INK4a

9p21

Gorlin syndrome

PTC

9q22.3

Binds/regulates b-catenin activity p16lnk4a cdki for cyclinD/cdk4/ 6; p19ARF binds mdm2, stabilizes p53 Receptor for sonic hedgehog

Juvenile polyposis

DPC4

18q21.1

Transduces TGF-b signals

Cowden syndrome BZS, LDD Tuberous sclerosis complex Familial prostate carcinoma Peutz-Jeghers syndrome

PTEN

10q23

Dual specificity phosphatase

TSC2

16

Cell-cycle regulator

Basal cell carcinoma, medulloblastoma Pancreatic, colon, hamartomas Glioblastoma, prostate, breast Renal, brain tumor

NKX3.1

8p21

Homeobox protein

Prostate

LKB1

19p13

Serine/threonine kinase

Familial gastric cancer

E-cadherin

16q22.1

Cell adhesion regulator

Ataxia telangiectasia

ATM

11q23

P13K-like kinase

HNPCC

MSH2

2p22

HNPCC

MLH1

3p21

HNPCC HNPCC HNPCC Bloom syndrome Fanconi anemia Complementation Gr A

PMS1 PMS2 MSH6 BLM

2q31 7p22 2p16 15q26.1

Mut S homologue, mismatch repair Mut L homologue, mismatch repair Mismatch repair Mismatch repair Mismatch repair DNA helicase

Hamartomas, colorectal, breast Breast, colon, skin, lung carcinoma Leukemias, lymphomas Colorectal cancer

FAA

16q24.3

Complementation Gr C

FAC

9q22.3

Xeroderma pigmentosum (seven complementation groups)

XPA XPB

9q34.1 2q21

Gene function Transcriptional regulator of cell cycle Transcriptional regulator Transcriptional regulator/ growth arrest/apoptosis Ras-GAP activity ERM protein/cytoskeletal regulator Regulates proteolysis

Involved in DNA cross-link repair Involved in DNA cross-link repair Binds damaged DNA Helicase, part of TFIIH

Cancer type Retinoblastoma, osteosarcoma Nephroblastoma Sarcomas, breast/ brain tumors Neurofibromas, sarcomas, gliomas Schwannomas, meningiomas Hemangiomas, renal, pheochromocytoma Colorectal cancer Melanoma, pancreatic

Colorectal cancer Colorectal cancer Colorectal cancer Colorectal cancer Multiple Leukemia Leukemia Skin

(continued)

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Tumor Suppression, Table 1 (continued) Associated cancer syndrome

Nijmegen breakage syndrome Familial breast cancer Familial breast cancer

Tumor suppressor gene XPC XPD XPE XPF XPG NBS1

Human chromosomal location 3p 19q12.3 ?11 16p13 13q23-33 8q21

BRCA1

17q21

BRCA2

13q12

Gene function ? Helicase, part of TFIIH Binds damaged DNA Structure-specific endonuclease Structure-specific endonuclease Involved in DNA double-strand break repair Transcriptional regulator/DNA repair Transcriptional regulator/DNA repair

Cancer type

Lymphomas Breast/ovarian tumors Breast/ovarian tumors

Metastasis

APC HNPCC hMSH2 hMLH1 hPMS1 hPMS2

KRAS

P53 DPC4

Tumor Suppression, Fig. 1 Multistep progression model for colorectal cancer. Tumorigenesis proceeds through a series of cellular alterations, including hyperplasia, benign polyps of increasing size and disordered growth, and carcinoma in situ with localized invasion.

Genetic alterations associated with this progression include inactivation of TSGs, e.g., APC, p53, and DPC4, mutational activation of the KRAS oncogene, and inactivation of one of the DNA mismatch repair genes

found to be mutated in sporadic cancers of the same histologic type as those seen in the relevant familial cancer cases. Progression to the cancerous condition is a multistep phenomenon. This is best illustrated by the colorectal cancer model (▶ multistep development; Fig. 1). Tumorigenesis proceeds through a series of cellular alterations, including hyperplasia of the colonic epithelium, benign polyps of increasing size and disordered growth, and ▶ carcinoma in situ with localized invasion. Distant metastases may occur. Accompanying these cellular alterations are genetic alterations, including activating mutation of the KRAS proto-oncogene

and loss-of-function mutations in multiple TSGs. In the case of TSGs, mutations in both alleles must occur for complete loss of function. Loss of function of multiple TSGs is a hallmark feature of most, if not all, cancers. TSGs span a broad range of functions (Table 1). These include kinases, phosphatases, cyclin-dependent kinase inhibitors, transcription factors, cell adhesion molecules, proteins involved in specific protein degradation pathways, and a variety of DNA repair processes. There is increasing evidence that tumor suppressor proteins and DNA repair proteins interact in functional networks (an example is given

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Tumor Suppressor Genes DNA damage (double-strand breaks)

ATM

p53

Apoptosis

p21 ↑

CHK2

14−3−3 ↑

BRCA1

NBS1

Recombinational repair

G1 G2 (Cell-cycle checkpoint)

Tumor Suppression, Fig. 2 Networking of tumor suppressor and DNA repair proteins. In response to DNA damage, ATM phosphorylates and activates p53 and the cell-cycle kinase CHK2. This kinase also enhances the activation of p53 via phosphorylation. Activated p53 upregulates expression of the cyclin-dependent kinase inhibitor, p21, and 14–3–3 proteins, resulting in cellcycle arrest at the G1 and G2 checkpoints, respectively.

The activated p53 protein may also induce apoptosis. Transient cell-cycle arrest allows for repair of damaged DNA by preventing the duplication and propagation of damaged DNA. ATM also plays an important role in DNA repair by phosphorylating and activating BRCA1 and NBS1 proteins. See Table 1 for identification of the genes. ! = activation; ┤ = inhibition

in Fig. 2). The ATM kinase phosphorylates and activates the tumor suppressor p53 and CDSI/ CHK2 proteins and the DNA repair proteins, BRCA1 and NBS1. These activations lead to cell-cycle checkpoint arrest and repair of DNA damage. Loss of function of any one of these factors compromises DNA repair, culminating in genomic instability and increased probability of progression to the cancer phenotype.

References

Clinical Aspects Identification of specific germline mutations in TSGs in affected families allows the recognition of those members who are carrying the mutant allele and, therefore, are at a significantly higher risk of getting cancer. Detection of mutant tumor suppressor proteins, e.g., p53, or loss of expression of such proteins may aid in cancer diagnosis and prognosis. Experimental investigations have established that restoration of TSG function in cancer cells that are defective for that function results in the suppression of tumor growth or death of the cancer cells. Clinical trials are in progress using ▶ gene therapy and pharmacologic approaches that aim to apply these procedures to the treatment of human cancers.

Eeles RA, Ponder BAJ, Easton DF et al (1996) Genetic predisposition to cancer. Chapman and Hall Medical, New York Harris CC (1996) Structure and function of the p53 tumor suppressor gene: Clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88:1442–1455 Lengauer C, Vogelstein B (1998) Genetic instabilities in human cancers. Nature 396:643–649 Stanbridge EJ (1990) Human tumor suppressor genes. Annu Rev Genet 24:615–657 Weinert T (1998) DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 94:555–558

Tumor Suppressor Genes Webster K. Cavenee Ludwig Institute for Cancer Research, UCSD, La Jolla, CA, USA

Synonyms Recessive oncogenes

Tumor Suppressor Genes

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Recombination Inherited genetic mutation

Point mutation or deletion CpG island methylation

Sporadic somatic mutation

Predisposed CH3

Tumor Suppressor Genes, Fig. 1 Chromosomal mechanisms for tumor suppressor gene inactivation. Left side, the first mutation (*) can occur in a single somatic cell and results in sporadic disease. Alternatively, it can occur in a germ cell (de novo mutation) or be inherited from an affected parent and results in heritable disease. Right side,

the first mutation can become completely inactivated by (from top to bottom) physical deletion or recombination of the wild-type chromosome, by a targeted second mutation or deletion of the remaining wild-type gene, or by methylation of the promoter of the wild-type gene, leading to the loss of expression

Definition

occurring mutant strains or those constructed by in vivo homologous recombination “gene knockout” techniques.

Tumor suppressor genes are genes whose products normally negatively regulate cell growth or cell behavior (Fig. 1).

Characteristics The hallmark of a tumor suppressor gene is that its function is lost during tumor initiation or progression. This typically occurs by one of a set of chromosomal processes called loss of heterozygosity but, in some cases, can occur by forming dominant negative forms of the tumor suppressor gene product. Their presence is usually inferred through the cytogenetic or molecular detection of subchromosomal loss. Upon molecular isolation, the genetic inference can be confirmed and dissected by demonstrating a restoration of growth regulation upon ectopic expression of the gene and/or by the formation of tumors or growth abnormalities in animals lacking the functional gene, either naturally

What Was the Evidence for Tumor Suppressors? The primary lines of evidence are genetic: One is that specific kinds of cancer can cluster in families. In most cases, the inheritance pattern is autosomal dominant which means that it is not sex linked, may be transmitted from either parent, and involves the transmission of a gene whose presence is sufficient to cause disease. In addition to familial clustering of the common cancers, two additional clinical observations provide strong epidemiological support for the contention that cancer has a genetic etiology: • First, some individuals and their families have an autosomal dominant transmission of cancer predisposition, not to a single tumor but to multiple tumors occurring independently at different body sites.

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• Second, individuals with a variety of multiorgan developmental defects often also develop specific rare tumors. A statistical argument can thus be made that the combined occurrence of multiple independent tumors or the routine association of developmental defects with tumors which are very rare in the general population is so unlikely as to suggest an etiologic relationship. The apparent dominant transmission of cancer traits is paradoxical in light of three observations. First, hybrid cells formed from the experimental fusion of highly malignant tumor cells with normal cells are not usually tumorigenic, suggesting that the normal phenotype is dominant in the presence of tumorigenic mutations. Furthermore, the occasional hybrid cell that regains tumorigenicity in these experiments has lost specific chromosomes originally contributed by the normal cell, implying that it is not gain of a dominant cancer trait but specific chromosomal loss that is responsible for the tumor phenotype. Second, if a single mutation was sufficient in itself to elicit a tumor, then families segregating for autosomal dominant forms of cancer would be expected to have no normal tissue in the diseased organ. This expectation is in direct contrast to the clinical description of these tumors as focal lesions surrounded by normal, functioning tissue of the same organ. Finally, epidemiological analyses of sporadic and familial forms of several human cancers have indicated that the conversion of a normal cell to a tumor cell requires multiple events. Retinoblastoma: The First Suppressor ▶ Retinoblastoma is a relatively rare tumor (1 in 20,000 births) of young children and occurs in both a sporadic and autosomal dominant inherited form. Based entirely on statistical data from epidemiology and clinical observations, several remarkable conclusions were made regarding the nature of events, leading to retinoblastoma tumor formation. First, the inherited mutation alone was not sufficient to cause the disease, since there are at least 107 retinoblast cells which are potential targets for retinoblastomas, each carrying the inherited mutation, yet on average, only three

Tumor Suppressor Genes

independent tumors form per affected individual. This also suggested that at a genetic level, mutations leading to retinoblastoma may be recessive, rather than dominant as suggested by the inheritance pattern. The hereditary tumors were proposed to arise through an initial germline mutation followed by a second mutation in a somatic cell. The rate at which somatic mutations occurred was similar in hereditary and sporadic cases, although sporadic tumors required two somatic mutations, each in the same retinoblast for tumor formation. Entirely consistent with this was the observation that hereditary cases usually occurred at an earlier age, were often bilateral, and had multiple tumors, whereas the sporadic cases were invariably unilateral and single tumors. Because of the small possibility that a second somatic mutation may never occur in hereditary cases, 5% of carriers do not develop any tumor. The nature of the two mutational targets in the genome was unknown at the time of these clinical observations, but cytogenetics and molecular genetics eventually led to the answer as well as to a general approach to other human cancers. Analysis of the chromosome band patterns from hereditary and sporadic retinoblastoma patients revealed a deletion of chromosome 13q14 (chromosome 13, q or long arm, band 1–4), suggesting that the gene for retinoblastoma (Rb) resided somewhere within this region. DNA from hereditary tumors was then analyzed with cloned DNA probes (termed “DNA markers”) that could distinguish the two copies, or alleles, of chromosome 13 within each cell. It was found that, in tumors of affected individuals, the region containing the suspected Rb gene on chromosome 13 was present in a mutant only state. This conversion from a heterozygous state to homozygosity for the mutation was termed loss of heterozygosity (LOH) and constituted the second hit required for tumor formation in hereditary cases. Furthermore, LOH on chromosome 13q14 also occurred in sporadic retinoblastoma. These data lent strong support to the idea that retinoblastoma tumor formation occurs by the unmasking of a recessive genetic defect. The discovery that LOH occurs in other hereditary and most sporadic cancers in humans marked

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Tumor Suppressor Genes, Table 1 Tumor suppressor genes, their primary biological functions and the types of tumors in which they have been found to be altered Gene RB1 p53 p16

Chromosomal location 13q14.2 17p13.1 9p21

p15

9p21

p18

1p32

▶ p21

6p21

E2F BRCA1 BRCA2 WT1 VHL PTCH TGFbR1 TGFbR2 DPC4 CDH1 APC MCC NF1 NF2 MSH2 MLH1 DCC PTEN

20q11 17q21 13q12–13 11p13 3p25–26 9q22.3 9q33–34 3p21.3 18q21.1 16q22.1 5q21 5q21 17q11.2 22q12 2p22 3p21.3 18q21 10q23.3

Function Cell-cycle regulator Genome-stability regulator Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Transcription factor Transcription factor Transcription factor Transcription factor Modulator of RNA polymerase Transcription repressor TGF-b receptor TGF-b receptor TGF-b pathway growth inhibitor Intercellular adhesion Cell signaling Cell Adhesion Cell signaling Cell signaling Mismatch repair protein Mismatch repair protein Differentiation factor Protein/lipid phosphatase

the simultaneous emergence of somatic cell cancer genetics and its coupling to the genetics of hereditary cancer. By identifying the region of chromosome 13q14 with the most consistent LOH in tumor DNA, the gene responsible for retinoblastoma was eventually isolated and its functionality assessed. Most importantly, the gene was shown to be mutationally inactivated in retinoblastoma tumors. When a normal copy of the gene was transferred to tumor cells, their growth and tumorigenic behavior was reduced. Thus, the conjoint application of epidemiology, cytogenetics, molecular genetics, and molecular biology allows the identification of a gene with tumor-suppressing function.

Cancer sites Retina, bone, bladder, breast, pancreas Brain, breast, leukemia, soft tissue Brain, melanocyte Leukemia Esophagus, lung, bladder, pancreas Prostate, lung Erythroleukemia Breast, ovary Breast, ovary Kidney Kidney, central nervous system Skin Colon, retina, liver, stomach Colon, retina, liver, stomach Pancreas, colon, bladder, liver Breast, ovary, liver, skin, endometrium Colon Colon Peripheral nervous system, skin Central nervous system Colon Colon Colon Brain, melanocytes, prostate, thyroid, breast

Are There Other Tumor Suppressors and What Cellular Role Do They Normally Play? Since the first suppressor was isolated, many others have been molecularly identified. As might be expected, these represent genes whose products are involved in many different aspects of cell growth and behavior. These include regulators of the cell cycle, growth and transcriptional regulators, DNA repair enzymes, differentiation factors, elements of cell ▶ motility, and regulators of cellular signaling. Thus, elucidation of the function and nature of tumor suppressors is not only of importance for understanding cancer etiology but also useful for dissecting normal cellular function.

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Clinical Relevance The intimate involvement of tumor suppressor genes in the etiology of most human cancers places them at the center of cancer research. Such knowledge has been exploited for the first prenatal and premorbid predictions of cancer occurrence, for molecular pathology approaches to tumor subtyping, for ▶ gene therapy approaches toward gene replacement, and as targets for agonist/antagonist development in rational drug design. Just as in research, the continued exploitation of tumor suppressor genes for clinical benefit to cancer patients is likely to assume a central role in modern therapies (Table 1).

Cross-References ▶ Allele Imbalance ▶ BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk ▶ CCCTC-Binding Factor ▶ CDKN2A ▶ Deleted in Pancreatic Carcinoma Locus 4 ▶ Gene Therapy ▶ Motility ▶ Neurofibromatosis 1 ▶ Neurofibromatosis 2 ▶ p21 ▶ Repair of DNA ▶ Retinoblastoma ▶ Von Hippel-Lindau Tumor Suppressor Gene

References Cavenee WK, White RL (1995) The genetic basis of cancer. Sci Am 272:50–57 Newsham I, Hadjistilianou D, Cavenee WK (1999) Retinoblastoma. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B (eds) The metabolic and molecular basis of inherited disease, 8th edn. McGraw-Hill, New York Perkins AS, Stern DF (1997) Molecular biology of cancer: oncogenes. In: DeVita VT, Hellman S, Rosenberg SA (eds) Cancer: principles and practice of oncology, 5th edn. Lippincott-Raven Publishers, Philadelphia

Tumor Typing

See Also (2012) Autosomal Dominant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 323. doi:10.1007/978-3-642-16483-5_489 (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) Chromosome Band. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 848. doi:10.1007/978-3-642-16483-5_1147 (2012) DCC. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1063–1064. doi:10.1007/978-3-642-16483-5_1524 (2012) DNA Repair. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1141. doi:10.1007/978-3-642-16483-5_1687 (2012) Gene Knockout. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1523. doi:10.1007/978-3-642-16483-5_2370 (2012) Loss of Heterozygosity. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2075–2076. doi:10.1007/978-3-642-164835_3415 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331

Tumor Typing ▶ Pathology

Tumor-Associated Macrophages Antonio Sica1, Alessandra Mancino1, Paola Larghi1, Luca Rubino2, Graziella Solinas1, Paola Allavena1 and Alberto Mantovani1 1 Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy 2 Department of Oncology, Humanitas Research Hospital, Humanitas Cancer Center, Rozzano, Milan, Italy

Definition Tumor-associated ▶ macrophages (TAMs) define a subset of myeloid cells that highly infiltrate solid tumors. Accumulating evidence clearly demonstrates, in various mouse and human

Tumor-Associated Macrophages

malignancies, including colon, breast, lung, and prostate cancer, a strict correlation between increased numbers and/or density of TAM and poor prognosis. Based on this, recruitment and activation of TAM are regarded as pivotal steps of tumor progression, and TAM are putative targets for therapeutic intervention.

Characteristics Experimental and clinical studies have revealed that chronic ▶ inflammation predisposes to different forms of cancer, including colon cancer, ▶ prostate cancer, and ▶ liver cancer, and that usage of ▶ nonsteroidal anti-inflammatory drugs can protect against the emergence of various tumors. In the late 1970s, it was found that a major leukocyte population present in tumors, the so-called TAM, promotes tumor growth. Over the years it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. In several experimental tumor models, the activation of an inflammatory response (most frequently mediated by macrophages) is essential for full neoplastic transformation and progression. This evidence strongly supports the idea that cancers originate at sites of chronic inflammation and suggests that the inflammatory circuits activated at the tumor microenvironment may represent suitable targets of novel anticancer therapies. Macrophages Macrophages (Mj) play an indispensable role in the immune system with decisive functions in both ▶ innate immunity and acquired immunity. In innate immunity, resident Mj provide immediate defense against foreign pathogens and coordinate leukocyte infiltration. Mj contribute to the balance between antigen availability and clearance through phagocytosis and subsequent degradation of senescent or apoptotic cells, microbes, and possibly neoplastic cells. Their role is essential for triggering, instructing, and terminating the adaptive immune response. Mj collaborate with T and B cells, through both cell–cell interactions

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and fluid phase-mediated mechanisms, based on the release of cytokines, chemokines, enzymes, arachidonic acid metabolites, and reactive radicals. Mj activation can be either proinflammatory or anti-inflammatory, thus contributing to tissue–cell destruction or to tissue regeneration and wound healing. These polar phenotypes are not expressed simultaneously but regulated in such a manner that Mj display a balanced, harmonious pattern of functions. Mj are critical effector cells in the acute innate response, for delayed-type hypersensitivity reactions and T cell-mediated immunity. In 1986 Mosmann et al. described two polarized sets of mouse T helper (Th) cells – Th1 and Th2 – with distinct cytokine secretion patterns. Th1 cells secreted interleukin 2 (IL-2), interferon-g (IFN-g), and lymphotoxin (LT, TNF-b). Th2 cells secreted IL-4, IL-5, and IL-6 and promoted B-cell proliferation and antibody secretion. Moreover, additional studies clarified that Th1 and Th2 cells may play opposite roles in pathological conditions, including infections and cancers. Although excluded from the original “type I–type II” paradigm, Mj’s role in the balance of polarized immune responses is being increasingly appreciated. Mj are able to secrete either IL-12 or IL-10, cross-regulatory cytokines crucial for the elicitation of IFN-g production and development of Th1 cells and IL-4/IL-13 secretion and Th2 cells proliferation correspondingly. The preferential production of IL-12 and IL-10 sets the basis for the M1/M2 Mj polarization paradigm, elsewhere defined as the elicitation of functionally distinct Mj populations, in response to the factors that dominate the inflammatory scene. In analogy with the Th1 and Th2 dichotomy, macrophages can be phenotypically polarized by the microenvironment to mount specific M1 or M2 functional programs. Chronic infections can tightly regulate the immune responses, being able to trigger highly polarized type I or type II inflammation and immunity. Classical or M1 macrophage activation in response to microbial products or IFN-g is characterized by high capacity to present antigen; high expression of proinflammatory cytokines, such as interleukin 12 (IL-12) and tumor necrosis factor-a (TNF-a) and consequent

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Tumor-Associated Macrophages Monocytes

M-CSF IL-4, IL-13, IL-10 Corticosteroids PGE VitD3 IG+TLR/IL-1R ligands IL-1ra decoy IL-1RII IL-10 CCL17 CCL18 CCL22 Polyamine Scavenger R Mannose R

GM-CSF LPS, IFNγ bacterial products

M2 macrophage

M1 macrophage

Scavenging matrix remodeling tissue repair angiogenesis immunesuppression

Bactericydal activity inflammatory cytokines immuno-stimulation

Tumor promotion

Tumor suppression

IL-1 TNF IL-12 CXCL9 CXCL10 CXCL11 RNI ROI

Tumor-Associated Macrophages, Fig. 1 Monocytes differentiate into polarized macrophage subsets when exposed to different cytokine milieu. In the presence of GM-CSF, IFN-g, LPS, and other microbial products, monocytes differentiate into M1 macrophages. In the presence of M-CSF, IL-4, IL-13, IL-10, and immunosuppressive agents (corticosteroids, vitamin D3, prostaglandins, and immunocomplexes (IG) in combination with IL-1 or

TLR ligands), monocytes differentiate into M2 macrophages. M1 and M2 subsets differ in terms of phenotype and functions. M1 cells have high microbicidal activity, immunostimulatory functions, and tumor cytotoxicity. M2 cells have high scavenging ability, promote tissue repair and angiogenesis and immunosuppression, and favor tumor progression

activation of a polarized type I response; and high production of toxic intermediates (▶ nitric oxide (NO), ▶ reactive oxygen species). Thus, M1 macrophages are generally considered potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, various signals (e.g., IL-4, IL-13, glucocorticoids, IL-10, immunoglobulin complexes/TLR ligands) elicit different M2 forms, able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, and promote ▶ angiogenesis, tissue remodeling, and repair. Figure 1 shows selected functions of M1 and M2 polarized macrophages. Microenvironmental signals expressed at the tumor microenvironment have the capacity to pilot recruitment,

maturation, and differentiation of infiltrating leukocytes and play a central role in the activation of specific transcriptional programs expressed by TAM. Tumor-Associated Macrophages To the extent that they have been investigated, differentiated mature TAM has a phenotype and function similar to type II or M2 macrophages. TAM has been shown to exert a negative effect on antitumor immune responses. TAM Recruitment TAMs are protumoral cells that are derived from circulating monocytes (Fig. 2) and are recruited to the tumor by a tumor-derived chemotactic factors,

Tumor-Associated Macrophages

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Blood Monocytes

Tumor cell Growth and survival factors

CC-Chemokines M-CSE VEGF

Promotion of metastasis

Chemotactic and survival factors TAM

Taming adaptive immunity

Matrix remodelling

Neoangiogenesis

Fibroblast

Vessel

Soild Tumor

Tumor-Associated Macrophages, Fig. 2 Tumorderived chemotactic factors (CC-chemokines, e.g., CCL2, macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF)) actively recruit circulating blood monocytes at the tumor site. In the tumor microenvironment, monocytes differentiate into tumor-associated macrophages (TAMs) that establish a

symbiotic relationship with tumor cells. The above tumor-derived factors positively modulate TAM survival. From their own, TAM secretes growth factors that promote tumor cell proliferation and survival; regulates matrix deposition and remodeling, thus favoring metastasis formation; activates neoangiogenesis; and promotes immunosuppression

originally identified as CC-chemokine ligand 2 (CCL2; also known as MCP-1). Following this observation, other chemokines active on TAM were detected in neoplastic tissues as products of either tumor cells or stromal elements. These molecules have an important role in tumor progression by directly stimulating neoplastic growth, promoting inflammation, and inducing angiogenesis. Evidence supporting a pivotal role for chemokines, in addition to CCL2, in the recruitment of monocytes to neoplastic tissues includes a direct correlation between chemokine production and monocyte infiltration in mouse and human tumors. Molecules other than chemokines can also promote TAM recruitment. In particular, tumor-derived cytokines such as ▶ vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF) promote

macrophage recruitment, as well as macrophage survival and proliferation, and their expression correlates with tumor growth. TAM Express Selected M2 Protumoral Functions The cytokine network expressed at the tumor site plays a central role in the orientation and differentiation of recruited mononuclear phagocytes, thus contributing to direct the local immune system away from antitumor functions. Immunosuppressive cytokines IL-10 and tumor growth factor-b (TGF-b) are produced by both cancer cells (ovary) and TAM. IL-10 promotes the differentiation of monocytes to mature macrophages and blocks their differentiation to dendritic cells (DC). Thus, a gradient of tumor-derived IL-10 may account for differentiation along the DC

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versus the macrophage pathway in tumors, resulting in tumor promotion. IL-10 promotes the M2c alternative pathway of macrophage activation and induces TAM to express M2-related functions. Under many aspects, TAM summarizes a number of functions expressed by M2 macrophages involved in tuning inflammatory responses and adaptive immunity, scavenges debris, and promotes angiogenesis, tissue remodeling, and repair. The production of IL-10, TGF-b, and PGE2 by cancer cells and TAM contributes to a general suppression of antitumor activities. TAMs are poor producers of nitric oxide (NO), and, in situ in ▶ ovarian cancer, only a minority of tumors and, in these, a minority of macrophages localized at the periphery scored positive for iNOS. Moreover, in contrast to M1-polarized macrophages, TAM has been shown to be poor producers of ROI, consistent with the hypothesis that these cells represent a skewed M2 population. Moreover, TAM was reported to express low levels of inflammatory cytokines (e.g., IL-12, IL-1b, TNF-a, IL-6). Activation of the transcriptional factor NF-kB is a necessary event promoting transcription of several proinflammatory genes. TAM displays defective NF-kB activation in response to M1-polarizing signals lipopolysaccharide (LPS) and TNF-a. Thus, in terms of cytotoxicity and expression of inflammatory cytokines, TAM resembles the M2 macrophages. Angiogenesis is an M2-associated function that represents a key event in tumor growth and progression. In several studies in human cancer, TAM accumulation has been associated with angiogenesis and with the production of angiogenic factors such as VEGF and platelet-derived endothelial cell growth factor. Additionally, TAM participates to the proangiogenic process by producing the angiogenic factor thymidine phosphorylase (TP), which promotes endothelial cell migration in vitro and whose levels of expression are associated with tumor neovascularization. Moreover, TAM accumulates in the hypoxic regions of tumors, and ▶ hypoxia triggers a proangiogenic program in these cells. Therefore, macrophages recruited in situ represent an indirect pathway of amplification of angiogenesis, in

Tumor-Associated Macrophages

concert with angiogenic molecules directly produced by tumor cells. On the antiangiogenic side, in a murine model, GM-CSF released from a primary tumor upregulated TAM-derived metalloelastase and angiostatin production, thus suppressing tumor growth of metastases. Finally, TAM expresses molecules that affect tumor cell proliferation, angiogenesis, and dissolution of connective tissues. These include epidermal growth factor (EGF), members of the FGF family, TGF-b, VEGF, and chemokines. In lung cancer, TAM may favor tumor progression by contributing to stroma formation and angiogenesis through their release of PDGF, in conjunction with TGF-b1 production by cancer cells. Macrophages can produce enzymes and inhibitors that regulate the digestion of the extracellular matrix, such as MMPs, plasmin, ▶ urokinase-type plasminogen activator (uPA), and the uPA receptor. Direct evidence has been presented that MMP-9 derived from hematopoietic cells of host origin contributes to ▶ skin carcinogenesis. Chemokines have been shown to induce gene expression of various MMPs and, in particular, MMP-9 production, along with the uPA receptor. Evidence suggests that MMP-9 has complex effects beyond matrix degradation including the promotion of the angiogenesis switch and release of growth factors. Modulation of Adaptive Immunity by TAM It has long been known that TAM have poor antigen-presenting capacity and can actually suppress T cell activation and proliferation. The suppressive mediators produced by TAM include ▶ prostaglandins, IL-10, TGF-b, and indoleamine dioxygenase (IDO) metabolites. Moreover, TAM is unable to produce IL-12, even upon stimulation by IFN-g and LPS. With this cytokine profile, which is characteristic of M2 macrophages, TAM is unable to trigger Th1-polarized immune responses but rather induce ▶ T regulatory cells (Treg). Treg cells possess a characteristic anergic phenotype and strongly suppress the activity of effector T cells and other inflammatory cells, such as monocytes. Suppression of T cell-mediated antitumor activity by Treg cells is associated with increased tumor growth and hence, decreased survival. For instance, in patients with

Tumor-Associated Stromal Progression

advanced ovarian cancer, an increase in the number of functionally active Treg cells present in the ascites was predictive of reduced survival. The complex network of chemokines present at the tumor site can play a role also in the induction of the adaptive immunity. Chemokines also regulate the amplification of polarized T cell responses. Some chemokines may enhance specific host immunity against tumors, but on the other hand, other chemokines may contribute to escape from the immune system, by recruiting Th2 effectors and Treg cells. Figure 2 summarizes symbiotic relationship between TAM and cancer cells. Conclusion Though the presence of TAM has been long considered as evidence for a host response against the growing tumor, it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. Molecular and biological studies have been supported by a large number of clinical studies that found a significant correlation between the high macrophage content of tumors and poor patient prognosis. TAM shares many similarities with prototypic polarized M2 mononuclear phagocyte population, in terms of gene expression and functions. In line with the known properties of M2 macrophage populations, several lines of evidence suggest that TAM promotes tumor progression and ▶ metastasis by activating circuits that regulate tumor growth, ▶ adaptive immunity, stroma formation, and ▶ angiogenesis. This hypothesis is now receiving new supporting evidence indicating that in vivo functional switching of infiltrating M2 macrophages toward an M1 phenotype provides therapeutic benefit in mice-bearing tumor xenograft. Identification of mechanisms promoting functional diversion of macrophages toward an M2 direction may disclose new valuable therapeutic targets against tumors.

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▶ Hypoxia ▶ Inflammation ▶ Innate Immunity ▶ Macrophages ▶ Macrophage-Stimulating Protein ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Nitric Oxide ▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Ovarian Cancer ▶ Prostaglandins ▶ Prostate Cancer ▶ Reactive Oxygen Species ▶ Regulatory T Cells ▶ Skin Carcinogenesis ▶ Urokinase-Type Plasminogen Activator ▶ Vascular Endothelial Growth Factor

References Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357:539–545 Mantovani A, Sozzani S, Locati M et al (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23:549–555 Sica A, Bronte V (2007) Altered macrophage differentiation and immune dysfunction in tumour development. J Clin Invest 117:1155–1166

See Also (2012) Arachidonic Acid. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 260. doi:10.1007/978-3-642-16483-5_379 (2012) Liver Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2063. doi:10.1007/978-3-642-16483-5_3393 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720 (2012) Xenograft. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3967. doi:10.1007/978-3-642-16483-5_6278

Cross-References ▶ Adaptive Immunity ▶ Angiogenesis ▶ Endothelins

Tumor-Associated Stromal Progression ▶ Stromagenesis

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Tumor–Endothelial Communication

Characteristics

Tumor–Endothelial Communication ▶ Tumor–Endothelial Cross-Talk

Tumor–Endothelial Cross-Talk Tobias Görge1, Anke Rattenholl2, Martin Steinhoff3,4 and Stefan W. Schneider5 1 Department of Dermatology, University of Münster, Münster, Germany 2 Applied Biotechnology Division, Department of Engineering and Mathematics, University of Applied Sciences Bielefeld, Bielefeld, Germany 3 UCD Charles Institute of Dermatology, University College Dublin, Belfield, Ireland 4 Department of Dermatology School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland 5 Hauttumorzentrum Mannheim (HTZM), Universitätsmedizn Mannheim, Mannheim, Germany

Synonyms Tumor–endothelial communication

Definition Research in the field of tumor–endothelial communication focuses on specific interactions and pathways that allow circulating tumor cells to interact with the endothelium – the inner lining of the vascular wall. These interactions lead to modifications of the endothelium that eventually facilitate both tumor cell adhesion and extravasation. These processes are frequently accompanied by microthrombotic events, and tumor–endothelial communication relies in many aspects on mechanisms known from the inflammatory response.

Neoplastic growth and survival of tumor cells in the host environment critically depends on effective mechanisms of tumor–host interactions. This dependency of tumor cell survival in the microenvironment is not only limited to the early phase of primary tumor growth, where obviously, e.g., nutritive supplies are needed, but persists throughout the entire life span of tumor presence that is actually characterized by continuous multiple tumor–host interactions. A characteristic hallmark of malignant tumor cells is their ability to spread via the host vascular system, settling up colonies distant from their place of origin, a process generally referred to as “▶ metastasis.” There is general agreement that in this process of metastasis via the vascular system – which is the most fearsome aspect of cancer as to the survival prognosis – tumor cells that have managed to leave their site of origin need to apply escape mechanisms, allowing avoidance of the host immunosurveillance and an eventual extravasation from the blood stream. The transmigration of circulating tumor cells into the host tissue is a highly regulated process, which up-to-date has not been completely understood. Circulating tumor cells extravasating to the hoststromal tissue will have to cross the natural barrier between blood and tissue: the endothelium. This interaction is a complex process that requires molecular mechanisms similar to those known from the proinflammatory and prothrombotic response. This encyclopedic entry will display the mechanisms that have coined the expression of tumor–endothelial communication. On the one hand, it summarizes the findings on tumor cell–endothelial interactions facilitating the transmigration of tumor cells; on the other hand, it will highlight the molecular mechanisms that are currently known for describing how tumor cells modify endothelial functions that eventually may lead to tumor-induced thrombotic events. It goes without saying that the current trend of linking thrombotic events to mechanisms of the inflammatory response also holds true for the understanding of tumor–endothelial communication.

Tumor–Endothelial Cross-Talk

There are multiple evidences that neoplastic cells from a variety of tumor entities actively modify the vascular endothelium. Tumor cells are capable of transforming the endothelial physiological quiescent disposition into a prothrombotic, proinflammatory, and proadhesive endothelium. One of the oldest documented observations of this phenomenon is generally accepted to date back to works by Trousseau in the late nineteenth century (1865). These studies showed that tumor cells are associated with thrombi of platelets and neutrophils. In addition, there are multiple clinical reports that a thromboembolic event of so far unknown origin might occasionally be the first hint to an ongoing metastatic tumor disease of a patient. So evidently, these observations reflect tumor–host cross-talk. Adhesion Molecules in Tumor–Endothelial Cross-Talk Mediating Extravasation The extravasation from the flowing blood stream is a highly regulated process that relies on a specific interaction of ligands on the circulating cell and their receptors on the vascular endothelium. Much of the current knowledge in this field derives from the studies on thrombosis and inflammation, where platelets and leukocytes are specifically directed to bind to the activated endothelium and vascular wall. So-called selectins are a family of adhesion molecules that are crucial in this process. There are L-, P-, and E-selectins that all recognize carbohydrate structures. While L-selectin is constantly present on leukocytes, Pand E-selectin become upregulated on activated endothelium. Absence of selectins severely delays or even prevents the adhesion of circulating cells. The lectin domain of selectins recognizes sialylated, fucosylated structures displayed mostly on mucin-type glycoproteins containing a terminal tetrasaccharide sLex and sLea. Next to platelets and neutrophils, selectin-ligands have been shown to be expressed by several tumor entities and thus support tumor–endothelial communication. In different mouse models of metastasis, it could be shown that P- and E-selectin deficiency reduces the amount of extravasation of tumor cells. It was also reported that heparin, apart from its properties as an anticoagulant,

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interferes with binding ligands for selectins and thus prevents metastatic tumor spread. The use of heparin for prevention of tumor metastasis has been shown to be beneficial in many human studies and has become an accepted option for the clinical treatment of cancer patients. In continuation of the initial selectin-mediated interaction of tumor cell and endothelium, integrins are recognized to play an important role in cell adhesion. Integrins are heterodimeric transmembrane proteins that consist of noncovalently bound alpha and beta chains. They play a crucial role in cellular processes such as cell adhesion and migration as well as in the control of cell differentiation, proliferation, and survival. Several binding partners for integrins on and below endothelial cells have been characterized so far, such as VCAM-1, ICAM-1, and extracellular matrix proteins such as fibronectin, vitronectin, and laminin. There is also a well-documented expression of integrins on several tumor entities. During the malignant transformation, the expression of integrins has been reported to alter and leads to different binding avidity. Research on integrin-mediated tumor extravasation currently focuses on alphavbeta3 integrin and alpha5beta1 integrin, as their inhibition could decrease metastasis in several studies. In summary, crossing the endothelial barrier, tumor cells rely on mechanisms that resemble those of cell arrest in the inflammatory and thrombotic response. Tumor–Endothelial Cross-Talk Leading to Prothrombotic Endothelial Activation Apart from modifying the vascular endothelium toward a proadhesive state, tumor–endothelial cross-talk also activates the prothrombotic activities of the vascular wall. The clinical observation is that of the so-called Trousseau’s syndrome, where especially visceral cancers lead to venous thrombotic events. In addition, there are several reports showing that progressive cancer is associated with elevated levels of circulating von Willebrand factor (VWF), a plasma surrogate marker for endothelial activation. The pathogenesis of the prothrombotic state in cancer is complex and probably multifactorial; however, some

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studies could elucidate molecular mechanisms critically involved in tumor-induced prothrombotic endothelial activation. A well-studied protein involved in endothelial activation is tissue factor (TF). Several tumor cells can directly release TF or stimulate circulating leukocytes to increase TF release, e.g., by the generation of prothrombotic TF-rich microparticles. Once released, TF activates coagulation factor VII, which in turn activates factorX, thus activating prothrombin to thrombin. On the endothelium, via activation of the thrombin receptor (proteinase-activated receptor 1/PAR-1), thrombin leads to an immediate release of VWF and P-selectin promoting thrombosis and inflammation. A similar mechanism has been described for cancer procoagulant (CP). CP is a single chain cysteine protease that has been identified in a variety of tumors. It can directly activate factorX and thus contribute to the generation of thrombin. Another direct mechanism of endothelial cells by metastatic tumor cells could be demonstrated. It was shown for human ▶ melanoma and ▶ colon cancer that metastatic tumor cell lines of these cancers are capable of generating and releasing matrixmetalloproteinase-1 (MMP-1). MMP-1 has been demonstrated to directly activate the thrombin-receptor on endothelial cells followed by VWF and IL-8 release. This mechanism does not rely on the presence of coagulation factors and is therefore proof of immediate tumor–endothelial communication. Conclusion The prothrombotic reaction of the endothelium upon contact with malignant circulating cells might be understood as a mechanism for prevention of further dissemination of the tumor, making trapped tumor cells more accessible to the host defense system. Indeed, it could be shown that in the absence of surrounding platelets, tumor cells were more prone to ▶ natural killer cell attacks than when protected in platelet emboli. However, it can also be speculated that tumor cell arrest in a thrombus might facilitate tumor cell extravasation. As antithrombotic treatment and depletion of circulating platelets has been demonstrated to positively influence tumor metastasis, specific

Tumor-Induced Platelet Aggregation

strategies of interference with tumor–endothelial cross-talk might eventually prove beneficial for the prevention of tumor cell metastasis.

References Borsig L (2004) Selectins facilitate carcinoma metastasis and heparin can prevent them. News Physiol Sci 19:16–21 Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420(6917):860–867 Goerge T, Barg A, Schnaeker EM et al (2006) Tumorderived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation. Cancer Res 66(15):7766–7774 Smorenburg SM, Van Noorden JCF (2001) The complex effects of heparins on cancer progression and metastasis in experimental studies. Pharmacol Rev 53:93–105

Tumor-Induced Platelet Aggregation ▶ Tumor Cell-Induced Platelet Aggregation

Tumor-Initiating Cells ▶ Cancer Stem-Like Cells ▶ Stem-Like Cancer Cells

Tumor-Reinitiating Cells ▶ Stem-Like Cancer Cells

Tumor-Repopulating Cells ▶ Stem-Like Cancer Cells

Tumor-Transforming 1 ▶ Securin

Tunicamycin

Tunicamycin Tatsushi Yoshida and Toshiyuki Sakai Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

Definition Tunicamycin is an antibiotic identified and isolated from the fermentation broth of Streptomyces lysosuperificus. Since the antibiotic interferes with the formation of viral and cellular surface coats, it was termed “tunicamycin” after the Latin word “tunica” for coats.

Characteristics Structure and Function Tunicamycin is a nucleoside antibiotic composed of uracil, a fatty acid, and two glycosidically linked sugars. The sugars are N-acetylglucosamine and an unusual 11-carbon aminodeoxydialdose, which has been named tunicamine. Tunicamycin is a white crystalline powder which is soluble in alkaline water, pyridine, and hot methanol; slightly soluble in ethanol and n-butanol; and insoluble in acetone, ethylacetate, chloroform, benzene, and acidic water. High-performance liquid chromatography (HPLC) of tunicamycin shows that the antibiotic is separated into ten different components that have molecular weights ranging from 802 to 858. These differences in molecular mass are attributable to differences in the acyl chain length of C13–C17 (Fig. 1). Tunicamycin inhibits the first step in the lipid-linked saccharide pathway. Tunicamycin specifically inhibits the formation of dolichyl (pyro)-phosphate N-acetylglucosamine (dolichylPP-GlcNAc) in the step of the transfer of GlcNAc1-P from UDP-GlcNAc to dolichyl-P. As a result of inhibition in the lipid-linked saccharide pathway, tunicamycin consequently inhibits the

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N-linked oligosaccharide formation of glycoproteins in the endoplasmic reticulum (ER) (Fig. 2). Antiviral Effect of Tunicamycin Tunicamycin strongly inhibits the multiplication of enveloped RNA and DNA viruses such as Newcastle disease virus, vesicular stomatitis virus, Semliki Forest virus, fowl plague virus, Sindbis virus, measles virus, influenza virus, Rous sarcoma virus, Rauscher murine sarcoma virus, and herpes simplex virus. Hemagglutinin and neuraminidase are glycoprotein components of the viral envelope. Tunicamycin specifically inhibits the biosynthesis of viral envelope glycoproteins. Tunicamycin possesses cytotoxic activity toward transformed mammalian cells infected with virus such as mouse 3T3 cells transformed by ▶ SV40 virus, Moloney sarcoma virus or polyomavirus, and human WI38 fibroblasts transformed by SV40. Parental cell lines are resistant to tunicamycin cytotoxicity prior to neoplastic transformation. Apoptosis Induction by Tunicamycin Tunicamycin induces ▶ apoptosis in various types of human cancer cells containing ▶ neuroblastoma, ▶ breast cancer, ▶ hepatocellular carcinoma, and ▶ cervical cancer. Studies have revealed the mechanism of tunicamycin-induced apoptosis. In eukaryotic cells, ER provides an environment for the synthesis and modification of membrane proteins and secreted proteins. These co- and posttranslational modifications including N-linked ▶ glycosylation are involved in subsequent protein folding and assembly. The accumulation of unfolded proteins causes ▶ endoplasmic reticulum stress, the term given to an imbalance between the cellular demand for ER function and ER capacity. Cells respond to ER stress by activation of the unfolded protein response (UPR). The UPR is mediated through three ER transmembrane receptors, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and pancreatic ER kinase (PKR)-like ER kinase (PERK). Under unstressed conditions, these sensor proteins are maintained in an inactivated state by an association with ER chaperone protein, glucose-regulated protein

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Tunicamycin

Tunicamycin, Fig. 1 Structure of tunicamycin

(GRP78) (also called Bip). The UPR is a cytoprotective response to reduce the accumulation of unfolded proteins and restore normal ER function; however, prolonged ER stress switches from pro-survival to proapoptotic signaling. Inhibition of N-linked glycosylation by tunicamycin leads to ER stress. Tunicamycin is a representative ER stressor used to elucidate the mechanism of ER stress and UPR followed by apoptosis. The accumulation of unfolded proteins in ER results in the release of GRP78 from IRE1, PERK, and ATF6. PERK phosphorylates the eukaryotic translation initiation factor-2a (eIF-2a) and allows the activating transcription factor 4 (ATF4). ATF4 transactivates C/EBPhomologous protein (CHOP). CHOP is an important factor in the induction of apoptosis by tunicamycin. CHOP-deficient mouse embryonic fibroblasts (MEF) provide partial resistance to apoptosis induced by tunicamycin treatment. MEF are not cells derived from cancer tissues but normal cells. MEF is often used to elucidate the mechanism of tunicamycin-induced apoptosis, since MEF leads to apoptosis by tunicamycin treatment and specific gene deficiency is easily provided in MEF. CHOP is a transcription factor and transactivates various genes. Tribbles-related protein 3 (TRB3) is a CHOP-downstream gene induced by CHOP at the transcriptional level. Knockdown of ATF4 and CHOP expression

represses tunicamycin-induced TRB3 upregulation. Furthermore, downregulation of TRB3 by small interfering RNA (▶ siRNA) partly blocks the apoptosis induced by tunicamycin treatment in human cervical cancer HeLa cells. Tunicamycin treatment also induces a p53-upregulated modulator of apoptosis (▶ PUMA) and NOXA in a tumor suppressor p53 gene-dependent manner. Both PUMAdeficient MEF and NOXA-deficient MEF partially reduce tunicamycin-induced apoptosis. These factors are key molecules of tunicamycininduced apoptosis. Tunicamycin Increases Effects of Anticancer Agents Many glycoproteins exist on the plasma membrane of tumor cells, including the efflux pump of anticancer agents. ▶ P-glycoprotein is a product of the MDR1 gene and protects cells against a broad spectrum of anticancer agents by functioning as an energy-dependent drug efflux pump. Inhibition of N-linked glycosylation of P-glycoprotein results in a reduced multidrugresistance phenotype of cancer cells. Tunicamycin treatment increases the cytotoxicity of doxorubicin, epirubicin, ▶ cisplatin, and vincristine against NIH-3T3 cells into which an exogenesis MDR gene had been introduced. The human epidermoid carcinoma KB-8-5-11 cell line contains the

Tunicamycin Tunicamycin, Fig. 2 Oligosaccharide formation and the action site of tunicamycin. (a) Lipidlinked oligosaccharide formation. Oligosaccharide is assembled onto the carrier lipid dolichol in the ER membrane. Tunicamycin inhibits the formation of dolichyl PP-GlcNAc. (b) N-linked oligosaccharide formation. Synthesized oligosaccharide (Glc)3(Man)9(GlcNAc)2 is transferred from the lipid dolichol to the asparagine side chain of a nascent polypeptide on the ER membrane. GlcNAc N-acetylglucosamine, Man mannose, Glc glucose

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amplified MDR1 gene. The sensitivity of this cell line to doxorubicin, epirubicin, cisplatin, and vincristine also increases by treatment with tunicamycin. Tunicamycin does not influence the uptake of these anticancer agents but reduces efflux of the agents in cells with a multidrugresistance phenotype. Human colon cancer cells with a strong multidrug-resistant phenotype mediated by high constitutive levels of the expression of P-glycoprotein increase daunorubicin accumulation by incubation with tunicamycin. Tunicamycin exposure reduces P-glycoprotein expression on the surface of the cell membrane. Tunicamycin also decreases the 50% inhibitory

concentration (IC50) of cisplatin in human pharyngeal carcinoma KB cells and human maxillary squamous cell carcinoma IMC-3 cells. Combined administration of tunicamycin with cisplatin injected by s.c. around the tumor inhibits tumor growth in C3H/He mice bearing cisplatinresistant squamous cell carcinoma in vivo and increases the in vivo apoptosis of tumor cells. Tunicamycin Enhances Death Ligand TRAILInduced Apoptosis TNF-related apoptosis-inducing ligand (TRAIL) is a type II membrane protein belonging to the TNF family, which preferentially induces

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apoptosis in a variety of tumor cells but not in normal cells in vitro and in vivo. Since some tumor cell lines are resistant to TRAIL, an agent that can overcome resistance to TRAIL has been sought. Tunicamycin sensitizes hormonerefractory prostate cancer cells to TRAIL-induced apoptosis. The combination of tunicamycin and TRAIL activates caspases under the condition in which a single agent hardly activates caspases. Tunicamycin enhances TRAIL activity through the induction of a TRAIL receptor, TRAIL-R2 (also called death receptor 5 (DR5)). Tunicamycin upregulates the transcription of TRAIL-R2 through a transcription factor, CHOP. Normal Cellular Toxicity of Tunicamycin The hepatotoxicity of tunicamycin has been reported in guinea pigs given a single dose of 400 mg/kg of tunicamycin. A periportal pattern of hepatocellular damage was observed with the death of many hepatocytes up to 72 h postinjection. Swollen hepatocyte cytoplasm protruded into many hepatic blood vessels and detached portions of hepatocytes producing emboli in pulmonary and cerebral capillaries. The toxic effects of tunicamycin have been examined in rats at gestation day 15. At 16 h postdosing, all pregnant rats had moderate to extensive vaginal bleeding and one fourth died. The other rats had free blood in the uterus and large decreases in red cell counts, hemoglobin, and packed cell volume. Tunicamycin toxicity has been reported in 6- to 8-week-old mice given a single injection of tunicamycin at a dose of 3 mg/g body weight intraperitoneally (i.p.) After 2 days, characteristic renal lesions were detected in tunicamycin-treated mice. Apoptosis was caused in the renal tubular epithelium. Moreover, tunicamycin treatment inhibited mammalian embryogenesis. Although early cleavage was normal, mammalian embryos did not undergo normal compaction and blastocyst formation. Trophoblast cell adhesion may be disrupted since tunicamycin is cytotoxic to these cells. In later development, tunicamycin inhibits kidney tubule formation when present during the embryonic induction of these structures.

Turban Tumor Syndrome

Tunicamycin induces apoptosis in a variety of malignant tumor cells and possesses cytotoxic activity to virus-transformed cells compared with parental normal cells. Moreover, tunicamycin enhances the effects of anticancer agents; however, tunicamycin also has cytotoxicity against normal cells and causes crucial damage during in vivo administration. The mechanism of tunicamycin-induced apoptosis through ER stress has been revealed, but it has not been completely elucidated yet. If tunicamycin is applied to cancer treatment, its usage should be restricted to local administration, but further studies are needed.

Cross-References ▶ Doxorubicin ▶ ER ▶ p53 ▶ TRAIL

References Eibein AD (1987) Inhibition of the biosynthesis and processing of N-linked oligosaccharide chains. Ann Rev Biochem 56:497–534 Ma Y, Hendershot LM (2004) The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 4:966–977 Noda I, Fujieda S, Seki M et al (1999) Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells. Int J Cancer 80:279–284 Shiraishi T, Yoshida T, Nakata S et al (2005) Tunicamycin enhances tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis in human prostate cancer cells. Cancer Res 65:6364–6370 Takatsuki A, Tamura G (1982) Inhibition of glycoconjugate biosynthesis by tunicamycin. In: Tamura G (ed) Tunicamycin. Japan Scientific Societies Press, Tokyo, pp 35–70

Turban Tumor Syndrome ▶ Cylindromatosis

Turcot Syndrome

Turcot Syndrome Paola Izzo Department of Molecular Medicine and Medical Biotechnology, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy

Definition Turcot syndrome (TS) is a rare inherited neoplastic disease characterized by the association of primary malignant neuroepithelial tumors of the central nervous system and colon cancers and/or multiple colorectal adenomas.

Characteristics Clinical Criteria The 130 or so Turcot syndrome (TS) cases described to date include various histopathologic types of ▶ brain tumors, e.g., glioma, medulloblastoma, and astrocytoma, associated with a broad spectrum of colorectal findings, from a single adenoma to typical adenomatous polyposis. Usually, polyps are fewer in number than in familial adenomatous polyposis (FAP [▶ APC gene in familial adenomatous polyposis]) but are larger in size, and multiple adenomas or colorectal cancers occur at an early age and undergo an earlier malignant transformation than in FAP or in hereditary nonpolyposis colorectal cancer (HNPCC). The clinical definition and the mode of inheritance of Turcot syndrome are controversial; some authors propose that TS is an allelic variant of FAP and support an autosomal dominant inheritance, while others postulate that TS is a disease independent of FAP with an autosomal recessive pattern of inheritance. Genetics Dominantly inherited cases have been associated with germ line mutations in either the tumor suppressor adenomatous polyposis coli gene (APC), usually mutated in FAP, or in the DNA

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mismatch repair (MMR) genes, which are usually mutated in HNPCC. Few recessive cases have been reported with the causative mutations found to be within the PMS2 gene, a minor MMR gene that is only rarely involved in HNPCC. In the first recessive case described, a germ line nonsense mutation (PMS2134) that was inherited from the healthy mother was found in one allele. The second recessive case was found to be a compound heterozygote for two frameshift germ line mutations within the PMS2 gene: a G deletion (1221delG) in exon 11 and a four-base pair deletion (2361delCTTC) in exon 14, both of which were inherited from the patient’s unaffected parents. This was the first evidence of recessive dominance of TS because two germ line mutations in PMS2 are not individually pathogenic, but become so when occurring together in a compound heterozygote. Interestingly, homozygous splice site mutation in the PMS2 gene, the c.9891G335–400 nm) per unit of time were marketed; these lamps can emit up to 10 times more UVA than that present in sunlight. Tanning devices have been equipped with fluorescent lamps that achieve a ratio of total UV, UVB, and UVA similar to tropical sun. The UV output and spectral characteristics of tanning appliances vary considerably as a result of differences in tanning appliance design (e.g., type of fluorescent tubes, materials that compose filters, distance from canopy to the skin), tanning appliance power, and tube aging. Evidence of the Carcinogenic Potential

A ▶ meta-analysis of epidemiological studies showed that ever-use of sunbeds is positively associated with an increased risk for melanoma (relative risk, 1.15; 95% confidence interval, 1.00–1.31), and there is a prominent and consistent increase in risk for melanoma in people who first used sunbeds in their twenties or teen years (relative risk, 1.75; 95% confidence interval, 1.35–2.26). Limited data suggest that the risk for squamous cell carcinoma is similarly increased after first use as a teenager (relative risk, 2.25; 95% confidence interval, 1.08–4.70). Four studies evaluated the risk for ocular melanoma and found an increased risk in the highest category of exposure together with a dose–response relationship in most studies. Data also suggest detrimental effects from the use of sunbeds on the skin’s immune response.

Uveal Melanoma

Radiation emitted by lamps used in tanning appliances (mainly UVA) significantly increase the carcinogenic effect of broad-spectrum UV radiation. Also, the few studies that have addressed the biological changes in the skin induced by indoor tanning have shown that they are similar to those induced by sunlight. UV-emitting tanning devices are classified Group 1 by the International Agency for Research on Cancer. Current evidence establishes a causal association for cutaneous malignant melanoma and for ocular melanoma (observed in the choroid and the ciliary body of the eye).

References AFSSET (2005) UV radiation. State of the knowledge on exposure and health risks. Agence française de Sécurité Sanitaire de l’Environnement et du Travail. Available at http://www.afsset.fr (in English) Griffiths HR, Mistry P, Herbert KE et al (1998) Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit Rev Clin Lab Sci 35:189–237 IARC (1992) Solar and ultraviolet radiation, vol 55, IARC monographs on the evaluation of carcinogenic risks to humans. International Agency for Research on Cancer, Lyon IARC (2006) Exposure to artificial UV radiation and skin cancer, vol 1, IARC working group reports. International Agency for Research on Cancer, Lyon IARC (2010) Radiation, vol 100D, IARC monographs on the evaluation of carcinogenic risks to humans. International Agency for Research on Cancer, Lyon Ullrich SE (2005) Mechanisms underlying UV-induced immune suppression. Mutat Res 571:185–205

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Definition Uveal melanoma is an intraocular melanocytic neoplasm that originates from within the uveal tract of the eye. The tumor is thought to arise most frequently from melanocytes within a nevus, but may also occur de novo. It is the most common primary intraocular malignancy in adults.

Characteristics Epidemiology Worldwide, the incidence of uveal melanoma is highest in Northern Europeans. Uveal melanoma has an incidence rate of around 6/1,000,000 in the United States. Similar to cutaneous melanoma, the incidence of uveal melanoma is much higher in whites than nonwhites. There is a slight male preponderance. The average age at diagnosis is 50–60, but any individuals of any age can be affected. Risk Factors Certain patient characteristics have been associated with increased risk of uveal melanoma, including light eye color, light skin color, and ability to tan. Individuals with ocular and oculodermal melanocytosis (nevus of Ota) are at increased risk of uveal melanoma. Interestingly, although these conditions can occur in any race, the increased rate of uveal melanoma appears to be primarily in whites.

Uveal Melanoma

Clinical Diagnosis

Justis P. Ehlers1 and J. William Harbour2 1 Cole Eye Institute, Cleveland Clinic, Cleveland, OH, USA 2 Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA

Signs and Symptoms

Synonyms Choroidal melanoma; Ciliary body melanoma; Intraocular melanoma

Uveal melanoma can be asymptomatic or associated with a variety of symptoms including blurred vision, visual field defects, distorted vision, flashes, and floaters. On clinical examination, uveal melanomas can range in pigmentation from light tan to dark brown, and they are typically dome or mushroom shaped (Fig. 1). Features associated with uveal melanoma on fundus examination include orange lipofuscin pigmentation on the tumor surface, exudative retinal detachment,

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Uveal Melanoma, Fig. 1 Elevated choroidal melanoma adjacent to the optic nerve

and “collar button” formation where the tumor herniates through the overlying Bruch’s membrane, which can cause intraocular hemorrhage. Ciliary body melanomas are located near the crystalline lens and can induce subluxation, cataract formation, and astigmatism (from tilting the lens). Dilated episcleral feeder vessels, so-called sentinel vessels, can be associated with an underlying ciliary body melanoma. In larger tumors, neovascular glaucoma and necrosis may occur. Pain may be present if these complications develop. Clinical features associated with increased risk of metastasis include advanced patient age, increased tumor size, and ciliary body involvement. Diagnostic Modalities Ophthalmoscopy remains the mainstay of diagnosis. Several adjunctive modalities can be used to further characterize the tumor and aid in diagnosis for ambiguous cases. Both A-scan and B-scan ultrasonography are critical for the complete evaluation of a suspected uveal melanoma. A-scan provides a single-dimensional image characterizing the echogenic interfaces within the tumor. This provides an accurate way to measure the tumor height as well as confirming the typical homogenous characteristics of uveal melanoma. B-scan provides a two-dimensional image that allows for a cross-sectional view of the tumor. Most tumors are dome or mushroom shaped, the latter being nearly pathognomonic for uveal melanoma (Fig. 2).

Uveal Melanoma

Fluorescein angiography is also a useful adjunct in to further characterize the tumor exclude other mimicking disorders. Common angiographic findings include early hyperfluorescence, pinpoint leakage, and occasionally a double circulation pattern from vessels within the tumor (Fig. 3). Fine-needle aspiration biopsy (FNAB) is required in about 5% of intraocular tumors for the purpose of confirming the diagnosis of a melanocytic tumor. In the setting of an experienced surgeon and cytopathologist, FNAB can yield a diagnosis in over 90% of cases. In addition, FNAB is increasingly being used to assess the metastatic potential of melanomas using molecular tools described below. Other imaging modalities such as CT and MRI may provide further information in selected settings, such as when orbital or optic nerve invasion of an intraocular tumor is suspected. Treatment Options Several treatment options exist for uveal melanoma. The appropriate choice is based on patient age, health and preference, and on tumor characteristics. Observation may be appropriate for small lesions where the diagnosis is in doubt and in elderly or infirmed individuals with limited life expectancy. Choroidal melanocytic tumors less than about 3 mm in thickness and less than about 12 mm in diameter are usually considered suspicious nevi until growth is documented, at which point treatment is usually recommended. Serial fundus photography and ultrasonography are used to detect tumor growth. Transpupillary thermal therapy (TTT) has been advocated as primary therapy for smaller tumors and as an adjunctive treatment following radiotherapy. Enthusiasm for primary TTT has waned as longer follow-up has revealed a high rate of local tumor recurrence. Most centers now reserve primary TTT for small tumors in patients who are unable to undergo surgery. The most widespread use of TTT today is as an adjunctive treatment following plaque or charged particle radiotherapy in tumors that are at high risk for recurrence, such as those located adjacent to the optic disc. In this setting, TTT appears to be

Uveal Melanoma

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Uveal Melanoma, Fig. 2 Ultrasound (B-scan) of dome-shaped (a) and mushroom-shaped (b) melanoma. Thin arrows identify the tumor; thick arrows identify (a) the optic nerve and (b) the detached retina

Uveal Melanoma, Fig. 3 Fluorescein angiogram showing double circulation. The thin arrow shows a normal retinal arteriole; the thick arrow shows the intrinsic tumor vessel

effective at hastening the regression and reducing local recurrence risk compared to radiotherapy alone. Complications from TTT include retinal vascular occlusion, retinal fibrosis, retinal neovascularization, cystoid macular edema, retinal tears, retinal detachment, and vitreous

hemorrhage. These complications are much more common with primary TTT than with adjunctive TTT. Enucleation was once the mainstay of treatment for uveal melanoma. Today, however, enucleation is usually reserved for tumors that are very large or have demonstrated extensive local invasion (e.g., transscleral or optic nerve invasion). Enucleation may also be preferred in eyes with poor visual potential. At the time of enucleation surgery, a spherical implant is placed in the orbit and attached to the extraocular muscles to imitate the movement of the eye. Subsequently, the patient is fit with a prosthetic eye, which is a shell painted to match the other eye. Plaque radiotherapy is the most common treatment for uveal melanoma. This treatment approach allows a large dose of radiation (85–100 Gy) to be delivered to the tumor over 4–5 days while minimizing radiation toxicity to the rest of the eye and surrounding structures. Various radioisotopes have been used, including 125 iodine, 106ruthenium, and 103paladium, but 125 iodine is the most common choice. The medium tumor trial of the Collaborative Ocular Melanoma Study compared 125iodine plaque radiotherapy to enucleation and found no

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Uveal Melanoma

Uveal Melanoma, Fig. 4 Uveal melanoma histopathology showing spindle cells (a) and epithelioid cells (b)

difference in survival. The plaque is surgically implanted on the scleral surface of the eye overlying the tumor. Treatment outcomes are significantly enhanced when intraoperative ultrasound is used to verify accurate placement of the plaque over the tumor. Local tumor control following plaque radiotherapy is greater than 90% in most centers. Complications include cataract, radiation retinopathy and papillopathy, neovascular glaucoma, vitreous hemorrhage, and rarely scleral necrosis. Incidence and severity of these complications are dose and location dependent. Focal delivery of high-dose radiotherapy can also be achieved with proton beam radiotherapy and other forms of charged particle therapy. A surgical procedure is required to place tantalum rings on the scleral surface overlying the tumor to direct the charged particle beam. Local tumor control appears to be slightly better with charged particle therapy than with plaque radiotherapy, especially in centers that do not use intraoperative ultrasonography to localize their plaques, but complications are also more common with charged particle therapy. Complications include eyelash loss, dry eye, cataract, neovascular glaucoma, and radiation retinopathy and papillopathy. Newer radiosurgery techniques such as Gamma Knife and CyberKnife are being evaluated for their role in uveal melanoma. Local tumor resection was once a popular alternative treatment that avoided radiotherapy and its

attendant complications. However, as more experience has been obtained, it has become clear that local resection has its own set of serious side effects, including a high rate of local tumor recurrence, retinal detachment, vitreous hemorrhage, and proliferative vitreoretinopathy. Today, local resection is usually reserved for selected tumors with small basal diameter and anterior location. There are many variations on this surgical technique, but most involve the creation of a partialthickness scleral flap, through which the tumor is resected en bloc. Adjunctive radiotherapy is often used postoperatively. Pathology The Callender classification is the most widely accepted histopathologic grading system for uveal melanoma and is based on the spindle and epithelioid cell types (Fig. 4). Spindle A cells have narrow nuclei with a longitudinal fold. Spindle B cells have larger nuclei with a defined nucleolus. Epithelioid cells are larger and polygonal in shape with large nucleoli. Uveal melanomas are usually described as predominantly spindle, epithelioid, or mixed. Spindle cells portend a better prognosis and epithelioid cells a worse prognosis. Other pathologic features that have been associated with worse prognosis include transscleral extension, increased number of mitotic figures, increased nucleolar area, and presence of looping extracellular matrix patterns.

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Uveal Melanoma, Fig. 5 Gene expression profiling is the most accurate predictor of metastasis currently available for uveal melanoma. (a) Unsupervised principal component analysis showing clustering of tumors (spheres) into class 1 (blue) and class 2 (red), with low and high risk for metastasis, respectively. (b) Heat map with

supervised analysis with only nine discriminating genes (rows), which classify all tumors correctly (columns). Since a small number of genes can be used, this test is being adapted for use as a clinical test that can be applied to enucleation and biopsy samples

Systemic Evaluation and Metastasis Metastatic uveal melanoma involves the liver in approximately 90% of cases. Other sites include lung, subcutaneous tissues, and bone. Systemic evaluation and monitoring should include liver function tests (especially lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transpeptidase) and imaging of the abdomen with CT or MRI. Median survival after clinical diagnosis of metastasis is about 5–7 months. Metastatic disease limited to the liver can occasionally be treated with partial hepatectomy or hepatic arterial chemoembolization. Systemic chemotherapy and immunotherapy have met with limited success.

that may predict metastasis with greater accuracy. The chromosomal alteration that is most closely linked to metastasis is loss of one copy of chromosome 3 (monosomy 3), which is found in about half of primary uveal melanomas. Various techniques have been employed to assess chromosome 3 status in clinical settings, including karyotype analysis, comparative genomic hybridization, fluorescence in situ hybridization, and loss of heterozygosity for polymorphic markers across the chromosome. Microarray gene expression profiling has identified two molecular subgroups of primary uveal melanoma, referred to as class 1 and class 2 (Fig. 5). Virtually all of the metastatic deaths occur in class 2 tumors. The class 2 gene expression signature is closely linked to monosomy 3, but there are some exceptions, and the former outperforms the latter in terms of predictive accuracy. Consequently, the gene expression-based classifier has been refined to require less than ten genes and a small number of tumor cells that can be obtained by FNAB. Clinical studies are now underway to determine the best combination of molecular testing approaches for stratifying patients based on

Prognostic Factors As mentioned in the previous sections, there have been many clinical and pathologic features of uveal melanoma that are statistically associated with metastasis. However, none of these features has demonstrated predictive accuracy sufficient for use in making treatment decisions in individual patients. This has led many researchers to investigate genetic features of the primary tumor

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metastatic risk for inclusion in clinical trials and implementation of preemptive, adjuvant systemic therapy. Systemic Therapy and Future Directions Recent work on tumor doubling times, diseasefree interval, and metastasis strongly implies that those uveal melanomas that have the capacity to disseminate (e.g., class 2 tumors) have already done so prior to ocular treatment in most cases. This would explain why successful local treatments have not been associated with a demonstrable improvement in survival. Consequently, it may only be possible to improve survival in uveal melanoma by identifying high-risk patients and treating them preemptively with systemic therapies that delay or prevent the development of clinical metastasis by maintaining micrometastases in a dormant state. The centerpiece of such a preventative strategy will be the newly emerging molecular prognostic tests described here. This strategy will also require the identification of new therapeutic approaches targeting the micrometastatic cell population.

Uvomorulin

References Ehlers JP, Harbour JW (2006) Molecular pathobiology of uveal melanoma. Int Ophthalmol Clin 46(1):167–180 Harbour JW (2003) Clinical overview of uveal melanoma: introduction to tumors of the eye. In: Albert DM, Polans A (eds) Ocular oncology. Marcel Dekker, New York, pp 1–18 Onken MD, Worley LA, Ehlers JP et al (2004) Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 64(20):7205–7209 Onken MD, Ehlers JP, Worley LA et al (2006a) Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas. Cancer Res 66:4602–4609 Onken MD, Worley LA, Davila RM et al (2006b) Prognostic testing in uveal melanoma by transcriptomic profiling of fine needle biopsy specimens. J Mol Diagn 8:567–573

Uvomorulin ▶ E-Cadherin

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V(D)J Recombination

Characteristics

Markus Müschen Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA

More than 30 years ago, Hozumi and Tonegawa discovered that immunoglobulin genes in B lymphocytes as opposed to any other somatic cell type undergo a complex rearrangement process in order to assemble a functional immunoglobulin variable region. This process, later termed V(D)J recombination, is in fact unique for B and T lymphocytes and critical for the expression of immunoglobulins and T cell receptors, respectively. Immunoglobulin and T cell receptor genes represent gene families in mammalians that are arranged in segments and hence require somatic recombination of individual variable (V), diversity (D), and joining (J) gene segments to assemble a coding immunoglobulin or T cell receptor gene. V(D)J recombination defines an early step during B- or T-lymphocyte development within the bone marrow or the thymus, respectively. In humans, three immunoglobulin and four T cell receptor gene loci are known (see Table 1). Each locus carries gene segments that together encode one protein chain. Immunoglobulins or T cell receptors typically represent the assembly of two heterodimers: for instance, immunoglobulins expressed on the cell surface of B lymphocytes comprise of two identical heterodimers of each one immunoglobulin heavy chain molecule and one immunoglobulin k or l light chain molecule.

Synonyms Immunoglobulin rearrangements; Somatic recombination of V, D, and J segments; T-cell receptor gene rearrangement

Definition Surface immunoglobulin (expressed on B lymphocytes) and T cell receptors (expressed on T lymphocytes) represent the central molecules for antigen recognition in adaptive immune responses. Variable (V), diversity (D), and joining (J) gene segments, which together encode immunoglobulin or T cell receptor variable chains, are present in every somatic cell. However, in order to acquire coding capacity, V, D, and J gene segments need to be assembled in a functional configuration. The mechanism for the assembly of V, D, and J gene segments, termed V(D)J recombination, represents a unique capacity of both B and T lymphocytes.

# Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3

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V(D)J Recombination

V(D)J Recombination, Table 1 Immunoglobulin and T cell receptor gene loci in the human Locus IGH, 14q32 IGK, 2p11.2 IGL, 22q11.2 TCRA, 14q11.2 TCRB, 7q34 TCRG, 7p14 TCRD, 14q11.2

V segments 123 76 74 49 64 14 8

D segments 27 None None None 2 None 3

Mechanism As opposed to meiotic recombination, V(D)J recombination represents a site-specific DNA recombination event in B or T lymphocytes. Sitespecific recombination is conferred by recombination signal sequences (RSS) immediately flanking V, D, and J gene segments. RSSs are composed of a conserved heptamer and nonamer and a non-conserved spacer of 12 or 23 bp in length. The length of the spacer determines the pairing of RSS motifs, which only allows synapse formation between RSS motifs of different spacer lengths. For the recombination process, RAG1 and RAG2 proteins (encoded by the recombinationactivating genes 1 and 2) are essential. RAG1 and RAG2 proteins are only expressed in B and T lymphocytes, and their expression is tightly controlled during their development. RAG1 and RAG2 form a heterodimeric complex that only recognizes synapses between two RSS motifs of different spacer lengths, i.e., only 12/23 RSS pairs. The 12/23 rule for RSS pairing ensures that V, D, and J segments are recombined in a coordinated manner to prevent mispairing of gene segments. The RAG1/RAG2 complex then introduces a DNA double-strand break exactly at the border of the heptamer element (Fig. 1). These DNA double-strand breaks are blunt ended and 50 -phosphorylated for signal ends, which facilitates selfligation and formation of signal joints within so-called excision circles (Fig. 1). In contrast, the broken-ended DNA of gene segments (coding joint, Fig. 1) are protected against immediate rejoining by the formation of a closed hairpin structure. Of note, even though the targeting by the RAG1/RAG2 complex is very precise, the sequence of coding joints, i.e., the junction

J segments 6 5 9 61 13 5 4

Gene product Ig heavy chain Ig k light chain Ig l light chain TCRa chain TCRb chain TCRg chain TCRd chain

between two rearranged gene segments, is extremely variable. This variability of coding joints is owed to further processing of the hairpin structure by the enzymes Artemis and DNA ligase IV. The diversity of coding joints is further increased by the activity of the lymphoid-specific terminal deoxynucleotidyl transferase (TdT): TdT is able to introduce additional nucleotides, the so-called N-nucleotides, into the junction between two gene segments in a template-independent manner. Classical RSS motifs containing the conserved heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences and a non-conserved 12 or 23 nucleotide spacer are exclusively found adjacent to immunoglobulin and T cell receptor gene segments. However, RSS-like motifs, so-called cryptic RSS sites, have been identified in a number of genes outside the immunoglobulin and TCR loci. Functional assays have shown that these cryptic RSS motifs (cRSS, Fig. 1) can indeed be targeted by the V(D)J recombinase machinery. Given that many genes, among them tumor suppressor and proto-oncogenes, harbor cryptic RSS sites, targeting by the V(D)J recombinase may predispose early B- and T-lymphocyte precursors to malignant transformation. In this regard, it is important to note that the RAG1 and RAG2 enzymes also possess transposase activity. Thereby, 50 -phosphorylated signal ends carrying a complete RSS motif on each end may attack unrelated DNA, preferentially at cryptic RSS sites (cRSS, Fig. 1). If both RSS motifs of the excised signal ends participate in this attack, this may lead to the integration of the RSS-flanked DNA fragments at positions staggered by 3–5 bp, resulting in target site duplication at the integration site (Fig. 1,

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Coding joint V

OH

7 23 9 HO

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cRSS O

HO

Signal ends

Closed signal joint in excision circle

Attack of cRSS motifs in unrelated DNA

Transposition of signal ends into unrelated DNA

V(D)J Recombination, Fig. 1 Schematic representation of RSS pairing during D-J segment joining. A simplified model of the IGH locus is shown including a group of V, D, and J gene segments. Each of these segments are flanked by either a recombination signal sequence (RSS) with a spacer length of 23 bp (V, light gray) or 12 bp (J, dark gray) or both types of RSS motifs (D). During the RAG1/RAG2mediated recombination process, two types of break intermediates are generated: a coding joint (top) with a junction between two rearranged gene segments and signal ends

(bottom) containing a fragment of excised DNA between two RSS motifs. These signal ends are processed by selfligation to a signal joint within a closed excision circle. These excision circles are very stable in lymphocytes but are not replicated during mitosis. On the other hand, broken-ended RSS motifs may attack unrelated DNA, preferentially at cryptic RSS sites (cRSS). Thereby, RAG1 and RAG2 can act as transposases, in that they catalyze the integration of the intervening DNA between two signal ends into the attacked unrelated DNA

bottom). It is obvious that integration of DNA that was excised during V(D)J recombination into unrelated loci carries the risk of oncogenic transformation. Even though classical transposition events are rarely seen in lymphoid malignancies, chromosomal translocations and deletions bearing the hallmarks of illegitimate V(D)J recombination are frequent events during malignant transformation of lymphocytes.

lymphoblastic leukemia (ALL) often coincides with the timing of activity of the V(D)J recombinase machinery. Therefore, it was an obvious hypothesis that aberrant or “illegitimate” V(D)J recombination may be the causative mechanism for many if not all subtypes of ALL. In many cases, ALL cells carry ▶ chromosomal translocations. The breakpoints of these gene rearrangements, however, exhibit the hallmarks of V(D)J recombination in only a few cases (Table 2). For instance, targeting to immunoglobulin (IGH, IGK, or IGL) or T cell receptor (TCRA, TCRB, TCRG, TCRD) loci would argue for involvement of V(D)J recombination. While

Clinical Aspects Malignant transformation of human B- and T-lymphocyte precursors toward ▶ acute

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V(D)J Recombination

V(D)J Recombination, Table 2 Frequent genetic aberrations in lymphoid malignancies related to illegitimate V(D)J recombination Locus BCL1

References Tsujimoto et al. (1998)

BCL2

Van Drager et al. (2000)

CCND2

Clappier et al. (2006) Clappier et al. (2006)

CDKN2A

Kitagawa et al. (2002) Cayuela et al. (1997) T cell lineage ALL Kitagawa et al. (2002) Cayuela et al. (1997) Wiemels et al. (2002) Kagan et al. (1989) Zutter et al. (1990) Finette et al. (1996) Chen et al. (1996) T cell lineage ALL Champagne et al. (1989) Cheng et al. (1990) Tuji et al. (2004) Aplan et al. (1990) Raghavan et al. (2001) Finger et al. (1989) Chen et al. (1990) Tycko et al. (1998) Retière et al. (1998)

CDKN2B CDKN2D E2A HOX11 HPRT LMO2 NOTCH1 SIL/SCL TAL1 TAL2 TCRB/ TCRG TTG1

Boehm et al. (1988) McGuire et al. (1989)

BCL1 and BCL2 gene rearrangements typically target the IGH locus, rearrangements of the CCND2, HOX11, LMO2, TAL1, TAL2, and TTG1 gene target at least one of the four TCR loci (Table 2). In addition, one would expect that breakpoints reflecting illegitimate V(D)J recombination exhibit traces of site-specific targeting in that they precisely flank RSS or RSS-like motifs. Virtually all genes involved in an IGH- or TCR-specific gene rearrangement show targeting of an RSS or RSS-like motif at least for one of the two translocation partners. In addition, genetic abnormalities were found, in which RSS or RSS-like motifs were targeted even though no immunoglobulin or TCR locus was involved. This applies mainly to intragenic/interstitial

Genetic aberration t(11;14)(q13; q32) t(14;18)(q32; q21) t(7;12)(q34;p13) t(12;14)(p13; q11) 9p21 deletion

Target IGH IGH

Malignancy Mantle cell lymphoma (B cell) Follicular B cell lymphoma

TCRB TCRA

T cell lineage ALL T cell lineage ALL

RSS

B and T cell lineage ALL

9p21 deletion 9p21 deletion t(1;19)(q23;p13) t(10;14)(q24; q11)

RSS RSS RSS TCRD

B and T cell lineage ALL T cell lineage ALL Pre-B cell ALL T cell lineage ALL

del Xq26-q27

RSS

Normal lymphocytes

t(11;14)(p13; q11)

TCRD

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del 9q34 del 1p32

RSS RSS

T cell lineage ALL T cell lineage ALL

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TCRD

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t(7;9)(q34;q32) inv(7)

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t(11;14)(p15; q11)

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deletions. Such deletions often occur in T cell lineage ALL cells, and deletions of the INK4 family genes CDKN2A, CDKN2B, and CDKN2D and within the HPRT, NOTCH1, and SIL/SCL loci (Table 2) are examples for such a second type of genetic aberration induced by illegitimate V(D)J recombination. Finally, addition of N-nucleotides (i.e., a junction sequence that cannot be attributed to either of the two fusion partners) by enzymatic activity of TdT represents a unique feature of V(D)J recombination. Indeed, junctional diversity compatible with the introduction of N-nucleotide was found in many cases, in which a gene rearrangement was mediated by V(D)J recombination, but not in all.

Valproic Acid

Applying these three criteria (involvement of IGH or TCR loci, site-specific targeting of RSS- or RSS-like motifs, and presence of N-nucleotides) to B and T cell lineage ALL, it appears that T cell lineage as opposed to B cell lineage ALL frequently arises through genetic aberrations that were caused by aberrant V(D)J recombination. With the exception of the ▶ E2A-PBX1 gene rearrangement, none of the classical chromosomal translocations in B cell lineage ALL are compatible with V(D)J recombination: ▶ BCR-ABL1, MLL-AF4, and TEL-AML1 gene rearrangements are not related to the IGH locus, do not show site-specific targeting at RSS-like motifs, and do not carry intervening nucleotides within the junction between the two fusion partners. The case of E2A-PBX1 is complicated because only the breaks of the E2A gene are sitespecific and localized at RSS-like motifs, while the breaks within the PBX1 gene are scattered over a large genomic region. Unlike BCR-ABL1, MLLAF4, and TEL-AML1 gene rearrangements, however, the E2A-PBX1 fusion carries N-nucleotides in almost all instances, which argues for a contribution of the V(D)J recombinase. ▶ Chromosomal translocations affecting one of the immunoglobulin loci in mature B cell lymphomas may also be owed to somatic hypermutation and/or immunoglobulin classswitch recombination. These two mechanisms are active in mature B cells during affinity maturation within germinal centers of tonsils, lymph nodes, and spleen. Indeed, frequent recurrent gene rearrangements in germinal center-derived B cell lymphoma most likely result from somatic hypermutation and/or class-switch recombination and not from V(D)J recombination. V(D)J recombinase-related genetic aberrations indeed seem to have distinct mechanisms in B cell lineage (pre-B ALL and B cell lymphoma) and T cell lineage (T cell precursor ALL) malignancies. According to a proposed model, chromosomal translocations in T cell lineage ALL are more frequent and are mainly caused by illegitimate V(D)J recombination between a TCR locus and a protooncogene locus, i.e., both loci were targeted by V (D)J recombination. Conversely, for translocations in B cell lineage malignancy, involvement of the V (D)J recombinase machinery is rare. In these rare

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cases, in which aberrant V(D)J recombination contributes to B cell lineage malignancy, V(D)J recombination typically targets only one of the two translocation partners: In t(1;19)(q23;p13) pre-B ALL, the E2A locus is targeted by the V(D)J recombinase but not its fusion partner PBX1. In both ▶ mantle cell lymphoma and ▶ follicular lymphoma, the IGH locus is targeted by aberrant V(D)J recombinase activity but not the BCL1 and BCL2 genes on the respective partner chromosome.

References Hiom K, Melek M, Gellert M (1998) DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 94:463–470 Hozumi N, Tonegawa S (1976) Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc Natl Acad Sci U S A 73:3628–3632 Marculescu R, Le T, Simon P et al (2002) V(D)J-mediated translocations in lymphoid neoplasms: a functional assessment of genomic instability by cryptic sites. J Exp Med 195:85–98 Schatz DG, Oettinger MA, Baltimore D (1989) The V(D)J recombination activating gene, RAG-1. Cell 59:1035–1048 Schlissel M, Constantinescu A, Morrow T et al (1993) Double-strand signal sequence breaks in V(D)J recombination are blunt, 50 -phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev 7:2520–2532

Valproic Acid Marta Kostrouchova Institute of Cellular Biology and Pathology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic

Synonyms 2-Propylpentanoic acid; Dipropylacetic acid

Definition Valproic acid is a short-chain branched fatty acid. It was first synthesized by B.S. Burton in 1882.

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During the last 40 years, valproic acid has been extensively used for the treatment of epilepsy, bipolar disorder, and other neurological disorders. In the human brain, valproic acid affects the function of the neurotransmitter gamma aminobutyric acid (GABA) (mainly as a GABA transaminase inhibitor). Valproic acid has histone deacetylase inhibitory activity and affects behavior and growth of various cancers and cancer cell lines. Valproic acid induces cell differentiation and ▶ apoptosis of diverse cancer cell lines, increases recognition of cancer cells by the immune system, inhibits cell proliferation, and decreases metastatic and angiogenic potential of cancer cells. The anticancer effect of valproic acid is based on its histone deacetylaseinhibiting activity, modulation of MAPK signaling, and beta-catenin pathway. Other mechanisms of action of valproic acid were also proposed. The relative safety of administration of valproic acid and its involvement in multiple pathways are making it a valuable lead in search for more potent and more specific drugs with anticancer activities. Molecular Formula: (CH3CH2CH2)2CHCOOH Molecular Mass 144.21

Characteristics The name valproic acid originates from its descriptive chemical name 2-propylvaleric acid (valeric acid is a synonym for pentanoic acid that was isolated from the flowering plant valerian (Valeriana officinalis)). Valproic acid was used as a solvent of neurologically active compounds, and its therapeutic potential was discovered by chance. Valproic acid is a relatively safe drug, with only infrequent major side effects that include hepatotoxicity, thrombocytopenia, and prolonged coagulation time. In about 5% of pregnant users, valproic acid can cause congenital anomalies such as spina bifida. Valproic acid inhibits proliferation and induces differentiation of neuroectodermal tumor cells. Valproic acid and its analogs modulate the behavior of various tumor cell types by inducing apoptosis and differentiation, inhibiting proliferation, decreasing angiogenic potency, and increasing immunogenicity of cancer cells.

Valproic Acid

Valproic acid induces apoptosis in two ways: through the effect on ▶ caspases and chromatin fragmentation and through its effect on membranes linked to phosphatidylserine externalization and release of cytochrome c from mitochondria. Valproic acid induced apoptosis in various animal cell lines including rat hepatoma cell line FaO; many human leukemia cell lines of B-, T-, and myeloid lineage; murine B-lymphoid cell lines; MV4-11 and KOCL-44; prostate carcinoma cell line; human thyroid cancer cells; and other cells. Treatment with valproic acid leads to inhibition of cell proliferation and induction of cell differentiation in different cell lines, mainly of neuroectodermal and leukemic origin. Valproic acid increased differentiation of human ▶ neuroblastoma cells (SJ-N-KP, AF8), which was documented by neurite extension and upregulation of neuronal markers. The decreased net proliferation rate after valproic acid administration was observed in ▶ prostate cancer cells – androgen receptor positive (LNCaP and C4-2) and androgen receptor negative (DU145 and PC3). In leukemic cell lines HL-60 and MOLT-4, valproic acid-induced cell differentiation was marked by an increase of CD 11b and co-stimulatory/adhesion molecule CD86. ▶ Hepatocellular carcinomas (HCC) that are resistant to conventional chemotherapeutics showed decreased proliferation in response to the treatment with valproic acid. A downregulation of anti- and upregulation of proapoptotic factors indicated that modulation of intracellular pro- and anti-apoptotic proteins is a key event in valproic acid-induced tumor cell death. The influence of valproic acid in the cell cycle was determined to be in the G1 phase. Valproic acid is a potent inhibitor of tumor angiogenesis. Valproic acid caused inhibition of proliferation, migration, and tube formation of endothelial cells and also caused a decrease of endothelial nitric oxide synthase (eNOS) protein level. The inhibition of angiogenesis in vivo was documented on the chicken chorioallantoic membrane assay and the Matrigel plug assay in mice. Valproic acid also caused disturbed vessel formation.

Valproic Acid

The indirect effect of valproic acid was observed in neuroblastoma cells. A higher production of anti-angiogenic molecules thrombospondin-1 and activin A was detected. Similarly the treatment of colon adenocarcinoma cell line Caco-2 caused significant reduction of ▶ vascular endothelial growth factor (VEGF) secretion, downregulation of protein expression and mRNA of VEGF, basic fibroblast growth factor (bFGF) protein level, and inhibition of the ubiquitin-proteasome proteolytic system activity. Valproic acid increases immunogenicity of cancer cells. Treatment of human ▶ hepatocellular carcinoma cells with valproic acid mediated recognition of cancer cells by cytotoxic lymphocytes via the immunoreceptor NKG2D. Valproic acid induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface and soluble and total MIC protein expression. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by natural killer cells. In primary human hepatocytes, valproic acid treatment did not induce MIC protein expression indicating that valproic acid mediates specific priming of malignant cells for innate immune effector mechanisms. Valproic acid was shown to modulate behavior of numerous tumors. Valproic acid was repeatedly used in patients with acute myeloid leukemia (AML) and combined with all-trans retinoic acid (ATRA). In some subtypes, the AML is connected with translocations that generate fusion genes including ▶ retinoic acid receptor alpha (RARa) which function as oncoproteins that inhibit differentiation pathways of myeloid lineage. ATRA was shown to partially release the differentiation block by binding to the RARa part of the fusion protein with subsequent increased expression of target genes (likely connected with disruption of binding of transcriptional corepressors and induced degradation of the fusion protein). Valproic acid markedly increased the efficacy of the treatment by ATRA and resulted in transient disease control in subsets of patients with AML. The antitumor efficacy of valproic acid was observed in medulloblastoma and supratentorial primitive neuroectodermal tumor (sPNET), which are the most common malignant brain tumors in

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children with poor prognosis. Two medulloblastoma (DAOY and D283-MED) and one sPNET (PFSK) cell lines were treated with valproic acid with resulting potent growth inhibition, cell cycle arrest, apoptosis, senescence, differentiation, suppressed colony-forming efficiency, and tumorigenicity in a time- and dose-dependent manner at clinically safe concentrations (0.6 and 1 mmol/l). Valproic Acid Affects Cell Behavior by Multiple Mechanisms Valproic Acid Inhibits Histone Deacetylase Activity

The effect of valproic acid on behavior of cancers and cancer cell lines initiated studies directed at its mode of action. Behavior of cells depends on their gene expression that in turn is regulated by the basic transcription machinery, by cell- and tissuespecific transcription factors and cofactors, and by the organization of chromatin. Posttranslational modification of chromatin, including histone acetylation, methylation, phosphorylation, ubiquitination, and other modifications, is a critical part of regulation of gene expression. Valproic acid was shown to downregulate the HDAC activity in teratocarcinoma and neuroblastoma cells. Nevertheless, this effect may have been caused by a direct action of valproic acid on enzymes involved in adding or removing acetyl residues on lysines of nucleosomal histones (mostly histones H3 and H4) or by a modulation of other targets. It was shown that valproic acid affects the in vitro deacetylation assay and acts as an HDAC inhibitor. Inhibition of HDAC activity was analyzed by measuring the acetylation of core histones H3 and H4 in the leukemic K562 and U937 cell lines. Valproic acid and its analogs (2-methyl2-n-propylpentanoic acid (2M2PP), 4-pentenoic acid (4PA), 2-methyl-pentenoic acid (2M2P), 2-ethylhexanoic acid (2EH), and valpromide (VPM)) were shown to inhibit class I HDAC (HDACs 1–3) and class II HDAC (HDACs 4, 5, and 7). Valproic acid was the strongest inhibitor. The mechanism by which valproic acid affects behavior of cancer cells differs from its antiepileptic effect since the efficacy of antineoplastic and antiepileptic effects differs between particular analogs.

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Valproic acid did not inhibit the activity of class II HDAC 6 and 10, in contrast to another HDAC inhibitor, trichostatin A (TSA). This implies a more selective effect of valproic acid on HDAC inhibition compared to TSA. Although valproic acid affects both class I and class II HDACs, the effect on these two classes differs. Valproic acid has been shown to inhibit the catalytic activity of class I HDACs and induce the proteasomal degradation of class II HDACs in contrast to TSA. Valproic acid and its analogs induce expression of multiple exogenous reporter genes which are associated with HDAC inhibition, i.e., SV40, p21, and gelsolin. Studies directed at the genome-wide expression pattern showed that valproic acid affects expression of selective groups of genes (by their induction or repression). Valproic Acid Interferes with MAPK Signaling

Valproic acid increases the DNA binding and transactivation activity of the transcription factor ▶ AP-1. In vitro studies showed that valproic acid specifically triggers the phosphorylation of ERK, the upstream modulator of AP-1, but does not act via the JNK (c-jun N-terminal kinase) and p38 pathways. Valproic acid and its derivatives (named above) activate MAPK. In clear contrast to their effect on inhibition of class I HDACs, the analogs of valproic acid have a stronger effect on MAPK activation than valproic acid itself. This may reflect a possibility that the effect on MAPK is not mediated by HDAC inhibition. Therefore, valproic acid-induced increases in AP-1 binding and function are likely due, at least in part, to activation of ERK followed by phosphorylation and increase in expression of c-Jun. Expression and phosphorylation of c-Jun in ERK pathway is required to direct cellular differentiation in poorly differentiated cells. Nevertheless, it seems likely that the way valproic acid affects cell behavior may to a large extent depends on the particular cell type. Valproic Acid Affects the Beta-Catenin Pathway

GSK-3 beta (glycogen synthase kinase-3 beta) is a negative regulator of the ▶ Wnt signaling

Valproic Acid

pathway, which regulates numerous processes, including cellular proliferation, cell migration, cell polarity, and organo- and carcinogenesis. GSK-3 beta phosphorylates beta-catenin and this leads to its rapid degradation. Inhibition of GSK-3 beta by Wnt signaling leads to stabilization and accumulation of the beta-catenin protein. Consequently, beta-catenin translocates to the nucleus where it activates transcription of Wnt-dependent genes by binding to factors Tcf/Lef (T-cell factor, lymphoid-enhancer factor). Valproic acid has been reported to inhibit GSK-3 beta-mediated phosphorylation of a peptide derived from CREB protein in human embryonic kidney 293 T and murine Neuro2A ▶ neuroblastoma cells and increase levels of beta-catenin in human neuroblastoma cells. Nevertheless, it was also proposed that valproic acid activates Wnt-dependent gene expression through the inhibition of histone deacetylase activity. The involvement of histone deacetylase inhibition by valproic acid in beta-catenin-dependent regulation is supported by its effect on the expression of E-cadherin. Expression of numerous tumor suppressor genes including E-cadherin is linked to hypermethylation of specific regions of DNA and may be partially reverted by increased histone acetylation. Valproic Acid May Influence Additional Pathways

Valproic acid was shown to be involved in other regulatory pathways. In many of them, it is likely that the effect is mediated primarily by the inhibition of histone deacetylase activity but some may be distinct. Valproic acid increases the levels of ▶ 5-lipoxygenase (5-LOX) protein in murine hippocampus. 5-LOX produces ▶ leukotrienes from arachidonic acid, and this is likely to be involved in the regulation of ▶ chromatin remodeling. Since increased levels of 5-LOX are linked to aging and neurodegeneration, it may be expected that the increase of 5-LOX expression may contribute to tumor cell aging and differentiation through chromatin remodeling. Valproic acid is likely to inhibit invasiveness of cancer cells at concentrations lower than those necessary for its antiproliferative effect. Increased expression of genes that inhibit invasiveness of

Valproic Acid

cancer cells was observed in response to treatment with relatively low doses of valproic acid (150 mmol/l). These genes included ▶ signal transducer and activator of transcription 6 (STAT6), RING1, RYBP, and PCDHGC3. Valproic acid may affect the regulation of gene expression by nuclear receptors which are critically influenced by HDACs. Moreover, some members of this superfamily of genes may be affected by valproic acid more directly. PPARd and PPARg (but not PPARa) were activated by valproic acid in Chinese hamster ovary cells and F9 teratocarcinoma cells. At least in the case of PPARd, this effect was not based on the binding and activation of the receptor by valproic acid as an agonistic ligand since it did not induce formation of heterodimers of PPARd with retinoid X receptor on DNA response elements. Activated expression of PPARg-dependent genes may significantly contribute to the anticancer activity of valproic acid. PPARg regulates the expression of the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) that is often mutated in cancers. PTEN, a lipid phosphatase, dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol (4,5)-diphosphate (PIP-2) and antagonizes the regulation by phosphatidylinositol-3 kinase (PI-3 K).

Cross-References ▶ Angiogenesis ▶ AP-1 ▶ Apoptosis ▶ Arachidonic Acid Pathway ▶ Acute Myeloid Leukemia ▶ Caspase ▶ Chemotherapy ▶ Chromatin Remodeling ▶ Epigenetic Modifications ▶ HDAC Inhibitors ▶ Hepatocellular Carcinoma ▶ Leukotrienes ▶ 5-Lipoxygenase ▶ Metastatic Colonization ▶ MAP Kinase

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▶ Neuroblastoma ▶ Prostate Cancer ▶ Peroxisome Proliferator-Activated Receptor ▶ Retinoic Acid ▶ Retinoids ▶ Signal Transducers and Activators of Transcription in Oncogenesis ▶ Valproic Acid ▶ Vascular Endothelial Growth Factor ▶ Wnt Signaling

References Altucci L, Clarke N, Nebbioso A et al (2005) Acute myeloid leukemia: therapeutic impact of epigenetic drugs. Int J Biochem Cell Biol 37:1752–1762 Blaheta RA, Michaelis M, Driever PH et al (2005) Evolving anticancer drug valproic acid: insights into the mechanism and clinical studies. Med Res Rev 25:383–397 Kostrouchova M, Kostrouchova M, Kostrouch Z (2007) Valproic acid: a molecular lead to multiple regulatory pathways. Folia Biol (Praha) 53:37–49 Minucci S, Pelicci PG (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6:38–51

See Also (2012) Arachidonic acid. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 260. doi:10.1007/978-3-642-16483-5_379 (2012) Cell cycle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) Chromatin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 825. doi:10.1007/978-3-642-16483-5_1125 (2012) Cofactor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 891. doi:10.1007/978-3-642-16483-5_1251 (2012) CREB-binding protein (CBP)/p300. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 993. doi:10.1007/978-3-642-164835_1370 (2012) ERK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1307–1308. doi:10.1007/978-3-642-16483-5_1987 (2012) Gamma-aminobutyric acid. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1494. doi:10.1007/978-3-642-164835_2312 (2012) Glycogen synthase kinase-3. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-164835_2448

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4782 (2012) HDACs. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1635. doi:10.1007/978-3-642-16483-5_2592 (2012) MAPK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) Nuclear receptor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2571. doi:10.1007/978-3-642-16483-5_4157 (2012) Posttranslational modification. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2966. doi:10.1007/978-3-642-164835_4696 (2012) TCF/LEF. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3625. doi:10.1007/978-3-642-16483-5_5705 (2012) Teratocarcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3651. doi:10.1007/978-3-642-16483-5_5729 (2012) Transcription factor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3752. doi:10.1007/978-3-642-16483-5_5901 (2012) Trichostatin A. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3783. doi:10.1007/978-3-642-16483-5_5973 (2012) Tumor suppressor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3803. doi:10.1007/978-3-642-16483-5_6056 (2012) Ubiquitin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3825. doi:10.1007/978-3-642-16483-5_6083 (2012) Wnt. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3953. doi:10.1007/978-3-642-16483-5_6255 (2012) Wnt/beta-catenin pathway. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3957. doi:10.1007/978-3-642-16483-5_6256

Vanadium Malay Chatterjee Department of Pharmaceutical Technology, Jadavpur University, Calcutta, West Bengal, India

Definition Vanadium, a member of group VB and in the 4th period of the periodic table, is a first transition series, d-block, grayish metallic element with an atomic number of 23 and forms different oxidation states of 1, 0, +2, +3, +4, and +5. In the oxidation states +3 (vanadic), +4 (vanadyl), and

Vanadium

+5 (vanadate) being most common, the oxidation state +4 is most stable in biological systems. Vanadium is an endogenous constituent of most mammalian tissues and is further considered a dietary micronutrient. It is an essential element for the proper growth and development of mammals owing to its diverse physiological and biochemical functions.

Characteristics The uniqueness of vanadium is that it is included in the list of 40 essential micronutrients required in small amounts for normal cell metabolism. Accordingly, it has been incorporated into many multinational pharmaceutical preparations for maintenance of normal health. Although micronutrients never have had pharmacological potencies, they can prevent the minor wear and tear of the essential critical molecules of the cell; vanadium may thus have a role in DNA maintenance reactions and may prevent genomic instability leading to ▶ cancers. The main source of vanadium intake for the general population is food, such as chicken, fish, grains, cereals, liver, spinach, black pepper, parsley, fruits, and vegetables. The total body pool of vanadium in humans is about 100 mg, with the actual daily dietary intake estimated to be 10–60 mg, depending on individual human diet. Studies have established vanadium as a novel biological regulator and have confirmed the biphasic effect of this trace element in biological systems, that is, essentiality at low concentrations, and toxicity at higher concentrations. Vanadium compounds can influence the behavior of enzymes, mimic growth factor activities, regulate carbohydrate and lipid metabolisms, modulate gene expression and signal transduction pathways, and in particular exhibit anticarcinogenic/ antineoplastic activities. The diverse biological action of vanadium results from its capacity to function as an oxyanion, oxycation, or prooxidant. Vanadium in Cancer Treatment Vanadium has a potential role in tumor growth inhibition and in prophylaxis against ▶ carcinogenesis in different experimental cancer models,

Vanadium

namely, liver cancer, ▶ colon cancer, ▶ breast cancer, and others and in various types of malignant cell lines. Vanadium compounds have been found potentially effective against murine leukemia, fluid and solid Ehrlich ▶ Aurora A tumor, murine mammary ▶ adenocarcinoma, HEp-2 human epidermoid carcinoma cells and human ▶ lung cancer, ▶ breast cancer, and ▶ gastric cancer. Synthetic complexes of vanadium with amino acids, peptides, proteins, organometallic and inorganic ligands, and pharmacologically active moieties are an area of current interest in anticancer research. A cytostatic effect is observed with vanadium(III)–L-cysteine complex on chemically induced ▶ leiomyosarcoma bearing Wistar rats. [VIII(Hcys)3].2HCl.2.5H2O or compound 1 exhibits significantly greater total antioxidant capacity along with inhibition of neutral endopeptidase activity as potent as thiorphan. Moreover, compound 1 prevents lung ▶ metastasis, thus proving its role as an antimetastatic agent. These beneficial effects of the above complexes, in combination with their low toxicity, provide evidence for their possible application in the treatment of human malignant diseases. A good candidate for the development of new vanadium derivatives with organic ligands is the flavonoid quercetin because of its own anticarcinogenic effect. The quercetin-vanadyl complex [VO(Quer)(2) EtOH](n) (QuerVO) stimulates ▶ extracellular signal-regulated kinase (ERK) phosphorylation, and this seems to be involved as one of the possible mechanisms for the biological effects of the complex. Organometallic vanadocene complexes have been found to be potent antiproliferative and antimitotic agents and block cell division at the G2/M phase of cell cycle in human cancer cells by disrupting bipolar spindle formation. Furthermore, vanadocenes as potent ▶ apoptosisinducing cytotoxic agents against human ▶ testicular cancer cells in vitro as well as in experimental mice model in vivo may therefore have potential utility in the treatment of testicular seminomas in humans. Vanadyl complexes of 1,10phenanthroline [VO(Phen)2+] and related derivatives possess strong antitumor chemopreventive activities against human nasopharyngeal carcinoma, and the observed effects are found to be

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superior to the chemotherapeutic drug, ▶ cisplatin. Bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) or Metvan [VO(SO4) (Me2-Phen)2] has been identified as the most promising multitargeted anticancer bisperoxovanadium (bpV) complex with apoptosisinducing activity in human leukemia cells, ▶ multiple myeloma cells, and solid tumor cells derived from ▶ breast cancer, ▶ glioblastoma, ▶ ovarian cancer, ▶ prostate cancer, and ▶ testicular cancer. It is highly effective against cisplatin-resistant ▶ ovarian cancer and testicular cancer cell lines. Metvan inhibits the constitutive expressions of [▶ matrix metalloproteinases (MMPs)]-2 and 9 proteins and its gelatinolytic activity in HL-60 cells, and leukemic cells from patients, and also inhibits the leukemic ▶ cell adhesion to the extracellular matrix proteins laminin, type IV collagen, vitronectin, and fibronectin and the invasion through Matrigel matrix. Metvan exhibits significant antitumor activity in severe combined immunodeficient mouse xenograft models of human malignant glioblastoma and breast cancer. The broad spectrum anticancer activity of Metvan together with favorable pharmacodynamic features and lack of toxicity of this oxovanadium compound may represent the first vanadium complex and a novel anticancer agent as an alternative to platinum-based ▶ chemotherapy.

Chemopreventive/Anticancer Mechanisms Several mechanisms for the cancer chemopreventive role of vanadium have been proposed. The biochemical basis of the chemoprotective effect of vanadium may primarily be attributed to the substantial elevation of phase II conjugating enzymes, which may lead to a move and shift of the metabolic profile that may reduce the intracellular concentration of carcinogen-derived reactive intermediates. However, it is not yet clear whether the activity of specific CYP isoforms is directly influenced/modulated by vanadium treatment. Ortho- and metavanadate have been found to drastically reduce the mutagenicity of metabolically activated carcinogens by modulation of protein phosphorylation through the inhibition of protein phosphotyrosine phosphatases (PTPases).

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This micronutrient is able to exert in vivo anticlastogenic effect through suppression of ▶ micronucleus formation, sister-chromatid exchange, and structural, numerical, and physiological chromosomal aberrations and may thereby prevent genomic instability. In aqueous solution, vanadium is found predominantly as oxoanions (e.g., VO43) and as such may exhibit nucleophilic character for the electrophilic agents to attack, thereby preventing DNA alkylation damage as per the “carcinogen interception mechanism,” although at the moment we cannot say if such processes take place within cells. Experiments on various cell lines reveal that vanadate exerts its antitumor effects through activation of protein tyrosine kinases (PTKs) and/or inhibition of cellular PTPases leading to an accumulation of phosphotyrosine residues in cellular proteins. Both effects activate signal transduction pathways leading either to apoptosis or to activation of tumor suppressor genes. The stimulatory effect of vanadate on the cellular phosphorylation status of cytosolic proteins may be mediated in a dose-dependent manner probably via a protein kinase C-dependent mechanism. Vanadyl state is readily converted to vanadate in presence of H2O2, and thus vanadate seems to be the species that inhibits specific PTPase and correspondingly increases the steady states of phosphorylation and thereby activates cytosolic PTKs at relatively low concentrations. Vanadium compounds have been shown to inhibit cell proliferation by limiting the expressions of several potential marker proteins, such as proliferating cell nuclear antigen, Ki-67 nuclear antigen, etc. In vitro antiproliferative activity of a variety of vanadium compounds may be exerted by their ability to interfere with the molecular interactions between GATA binding protein 1 and ▶ nuclear factor kappa B (NF-kappaB) transcription factors and target DNA elements. Vanadium complexes with a +5 oxidation state and their discrete anionic units appear essential for inhibition of tumor cell growth with induction of apoptosis, whereas a +4 oxidation state appears to be important in inhibiting transcription factors/DNA interactions. Vanadium-induced apoptogenic signals are mediated through upregulation and overexpression

Vanadium

of p53 tumor suppressor and proapoptotic Bax and by downregulation of Bcl2. Reactive oxygen species generated by Fenton-like reactions and/or during the intracellular one-electron reduction of V (V) to V(IV) by, mainly, NADPH participate in the majority of the vanadium-induced intracellular events. ROS/H2O2 generated by vanadate triggers ▶ DNA damage and also activates ▶ mitogenactivated protein kinases (MAPKs) signal transduction pathways leading to an increased p53 protein expression and p53 phosphorylation, respectively. This in turn induces p53 transactivation, which subsequently leads to cell apoptosis. p53 is also an important transrepressor of inducible ▶ nitric oxide synthase expression and thereby attenuates excessive nitric oxide production in a regulatory negative feedback loop. Future Direction Given what is known of vanadium effects on animal physiology, individual responses to vanadium treatment might be influenced by plasmaand cellular-binding proteins and genetic predisposition. Nonetheless, the potential therapeutic advantage of vanadium compounds and the available options for mitigating toxicities suggest that further development should yield safe and effective pharmacological formulations as antineoplastic agent.

References Chakraborty T, Chatterjee A, Rana A et al (2007) Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: chemopreventive role of vanadium on this process. Biochim Biophys Acta 1772:48–59 Chatterjee M, Bishayee A (1998) Vanadium – a new tool for cancer prevention. In: Nriagu JO (ed) Vanadium in the environment, part two, health effects. Wiley, New York, pp 347–390 Evangelou AM (2002) Vanadium in cancer treatment. Crit Rev Oncol Hematol 42:249–265 Hamilton EE, Fanwick PE, Wilker JJ (2006) Alkylation of inorganic oxo compounds and insights on preventing DNA damage. J Am Chem Soc 128:3388–3395 Ray RS, Ghosh B, Rana A et al (2006) Suppression of cell proliferation, induction of apoptosis and cell cycle arrest: chemopreventive activity of vanadium in vivo and in vitro. Int J Cancer 120:13–23

Vascular Disrupting Agents

Vascular Disrupting Agents Howard W. Salmon1 and Beth A. Salmon2 1 Department of Radiation Oncology, North Florida Radiation Oncology, Gainesville, FL, USA 2 Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA

Synonyms Vascular-targeted therapies; Vascular targeting agents; VDAs

Definition Vascular disrupting agents (VDAs) disrupt tumor blood flow by specifically targeting vascular abnormalities associated with solid tumor development. The result is tumor vessel occlusion followed by extensive tumor necrosis.

Characteristics Tumor growth is limited by the diffusion of oxygen and nutrients from blood vessels into the surrounding tissue. In order to grow beyond a size of 1 mm3, tumors are dependent upon their ability to induce ▶ angiogenesis (the formation of new blood vessels). As the tumor outgrows its blood supply, angiogenic factor (i.e., VEGF and bFGF) expression is induced, which stimulates the formation of new blood vessels. The growth rate of tumors is such that angiogenesis must be continuously induced to keep a steady supply of oxygen and nutrients to the tumor tissue, resulting in a state of endothelial proliferation not normally present in the adult body. The vessels produced by this elevated angiogenic state are also abnormal in structure: tortuous vessels that experience transient interruptions in blood flow and are often leaky with reduced or nonexistent pericyte.

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The abnormality of the tumor vasculature provides a novel target for anticancer therapy, with the idea of interfering with tumor growth through the pharmaceutical disruption of the tumor blood supply (termed vascular targeting). Several vascular targeting agents have since been identified and have begun entering clinical trials. The vascular targeting approach can be broadly divided into categories: the anti-angiogenic and the vascular disrupting agents (VDAs). The anti-angiogenic agents prevent the formation of new blood vessels thereby slowing tumor growth, while VDAs target and disrupt the existing tumor vasculature, which results in acute destruction of tumor tissue. The angiogenic process is a multistage event including the upregulation of pro-angiogenic factors, increased endothelial cell proliferation, degradation of the vessel basement membrane, and endothelial cell migration and new tube formation. Each of these stages is a potential target for anti-angiogenic agents, but agents inhibiting pro-angiogenic factors are perhaps the most common. The most studied angiogenic factor involved in tumor pathophysiology is VEGF, and several agents have been developed which either prevent the binding of VEGF to its receptors or inhibit activation of the receptor. The agent bevacizumab is an anti-VEGF antibody that prevents VEGF from binding and activating its receptors and has had therapeutic success in clinical trials. The tyrosine kinase ZD6474 has been shown to inhibit angiogenesis by selectively preventing activation of the VEGFR-2 receptor and is also undergoing testing in clinical trials. Vascular Disrupting Agents The VDAs can be further divided into two basic categories: biological agents and small molecules. Biologic approaches, such as antibodies and fusion proteins, utilize antigens specific to tumor endothelium to target and destroy these cells, while the small molecules rely upon physiological differences to selectively destroy tumor, not normal endothelial tissue. The Biologic Approach The concept of using fusion proteins to target and disrupt tumor vasculature relies upon two

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Vascular Disrupting Agents

Flavonoids such as flavone acetic acid (FAA) and its fused tricyclic analog 5,6-dimethylxanthenone4-acetic acid (DMXAA) have been shown to induce extensive hemorrhagic necrosis in tumors as a result of vascular collapse. Mechanistically, the action of this class of agent is believed to be largely indirect, through the induction of cytokines, particularly TNFa. This view is supported by experimental evidence showing that antibodies to TNFa could inhibit FAA-induced vascular collapse. It is of interest to note that although both FAA and DMXAA selectively damage tumor blood vessels in preclinical tumor models, only DMXAA induces TNFa in both human and mouse macrophages. Consequently, DMXAA is currently considered to be the lead agent of this class of VDAs.

anti-vascular properties as early as the 1930s. However, the high toxicity of these agents limited their usefulness as vascular targeting therapies. Agents with much more favorable therapeutic indexes, such as combretastatin A4 phosphate (CA4P) and ZD6126, have rekindled interest in the use of this group of VDAs. These agents have shorter half-lives and bind tubulin in a reversible manner, which reduces their toxicity to normal tissues. Tubulin-binding agents bind tubulin subunits and prevent their polymerization. The result is depolymerization of the microtubules, reorganization of the actin cytoskeleton, and increased cellular permeability, which has been shown to be particularly destructive to dividing endothelial cells. In vivo, VDA treatment induces rapid disruption of blood flow and collapse of the vascular network. Although the precise mechanism of tumor blood flow shut down and vessel disruption is not fully understood, in vitro experiments have demonstrated endothelial cell shape changes and interruption of vascular endothelial-cadherin (VEcadherin) signaling as a result of tubulin-binding agent treatment. In vivo endothelial cell shape changes could augment vessel occlusion, while disruption of VE-cadherin results in reduced endothelial cell adhesion and could lead to vessel disruption. The tumor cells downstream of the disrupted blood vessels die due to a lack of oxygen and nutrients and the buildup of toxic biological by-products. After VDA treatment, necrosis is induced throughout the central portion of the tumor. The tubulin-binding VDAs should not be confused with other microtubule-binding anticancer agents such as the taxanes. These agents bind to microtubules and prevent depolymerization, contrasting with the VDAs which bind the tubulin subunits to prevent microtubule elongation. Treatment with these agents has not been shown to result in tumor vascular collapse, and consequently, anticancer therapies like taxanes are designed to directly target and destroy tumor cells, not the tumor vasculature.

Tubulin-Binding Agents The tubulin-binding agents colchicine and the vinca alkaloids were recognized to have

VDAs as Adjuvants to Conventional Therapy A common characteristic observed after VDA treatment is the presence of a so-called viable

necessary components: a ligand that selectively binds tumor endothelium and a conjugated toxin. The result is precise delivery of the toxic agent to only the tumor endothelium and not normal tissues. Two fusion protein VDAs that have produced positive preliminary results have utilized angiogenic proteins to specifically target the tumor endothelium. Testing of a VEGF121/rGelonin fusion toxin in nude mice bearing PC-3 tumors revealed specific localization of the conjugate to the tumor vasculature and resulted in tumor vessel thrombosis. The fusion complex of antibody against fibronectin conjugated to tissue factor was also shown to specifically target the tumor endothelium, and treatment of tumor-bearing mice with this agent resulted in disruption of tumor blood flow. Small Molecule VDAs ▶ Flavonoids and tubulin-binding agents are two classes of small molecule VDAs that are currently being evaluated. The flavonoids are believed to induce endothelial cell death primarily through the induction of inflammatory cytokines such as TNF-a. The tubulin-binding agents’ primary mechanism of action is the disrupting of the microtubule network of the tumor endothelium. Flavonoid Agents

Vascular Disrupting Agents

rim of tumor cell existing at the tumor periphery. This thin layer of viable tumor tissue remains at the tumor periphery, presumably because this tumor tissue is supported by the normal vasculature, which is not affected by VDA treatment. The cells of this region are able to survive regardless of VDA dose or treatment schedule. These tumor cells continue to divide and rapidly repopulate the tumor, limiting the success of VDAs as single agents. While VDA treatment does not entirely eradicate the tumor, a large proportion of the center of the tumor is killed. In an untreated tumor, the cells of this central region are typically hypoxic, slowly dividing, poorly perfused, and located in a high interstitial fluid pressure and low-pH environment, making them more resistant to conventional anticancer therapies. As VDAs destroy this typically resistant region, addition of VDAs to current treatment regimens could improve treatment outcome when used in combination with this therapies. Several VDA and conventional therapy combinations have been tested in the preclinical setting and demonstrated beneficial treatment outcome over either therapy alone. These studies found improved treatment outcome when the VDAs CA4P or ZD6126 were combined with radiation. VDA combination with radiation was also found to effectively treat large tumors successfully, more so than smaller tumors. The combination of ZD6126 with the anti-angiogenic agent ZD6474 produced improved treatment outcome in the HT29 and OW1 tumor models over either treatment alone. A few combinations are currently being tested in various clinical trials. VDAs such as ZD6126 and CA4P have been shown to produce maximum effective at doses well below the maximum tolerated dose. Neither does treatment with these agents alone result in significant growth delay, presumably due to the rapidly dividing tumor cells at the tumor periphery which survive VDA treatment. Consequently, there have been difficulties evaluating VDA treatment efficacy in the clinical setting. Preclinical evaluations of efficacy centered on the percent-induced tumor necrosis, but these

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measurements are not practical in the clinical setting as they require highly invasive procedures that are not applicable to tumors in all locations. The viable tissue that causes difficulties with the evaluation of treatment efficacy also limits the usefulness of VDAs as single agents. However, while the effects of these VDAs have been extensively studied for the tumor as a whole, little data exists that describes how the characteristics of the tissue that survives VDA treatment change during the phase of VDA-induced damage. Changes induced in this region by VDA treatment have the potential to negatively impact the efficacy of agents used in combination with VDAs. By understanding the VDA effects upon the surviving tumor tissue, insight may be obtained into possible means of overcoming these limitations. For example, decreases in perfusion as a result of VDA treatment that do not result in tumor death may inhibit the delivery of chemotherapeutic agents to the affected region and decrease the radiosensitivity of tumor cells in the affected area. A few studies have reported varying combination treatment efficacies with differing treatment schedules. Therefore, a better understanding of the effects of VDA treatment on the surviving tumor tissue could lead to more effective combination treatment strategies.

Cross-References ▶ Vascular Targeting Agents

References Horsman MR, Siemann DW (2006) Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies. Cancer Res 66(24):11520–11539 Siemann DW, Chaplin DJ, Horsman MR (2004) Vasculartargeting therapies for treatment of malignant disease. Cancer 100(12):2491–2499 Thorpe PE (2004) Vascular targeting agents as cancer therapeutics. Clin Cancer Res 10(2):415–427 Tozer GM, Kanthou C, Baguley BC (2005) Disrupting tumour blood vessels. Nat Rev Cancer 5(6):423–435

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Vascular Endothelial Growth Factor Dieter Marmé Tumor Biology Center, Institute of Molecular Oncology, Freiburg, Germany

Synonyms Vascular permeability factor; VEGF; VPF

Characteristics VEGF Family Four VEGF family members have been described in mammals, VEGF-A through VEGF-D. VEGF-E, the fifth member of the family, is coded by the Orf virus. An additional relative is the placenta-derived growth factor, PlGF. Whereas the VEGFs are potent growth promoting and vascular permeability enhancing factors, PlGF is incapable of inducing permeability. All members exert their biological functions as homodimers. VEGFs act almost exclusively on endothelial cells. VEGF is expressed by almost all cell types with the exception of endothelial cells which express only marginal amounts of the growth factor. Expression is controlled by a number of different mechanisms. Extracellular signals such as growth factors and cytokines are able to induce transcription of the VEGF gene. Activated oncogenes, such as ras, raf, or src, as well as inactivated tumor suppressor genes, such as p53 or von Hippel-Lindau (VHL), contribute to enhanced transcription. The newly identified p53 analogue p73 in its wild-type form can cause repression of VEGF transcription. Hypoxia, a physiological signal in early embryonic development and a pathophysiological signal in many tumors, causes enhanced production of VEGF mRNA and also stabilizes the VEGF mRNA. The pattern of VEGF expression is strictly controlled by some of these factors in a time- and tissue-specific fashion during embryonic development and physiological angiogenesis. In pathological situations, VEGF

Vascular Endothelial Growth Factor

expression proceeds with no specific control, exceeds physiological concentration, and occurs at wrong times and locations. VEGF Receptor Family All VEGF family members mediate their signals through a family of distinct high-affinity receptors, VEGF-R1 through VEGF-R3. All three VEGF receptors are tyrosine kinases that become stimulated upon ligand binding, with VEGF-R1 tyrosine kinase being much less activated than VEGF-R2 and VEGF-R3. VEGF-R1 is expressed as two distinct forms: the entire transmembrane receptor VEGF-R1 and a soluble variant sVEGF-R1 generated by alternatively spliced mRNA. VEGF-R1 and its soluble variant bind VEGF-A, VEGF-B, and PlGF. VEGF-R2 has high affinity for VEGFA, VEGF-C, VEGF-D, and VEGF-E. VEGF-R3 binds VEGF-C and VEGF-D. All three VEGF receptors are exclusively expressed on the surface of endothelial cells. VEGF-R2 is the most important VEGF receptor mediating a proliferative response to endothelial cells. Upon transfection into non-endothelial cells, VEGF-R2 becomes autophosphorylated in response to VEGF binding, but is no more mitogenic. This indicates the involvement of cell type-specific signaling mechanisms. The endothelial cell proliferation and survival in response to VEGF requires the association of cell surface adhesive molecules. Activated VEGF-R2 associates with integrins anb3. VE-cadherin colocalizes with VEGF-R2 and upon stimulation by VEGF becomes associated with VEGF-R2, b-catenin, and PI-3-kinase. This leads to activation of PKB/Akt and initiation of a survival signal. Disruption of VE-cadherin leads to prevention of VEGF-mediated cell survival. VEGF receptor expression is under tight control in embryo development and normal physiology. In pathological situations, such as tumor angiogenesis, VEGFR1 and VEGF-R2 are transcriptionally upregulated in response to VEGF and thus generate an amplification mechanism to enhance this fatal process. VEGF in Vasculogenesis and Physiological Angiogenesis VEGF is widely and abundantly expressed in many tissues during fetal development and has

Vascular Endothelial Growth Factor

been implicated in the process of vasculogenesis, i.e., the de novo formation of the vascular system. Mice deficient for VEGF die at day 8.5–9.0 and show a delayed differentiation and an impairment of both vasculogenesis and angiogenesis, i.e., the sprouting of new capillary vessels from pre-existing vasculature. Similarly, the receptors VEGF-R1 and VEGF-R2 are strongly expressed in the developing embryo. In particular, VEGF-R2 is expressed in the hemangioblasts, the common precursor to both endothelial cells and hematopoietic lineages. VEGF-R1, being nonessential for endothelial development, is required at a later stage of the organization of the embryonic vasculature. Disruption of the VEGF-R2 gene interferes with endothelial cell development, leading to death of embryos at days 8.5–9.5. Disruption of the VEGF-R1 gene permits endothelial cell differentiation but results in thin-walled vessels of larger than normal diameter, and the embryos die at day 9. In the adult organism, large amounts of VEGF are also found in the female reproductive tissues in association with hormonally regulated angiogenesis that takes place in the ovary and endometrium at specific stages of the menstrual cycle and in pregnancy. Strong expression of VEGF can be detected in several tissues of the adult in the absence of angiogenesis, particularly the kidney, lung, adrenal gland, and heart. However, some of these tissues express only low levels or no VEGF receptors which might explain the absence of angiogenesis. As VEGF has been shown to be a survival factor for vascular endothelial cells, it might also be that this function, requiring only low levels of VEGF receptors, is important for maintaining vascular homeostasis without angiogenesis to occur.

Clinical Relevance VEGF in Pathophysiological Angiogenesis

VEGF, as well as its receptors VEGF-R1 and VEGF-R2, is strongly overexpressed at both the mRNA and protein levels in almost all malignant tumors. Tumor metastases exhibit overexpression of VEGF similar to that found in the primary

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tumors from which they arose. Elevated VEGF levels have been found in the blood of tumor patients, correlating in many cases with poor clinical prognosis of the disease. Accordingly, the soluble variant of the VEGF-R1 is also found to be elevated in the blood of tumor-bearing patients indicating the presence of activated tumor endothelium in the diseased areas. Thus, VEGF and the soluble variants of VEGF-R1 could be regarded as surrogates for pathological tumor ▶ angiogenesis. In addition to the intimate involvement of VEGF and its receptors in tumor angiogenesis, VEGF is also capable of increasing vascular permeability. VEGF-induced leakage of plasma from hyperpermeable microvessels results in fluid accumulation within tumors. This is also favored by the fact that tumors in general lack lymphatic vessels and hence are unable to drain extravasated proteinaceous fluid effectively. This is in particular obvious in ▶ brain tumors, showing increased intracranial pressure, and in tumors metastasizing to body cavities leading to substantial accumulation of fluid. VEGF and both of its receptors are also overexpressed in a number of pathological entities that involve angiogenesis, but are not associated with neoplasia. These include diabetic and other retinopathies, rheumatoid arthritis, and psoriasis. In all of these examples, overexpression of VEGF and its receptors is accompanied by increased microvascular hyperpermeability and pathological angiogenesis.

VEGF/VEGF Receptor System: Therapeutic Opportunities As VEGF and its receptors are intimately involved in the pathology of many diseases, such as cancer, rheumatoid arthritis, and diabetic retinopathies, considerable efforts have been made to interfere therapeutically with this signaling system. Monoclonal antibodies against VEGF and the binding domain of its receptor VEGF-R2 have been generated. Animal experiments show efficacy against tumor vascularization, tumor growth, and metastasis formation. Both antibodies have been humanized (humanized antibodies) and are being evaluated

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Vascular Endothelial Growth Factor Receptor (VEGFR) Inhibitors

in the clinic. Low molecular weight compounds were developed to inhibit the tyrosine kinase activities of VEGF-R1 and VEGF-R2. These compounds show considerable activity against growth of highly vascularized tumors and also inhibit metastasis formation. Some of these inhibitors also show activity against other non-VEGF receptor tyrosine kinases. Clinical evaluation is being carried out to demonstrate whether the additional non-VEGF receptorrelated activity is beneficial or not. Furthermore, a combination of anti-VEGF strategies together with low-dose cytotoxic strategies has revealed synergistic effects in animals. This indicates that antiangiogenic therapy could be useful to enhance the therapeutic potential of conventional chemotherapeutic drugs.

Vascular Endothelial Growth Factor Receptor (VEGFR) Inhibitors ▶ Receptor Tyrosine Kinase Inhibitors

Vascular Maturation ▶ Vascular Stabilization

Vascular Permeability Factor ▶ Vascular Endothelial Growth Factor

Cross-References

Vascular Remodeling

▶ Angiogenesis ▶ Brain Tumors ▶ Von Hippel-Lindau Disease

▶ Vascular Stabilization

References Carmeliet P (2000) Mechanisms of angiogenesis and arteriogenesis. Nat Med 6:389–395 Siemeister G, Martiny-Baron G, Marmé D (1998) The pivotal role of VEGF in tumor angiogenesis: molecular facts and therapeutic opportunities. Cancer Metastasis Rev 17:241–248 Veikkola T, Karkkainen M, Claesson-Welsh L et al (2000) Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60:203–212

Vascular Stabilization Süleyman Ergün1, Derya Tilki2 and Nerbil Kilic3 1 Institut für Anatomie und Zellbiologie, JuliusMaximilians-Universität Würzburg, Würzburg, Germany 2 Martini-Klinik, Prostatakrebszentrum, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 3 Kantonspital St. Gallen, St. Gallen, Switzerland

See Also (2012) Humanized antibodies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1760. doi:10.1007/978-3-642-164835_2863 (2012) Monoclonal antibody. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Monoclonal antibody therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 2367-2368. doi:10.1007/978-3-642-164835_3823 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331

Synonyms Functional vascular stabilization; Structural vascular stabilization; Vascular maturation; Vascular remodeling

Definition Vascular stabilization stands for the basic steps of the morphogenetic processes, finally leading to

Vascular Stabilization

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Vascular Stabilization, Fig. 1 From the primitive vascular plexus to the hierarchy of normal vascular system: The primitive dense vascular plexus is mainly composed of unstable nascent blood vessels of approximately equal state of vascular stabilization (a, left). Cross section shows that BM is not regularly structured and pericytes (red) are not tightly connected to the endothelial layer (green). Vascular stabilization during embryogenesis and

fetal development has led to a decrease of vascular density (a, right). In cross section (a, right) a dense BM (gray) is visible, endothelial cells (green) build a closed layer, and mural cells (red) are enclosed by the same BM and are tightly organized around the endothelial layer. These processes serve as the basis for the first step of vascular hierarchy in the adult as demonstrated in (b)

vascular maturation such as establishment of interendothelial contacts, development of a regularly structured basement membrane enveloping both endothelial cells and pericytes, and integration of one layer of peri-endothelial cells (pericytes or smooth muscle cells) into the vascular wall.

the rest serve as the source for hematopoiesis. The primitive vascular network developed in the chorioallantoic membrane subsequently connects to the cardiac system and serves as the basis for a functioning blood circulation. However, this system comprising nascent blood vessels will undergo enormous remodeling processes containing several sequential steps which are precisely coordinated timely and spatially. The essential step of the vascular remodeling in the early phase of vascular development is structural stabilization, which involves the development of interendothelial contacts and basement membrane and integration of peri-endothelial cells into the vascular wall (Fig. 1). Accompanied by organogenesis and the demand on blood perfusion, further processes of vascular maturation will lead to the development of the vascular wall containing several layers of smooth muscle cells and connective tissue, depending on perfusion pressure and exchange processes between the vascular and interstitial compartments. The factors and mechanisms regulating these processes are also involved

Characteristics Normal Vascular Hierarchy The adult vascular system is hierarchically organized in large-sized, middle-sized, and microvessels at both the arterial and venous sides. The lumen of all blood vessels is lined by endothelial cells (EC). EC are of mesodermal/mesenchymal origin and differentiate from hemangioblasts; they are probably the common precursor of endothelial and hematopoietic cells. The first hemangioblasts are visible in the blood islands of chorioallantoic membrane, and the peripheral cells of these islands differentiate to endothelial cells, while

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Vascular Stabilization

Vascular Stabilization, Fig. 2 Vascular destabilization and initiation of angiogenesis: A normal stabilized capillary with a dense BM (gray) enclosing both endothelial cells (green) and pericyte (red) (a) will be destabilized by the action of pro-angiogenic factors as shown by VEGF and Ang2 leading to endothelial fenestration (fn), opening of interendothelial contacts (iec), development of

transendothelial gaps (gap), degradation of BM, and finally detachment of pericytes from the endothelial layer (b). These morphogenetic events are accompanied by an abnormal vascular leakiness. The further duration of pro-angiogenic action would finally lead to the sprouting of new nascent and unstable blood vessels (c), a process defined as angiogenesis

in the regulation of vascular permeability, which also depends on organ-specific requirements. The end of these processes is the construction of a vascular hierarchy comprised of large- and mid-sized arteries and veins macroscopically visible, as well as arterioles, capillaries, and venules making up the microvascular part of the blood vessels (Fig. 1). As far as we can observe, the vascular stabilization and the final vascular maturation result in a decrease of vascular density as blood vessels which are not included in the organ perfusion by blood undergo regression and successively disappear (Fig. 1).

probably induced by a switch of the angiogenic balance toward the activation of angiogenic sprouting. The further duration of this process is mediated by pro-angiogenic factors such as VEGF (▶ vascular endothelial growth factor), FGF-2 (▶ fibroblast growth factors), and Ang2. It would lead to complete destabilization and disintegration of the endothelial layer. The detachment of endothelial cells from the basement membrane results in the migration and subsequent proliferation of these cells. Finally, new capillary sprouts formed by the new endothelial cells are nascent and structurally unstable. The maintenance of pro-angiogenic activation would hold these new vessels in structural instability and in an abnormal leaky state. This phenotype of vessels dominates in the tumor vascular bed. Particularly, the concerted action of VEGF and Ang2 at certain sites of tumor vasculature has been shown to be very effective in keeping the blood vessels unstable and angiogenesis ongoing. Vascular destabilization accompanied by abnormal vascular leakiness is also frequently observed during inflammation. It is well known that several cell types involved in the inflammatory process such as macrophages or lymphocytes produce high amounts of pro-angiogenic factors including VEGF and bFGF. Also Ang2 which is involved in vascular destabilization has been detected in several inflammatory processes.

Vascular Destabilization and Activation of Angiogenesis The initiation of ▶ angiogenesis is marked by structural destabilization of the vascular wall, generally accompanied by an abnormal vascular permeability. This is also a common sign of the tumor vascular bed. Looking at the structure of tumor vessels, a structural instability marked by the loosening of pericytes/smooth muscle cells from the endothelial layer, opening of interendothelial junctions (▶ tight junctions), development of endothelial fenestration and/or transendothelial gaps, and degradation of the vascular basement membrane are frequently observed alterations of the vascular wall in this initial phase of angiogenesis (Fig. 2). This initial destabilization is

Vascular Stabilization

Thus, inflammation seems to provide high amounts of VEGF and Ang2, and the concerted action of both is the main mediator of vascular destabilization. Vascular Stabilization While it has been assumed for several years that new vessels provided by angiogenesis are lined only by endothelial cells and would not be able to enter further steps of vascular maturation, detailed studies in the last decade have revealed without doubt that some of the new vessels developed by physiologic or pathologic angiogenesis as in tumors (▶ cancer) do exhibit a basement membrane and mural cells such as pericytes and/or smooth muscle cells. As of now, only a few factors have been characterized as being involved in these processes. To give a systematic overview of the vascular stabilization, it makes sense to evaluate the following morphogenetic steps: 1. Interendothelial contacts: Like cell-cell contacts between epithelial cells, TJs or zonula occludens, adherence junctions (AJ) or zonula adherens, and ▶ gap junctions (GJ) are the main cell-cell contact types between endothelial cells, but in contrast to epithelial cells, ▶ desmosomes are absent between endothelial cells. Also, in endothelial cells, TJs are localized at the boundary between the apical and basolateral sides of endothelial cells, and thus, they are important for the barrier function. The members of the claudin family, occludin and junctional adhesion molecule-A (JAM-A), as well as their cytoplasmic interaction partners such as the members of the ZO (zonula occludens proteins) are the main players that govern the establishment of endothelial TJs. Also ESAM, the endothelial cell-selective adhesion molecule, is localized at the TJ and regulates the paracellular permeability of the endothelial barrier. VE-cadherin (vascular endothelial cadherin) (▶ adherens junctions) is essential for the establishment of adherence zones between endothelial cells. The main intracellular partners of VE-cadherin are band g-catenin. They link a-catenin, which

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anchors this complex to actin. A further intracellular partner of VE-cadherin is p120, a substrate of src. Of particular impact for vascular stabilization versus destabilization is the interaction of VE-cadherin with endothelial signaling proteins. VE-cadherin, VEGFR-2 (VEGF receptor type 2, KDR), and Src-kinase form a complex which is obviously essential for the maintenance of the endothelial barrier controlling the paracellular transport of molecules and cells through the endothelial layer. In contrast, the interaction of VE-cadherin with VE-PTP (vascular endothelial-specific receptor-protein tyrosine phosphatase) strengthens the AJ between endothelial cells. Gap junctions (GJ) represent intercellular channels mediating exchange of ions and small molecules between neighboring cells by diffusion. The establishment of GJ is governed by ▶ connexins; ~20 connexins (Cx) have been identified until now. Expression studies demonstrate the presence of Cx37, Cx40, and Cx43 between endothelial cells, but the role of connexins in the interendothelial communication is not sufficiently understood. Considering the fact that the expression pattern of these connexins in the blood vessels depends on the vessel type and the position in the vascular tree, a regulating role of the connexins in differentiation or maturation of blood vessels can be postulated. Although there are several excellent studies regarding the expression and the role of each factor involved in cell-cell contact between endothelial cells, it is not clarified at exactly which stage of vascular morphogenesis these contacts are established. Particularly the determination of temporary and spatial sequences of these processes needs to be evaluated. Based on studies, it can be postulated that in the first step of vascular morphogenesis, where the nascent vessels are lined only by endothelial cells, the first provisional interendothelial contacts are mediated by ▶ cell adhesion molecules like integrins, VCAM, ICAM, and CEACAM1. Particularly, CEACAM1 has been shown to be involved in vascular stabilization in a dual way: CEACAM1 overexpression in endothelial cells induces a signaling leading to vascular

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stabilization, while its downregulation in epithelial cells as it occurs in several tumors leads to vascular destabilization. In a second step, the special cell-cell contacts mentioned above will be established between endothelial cells serving the basis for the entering of nascent and still unstable vessels into the stabilization process. 2. Construction of the vascular basement membrane (BM): The BM (or electron microscopically basal lamina) is an extracellular matrix structure of flexible thickness of 40–120 nm underlying all epithelial cell sheets and tubes formed by endothelial cells. In general, the assembly of BM is mainly provided by collagen type IVand laminin, which have the capacity to self-assembly, but several other molecules like perlecan, nidogens, and collagen type XVIII are identified components of BM and necessary for its regular construction. In transmission electron microscopic studies of the normal vasculature, the basal lamina (corresponding structure to basement membrane) shows an amorphous and dense structure. In small blood vessels, BM encloses both endothelial cells and pericytes. The dense structure of BM is mostly not present in the basal lamina of tumor blood vessels, indicating a degradation or insufficient construction of the BM. This is one of the essential parameters leading to the loss of endothelial anchoring to the extracellular matrix and to detachment of pericytes from the endothelial layer with subsequent migration and proliferation of endothelial cells. The components of wellstructured BM signal an inhibitory effect on angiogenesis, while the direct contact of endothelial cell to the “provisional” matrix components such as collagen type I, fibronectin, and vitronectin accelerates the proliferation and migration of endothelial cells. BM collagens contain cryptic domains with anti-angiogenic activity (▶ antiangiogenesis) if they are released from their mother substance as demonstrated by ▶ endostatin, a fragment of collagen type 18, and tumstatin, a fragment of collagen type IV. Endostatin has been shown to stabilize newly formed endothelial tubes and

Vascular Stabilization

to strengthen the endothelial barrier by protecting the basement membrane and interendothelial contacts in normal structure. Taken together, these data suggest that a wellconstructed vascular basement membrane stabilizes the newly formed endothelial tubes and reduces the angiogenic potency. Indeed, in experimental models and in preclinical treatment with different types of angiogenic inhibitors (▶ antiangiogenesis), stabilization of the vascular wall and the decrease of vascular leakiness are frequently observed phenomena. 3. Integration of vascular mural/peri-endothelial cells into the vascular wall: The assembly of pericytes or smooth muscle cells to the vascular wall is an essential step of vascular stabilization. Factors involved in this process are Ang1 and Tie-2 system, TGFb, PDGF, and their receptors. Although the vascular mural cells are of multiple origins, their presence and interaction with endothelial cells are crucial for vascular stabilization. Ang1 is not only involved in the assembly of pericytes into the vascular wall, but it also mediates cell-cell and cell-matrix interaction, leading to a significant reduction of vascular leakiness. Ang1- and Tie-2 knockout mice are lethal because they lack vascular plasticity and remodeling resulting in disorganization of the primitive vascular plexus and apoptosis of endothelial cells. In contrast, Ang1 overexpression in mice blocks the VEGF-induced vascular leakiness and increases the diameter of blood vessels. To reduce the role of pericytes in vascular morphogenesis only to a mechanical supportive function for the endothelial cells would be very simplifying and not appropriate. Via direct signaling facilitated by cell-cell contacts between endothelial cells and pericyte processes through the basement membrane, pericytes influence the maturation and quiescence of endothelial cells. But these actions of pericytes on endothelial cells can only be effective when pericytes are well integrated into the vascular wall enclosed by the same basement membrane as endothelial cells (Fig. 3).

Vascular Stabilization

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Vascular Stabilization, Fig. 3 Vascular stabilization: The wall of a newly formed unstable blood vessel is constructed by endothelial cells (green) with provisional interendothelial contacts, provisional BM without the regular dense organization, and mural cells (red) which are present but not regularly integrated into the vessel wall (a). Several endogenous factors as shown by Ang1, TGFb,

PDGF, and endostatin promote the structural stabilization and serve in a concerted interplay with other factors such as VE-cadherin, occludin, claudins, collagen IV, laminin, and integrins as the basis for establishment of durable interendothelial contacts, a regular BM, and mural cells tightly integrated into the vascular wall, a process named vascular stabilization

Clinical Implications In several diseases, the vascular dysfunction is either an accompanied clinical problem making therapeutic handling much more difficult such as in diabetic retinopathy and microangiopathy, or it is an essential prerequisite for the full development of diseases such as cardiovascular failure or tumor growth and metastasis. Since cardiovascular diseases rank first and tumor second in the list of fatal diseases worldwide, the therapeutic managing of vascular morphogenesis is a big challenge in medicine. The role of vascular stabilization in the process of vascular morphogenesis, plasticity, and remodeling is still not sufficiently understood because it was neglected for a long time. In the last few years, the mechanisms of vascular stabilization, particularly the interaction between endothelial and vascular mural cells, have received more attention in both cardiovascular and tumor angiogenesis research. While antiangiogenesis targets the tumor vasculature to cut blood supply to the tumor tissue, the pro-angiogenic therapeutic strategies deal with the creation of new vessels, which should have the capacity, as far as possible, to achieve a structural stabilization and functional normalization. Therapeutic angiogenesis and vascular stabilization: “One is as old as one’s arteries” is a

frequently cited statement from Virchow and underlines the impact of the arteries in determining the life span and also the quality of life. Cardiovascular disorders such coronary artery disease or the sclerotic changes of the peripheral arteries are mostly caused by degenerative alteration of the vascular wall at the arterial part of the vascular system. In all these disorders, the formation of new vessels, for example, the initiation of the growth of collateral vessels, is desirable, but it is difficult to bring the new vessels in a stable state, fulfilling the specific tissue demands on vascular perfusion and permeability. Although therapeutic angiogenesis in preclinical studies shows promise, no clear success has been achieved in controlled clinical phase II and III studies until now. These results demonstrate how complex the processes are which govern the morphogenesis of blood vessels, but they also show how difficult the way is from experimental models to human trials. Despite these ups and downs, the therapeutic angiogenesis still remains a fascinating perspective, highly promising for the treatment of cardiovascular and ischemic disorders. Anti-angiogenic tumor therapy and vascular stabilization: The destabilization of blood vessels accompanied by abnormal vascular leakiness is one of the earliest signs of angiogenic activation.

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Vascular Stabilization, Fig. 4 Vascular stabilization and tumor growth: Stabilization of tumor vessels occurs mostly in the tumor center but significantly extends to the marginal zone under anti-angiogenic therapy, e.g., with endostatin. This is accompanied by a dramatic regression of a part of the blood vessels, resulting in a significant reduction of vascular density. This process apparently changes the perfusion of tumor tissue. In the marginal

tumor zone with unstable blood vessels, there is a nearly equal perfusion of the whole vascular bed as marked by arrows. In contrast, in the tumor center, the main route of blood flow is served by stabilized vessels, while unstable blood vessels were successively cut of function and underwent regression. This may result in further necrosis of tumor tissue. Green, endothelial lining; gray, vascular basal lamina

This vascular phenotype dominates the tumor vascular bed. Although it was believed for a long time that tumor vessels lack BM and pericytes, detailed studies in the last 5–6 years have revealed that in tumor vessels also, endothelial tubes are underlined by a BM and that pericytes are organized around these tubes. But in contrast to normal vasculature, the BM of tumor blood vessels is not well structured, the interendothelial contacts are not strong enough, and the pericytes are not tightly connected to the endothelial tubes. This vascular phenotype has a high plasticity, making it highly susceptible to pro-angiogenic factors such as VEGF and Ang2. While the link between vascular destabilization and angiogenic activation, as well as tumor vascularization, has been known for several years, well studied, and understood to a great extent, the impact of vascular stabilization on tumor vascularization, tumor growth, and metastasis (cancer)

was mostly neglected until a few years ago. Based on emerging data from anti-angiogenic therapy, a “normalization” of tumor vessels has been postulated. According to this hypothesis, the anti-angiogenic therapy would create a “therapeutic window,” enabling a more effective use of chemo- or radiation therapy for tumors. This suggests an indirect relation between vascular stabilization and tumor therapy, but what is the role of vascular stabilization per se on tumor angiogenesis and tumor growth and metastasis? The best known factor leading to vascular stabilization is Ang1. Ang1 overexpression blocks the VEGF-induced vascular leakiness by stabilization of blood vessels. In mice overexpressing Ang1, tumor vascularization was seen to be less active than those overexpressing Ang2, or tumor growth was suppressed. But there are also studies suggesting that vascular stabilization, for example, by Ang1, results in the acceleration of tumor

Vascular Targeting Agents

growth. These controversial findings demonstrate clearly that this aspect of tumor angiogenesis has not been studied sufficiently. Hypothetically, one would expect that vascular stabilization reverses the angiogenic phenotype of blood vessels to a more quiescent phenotype by reducing susceptibility of tumor vessels to pro-angiogenic factors. This in turn would cause a reduction of vascular density and part starvation of tumor tissue, resulting in tumor necrosis (Fig. 4). This has been observed in several experimental tumor models and experimental tumor treatments with different antiangiogenic substances. It is imaginable that we principally need a two-step approach of antiangiogenic tumor therapy: The first step should aim to achieve a disruption of new nascent and unstable blood vessels and a suppression of endothelial proliferation, and the second would deal with the stabilization of tumor vessels switching tumor vasculature from the angiogenic to quiescent phenotype. Both these steps would serve the basis for a therapeutic “window” enabling the combination of angiogenesis inhibitors and chemotherapeutic drugs (▶ Chemotherapy) and/or radiation (▶ Radiation oncology) in the final step of tumor therapy. Taken together, emerging data recommend a stronger consideration of mechanisms that govern vascular stabilization in future anti-angiogenic tumor therapy strategies.

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Vascular Targeted Therapies ▶ Vascular Targeting Agents

Vascular Targeting Agents Michael R. Horsman1 and Dietmar W. Siemann2 1 Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark 2 Department of Radiation Oncology, University of Florida, Gainesville, FL, USA

Synonyms Angiogenesis-inhibiting agents; Vascular disrupting agents; Vascular targeted therapies; VTAs

Definition Vascular targeting agents (VTAs) are primarily cancer therapies that are specifically designed to target the vasculature of tumors and as a consequence will inhibit tumor growth and development. They may also be used to treat other pathophysiological conditions in which the tissue vasculature plays a role.

References

Characteristics

Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 84:869–901 Ergun S, Tilki D, Oliveira-Ferrer L et al (2005) Significance of vascular stabilization for tumor growth and metastasis. Cancer Lett 18:180–187 Jain RK (2005) Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307:58–62 Kalluri R (2003) Angiogenesis: basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 3:422–433 Thurston G, Rudge JS, Ioffe E et al (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 6:460–463

Background In cancer, the vascular supply to tumors is critical. For most solid tumors to grow beyond a size of a few millimeters, it is necessary for them to develop their own functional blood supply, which they do from the already established normal tissue vasculature by a process called ▶ angiogenesis. This process begins with the tumor cells secreting various angiogenic growth factors. These factors are upregulated by different environmental changes such as ▶ hypoxia, loss of tumor suppressor function, or ▶ oncogene

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activation. Of these angiogenic factors, the most potent and specific is ▶ vascular endothelial growth factor (VEGF), which is not only crucial for endothelial cell proliferation and blood vessel formation but also induces significant vascular permeability and plays a key role in endothelial cell survival signaling in newly formed vessels. These growth factors react with various receptor kinases on the endothelial cells and then put in motion a series of physical events that include destruction of the basement membrane of the normal endothelial cells, migration of endothelial cells into the extracellular matrix in the form of a sprout, division of endothelial cells away from the sprout tip, the formation of solid strands of endothelial cells in the extracellular matrix, the development of a lumen within the strands, fusion with other sprouts to form loops, and the formation of new sprouts and loops from these primary loops. All this ultimately results in the establishment of a functional vascular supply for the tumor. Once this occurs not only can the primary tumor begin to grow, but the tumor cells have a means of entering the systemic circulation and so move to other areas in the body and then ultimately form ▶ metastases. This importance of the tumor vasculature makes it an excellent target for therapy, and two major VTA approaches have now evolved. The first is based on controlling the development of the tumor blood vessels by inhibiting the angiogenesis process, while the second involves a disruption of the already established tumor blood vessels. It has been demonstrated that vascular effects are involved in the action of other therapies, including certain types of ▶ chemotherapy, radiotherapy, and inhibitors of epidermal growth factor receptors or cyclooxygenase-2. But in these situations the vasculature is far from being their principal target, and as such it is technically incorrect to classify them as VTAs. Mechanisms Although angiogenesis inhibitors (AIs) and ▶ vascular disrupting agents (VDAs) both target the tumor vascular supply, they are two distinct approaches. AIs are designed to prevent further development of the tumor neovascular network.

Vascular Targeting Agents

The complex process of tumor angiogenesis offers many possible targets for ▶ antiangiogenesis strategies. These strategies vary from regulation of angiogenic factor expression in tumors to endogenous inhibitors of angiogenesis. Based on their biological activities, these strategies can be categorized into several broad classes. One class of agents specifically targets the angiogenic growth factors, which play the most significant role in neovascularization, especially VEGF. VEGF has been targeted by a variety of strategies, including inhibitors of endothelial cell receptor signaling that interfere with associated ▶ receptor tyrosine kinase activities (e.g., SU5416, sorafenib, sunitinib, vandetanib, and PTK787/ZK 222584), as well as monoclonal antibodies directed against proangiogenic growth factors (e.g., bevacizumab/ Avastin and DC101). Bevacizumab/Avastin, a recombinant humanized monoclonal antibody to VEGF, is the first anti-angiogenic therapy to have demonstrated a survival advantage when given to patients with cancer. It is currently being investigated in a variety of tumor types. A second class of agents includes those designed to inhibit endothelial cell functions, basement membrane degradation, endothelial cell migration, proliferation, and tube formation. One example is ▶ endostatin, a naturally occurring fragment of collagen XVIII, which has been identified as a potent endogenous inhibitor of endothelial cell function. A third class consists of agents that target survival factors of neovascular blood supply, such as integrin antagonists. Integrins are heterodimeric transmembrane proteins that control cell motility, differentiation, and proliferation via interactions with extracellular matrix molecules. The avb3 integrin is an attractive target for anti-angiogenic therapy because it is almost exclusively present on the cell surface of activated endothelial cells and is considered a survival factor for angiogenic vessels in tumors. Finally, nonspecific therapies that impact new vessel development are also being actively considered. Thalidomide and its analogues are one such group of agents that inhibits angiogenesis, but the mechanism of action is poorly understood. VDAs are agents that cause direct damage to the already established tumor endothelium. These include physical treatments like hyperthermia or

Vascular Targeting Agents

▶ photodynamic therapy, which have been well documented to induce direct tumor cell killing and an indirect effect through the induction of vascular damage. They also include biological response modifiers (BRMs), i.e., substances, either natural or synthesized, that boost, direct, or restore normal immune defenses such as interferons, interleukins, thymus hormones, and monoclonal antibodies or cytokines like tumor necrosis factor (TNF) and interleukins; certain established chemotherapeutic drugs such as vinca alkaloids and arsenic trioxide; and various ligand-based approaches that use antibodies, peptides, or growth factors that can selectively bind to tumor vessels. But more commonly VDAs involve the use of small molecule drugs, of which there are two major classes of agents. The first includes flavone acetic acid and its derivative Vadimezan (DMXAA, ASA404), which have a complex mechanism of action that is poorly understood, but their main effect on vascular endothelial cells is thought to involve a cascade of direct and indirect effects, the latter involving the induction of cytokines, especially TNF-a, leading to the induction of hemorrhagic ▶ necrosis. A second group includes the tubulin-binding agents combretastatin A-4 phosphate (CA4P, fosbretabulin, Zybrestat), ombrabulin (AVE8062), Plinabulin (NPI-2358), MN-029, and OXi 4503. These tubulin-depolymerizing agents are believed to selectively disrupt the cytoskeleton of proliferating endothelial cells, resulting in endothelial cell shape changes and subsequent thrombus formation and vascular collapse. Since they preferentially target dividing endothelial cells, this accounts for their tumor specificity. Both types of small molecular drugs have been shown to have potent anti-vascular and antitumor efficacy in a wide variety of preclinical models, and the lead agents are undergoing clinical evaluation. Since AIs and VDAs induce vascular effects by very different mechanisms, their antitumor activity and optimal application will be very different. Generally, AIs are given as a chronic administration and essentially slow tumor development. There are examples where tumor growth can be completely inhibited or the treatment of established tumors can result in tumor regression,

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but these tend to be exceptions rather than the norm. As a result AIs are probably best suited for early stage or metastatic disease. With VDAs the administration is of a more acute type to induce substantial vascular shutdown. Antitumor effects should also be possible with lower doses given over a prolonged period, but that would probably increase the risk of normal tissue vessel damage and defeat the potential benefit. Following treatment with VDAs, tumor shrinkage has been observed, but this appears to be tumor and drug dependent, and although significant it is generally modest, and thus tumor growth is only temporarily delayed. There is good evidence that VDAs have a superior effect on bulky disease. Given the key differences between AIs and VDAs, it should be clear from a therapeutic perspective that targeting the tumor vasculature with AIs and VDAs is complimentary and not redundant. Such approaches are being actively pursued. Clinical Aspects It is clear that neither AIs nor VDAs, whether given alone or even in combination, can induce tumor control. Therefore, their clinical potential as anticancer therapies requires that they must be combined with other cancer therapies, and numerous preclinical studies have demonstrated that when this is done significant improvements in tumor response are possible. For AIs, this has been demonstrated when they have been combined with conventional treatments like radiation (▶ ionizing radiation therapy) and chemotherapy, including ▶ alkylating agents (e.g., ▶ cisplatin, carboplatin, melphalan, cyclophosphamide, and dacarbazine), nitrosoureas (e.g., BCNU), antimetabolites (e.g., ▶ gemcitabine, 5-▶ fluorouracil, and pemetrexed), anthracyclines (e.g., doxorubicin), topoisomerase inhibitors (▶ topoisomerase enzymes as drug targets) (e.g., ▶ irinotecan, topotecan, and etoposide), taxanes (e.g., ▶ paclitaxel and ▶ docetaxel), corticosteroid hormones (e.g., prednisone), and bioreductive drugs (e.g., ▶ mitomycin C). AIs have to a lesser extent also been combined with hyperthermia and photodynamic therapy. With VDAs the combinations have also included radiation and various

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chemotherapy agents, such as alkylating agents (e.g., cisplatin, carboplatin, melphalan, cyclophosphamide, and chlorambucil), antimetabolites (e.g., 5-fluorouracil), anthracyclines (e.g., doxorubicin), topoisomerase inhibitors (e.g., irinotecan and etoposide), taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids (e.g., vincristine and vinblastine), and bioreductive drugs (e.g., mitomycin C, tirapazamine, and AQ4N). Less conventional therapies combined with VDAs include hyperthermia, radioimmunotherapy, and antibody/clostridiadirected enzyme prodrug therapy. One important issue here concerns the pathophysiological effects induced by VTAs. As a result of targeting tumor vasculature, VTAs modify the tumor pathophysiology, and these include changes in vascular density, blood perfusion, oxygenation, metabolic activity, intracellular/extracellular pH, and interstitial fluid pressure. Many of these pathophysiological changes can have a profound influence on the activity of the other combination therapy, and since the reported changes include both increases and decreases, the effects can be in both a negative and positive fashion. For example, increasing tumor oxygenation status will enhance tumor radiation response, while decreasing oxygenation will reduce it. With the AIs these pathophysiological changes can be highly variable between the different drug types. The same AI can also produce completely opposite pathophysiological effects in different tumor types, even when administered using similar drug doses and treatment schedules. VDAs are more consistent in the pathophysiological changes induced. Essentially they all decrease tumor blood perfusion and as such will make the microenvironmental conditions, especially oxygenation and pH, worse. These effects suggest that timing and sequence between the different treatments must be considered in any clinical application, especially when combining VTAs with therapies that already have some beneficial effect in patients. Despite these potential limitations, preclinical studies with VTAs in combination with other therapies do show an enhanced tumor response without any significant increased damage in dose-limiting normal tissues.

Vascular Targeting Agents

Numerous VTAs are currently under clinical evaluation. With AIs these include both specific and nonspecific inhibitors of angiogenesis, and the phase of testing ranges from Phases I to IV. The most popular agents in clinical testing are the anti-VEGF antibodies (e.g., Bevacizumab), followed by receptor kinase inhibitors (e.g., SU5416, sunitinib, sorafenib, vandetanib, and PTK 787/ZK 222584) and the nonspecific inhibitor thalidomide. Far fewer trials are underway with the VDAs, and most agents that are being investigated are either in Phase I or II, although there are a limited number of Phase III trials. The lead agent in this series is CA4P. Experimental studies strongly support the concept that the application of angiosuppressive and vascular disrupting strategies as adjuvants to standard anticancer therapy can improve treatment outcomes. Lead vascular targeting agents are now under active investigation in such settings in patients. Ultimately, it is possible to envisage future treatment protocols consisting not only of the current mainstays of cancer management, surgery, radiotherapy, and chemotherapy but also of “vascular targeted therapy” consisting of a battery of tumor vessel directed agents.

Cross-References ▶ Alkylating Agents ▶ Angiogenesis ▶ Antiangiogenesis ▶ Chemotherapy ▶ Cisplatin ▶ Docetaxel ▶ Endostatin ▶ Fluorouracil ▶ Gemcitabine ▶ Hypoxia ▶ Ionizing Radiation Therapy ▶ Irinotecan ▶ Metastasis ▶ Mitomycin C ▶ Necrosis ▶ Oncogene ▶ Paclitaxel ▶ Photodynamic Therapy

Vasculogenic Mimicry

▶ Receptor Tyrosine Kinases ▶ Topoisomerases ▶ Vascular Disrupting Agents ▶ Vascular Endothelial Growth Factor

References Clémenson C, Chargari C, Deutsch E (2013) Combination of vascular disrupting agents and ionizing radiation. Crit Rev Oncol Hematol 86:143–160 Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676 Folkman J (1976) The vascularisation of tumors. Sci Am 234:58–71 Horsman MR, Siemann DW (2006) Pathophysiological effects of vascular-targeting agents and the implications for combination with conventional therapies. Cancer Res 66:11529–11539 Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727–739 Siemann DW, Horsman MR (2009) Vascular targeting therapies in oncology. Cell Tiss Res 335:241–248

See Also (2012) Bioreductive drug. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 415. doi:10.1007/978-3-642-16483-5_645 (2012) Tumor suppressor. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3803. doi:10.1007/978-3-642-16483-5_6056

Vascular-Targeted Therapies ▶ Vascular Disrupting Agents

Vasculogenic Mimicry Baocun Sun, Shiwu Zhang and Danfang Zhang Department of Pathology, Tianjin Cancer Hospital and Tianjin Cancer Institute, Tianjin, People’s Republic of China

Definition The generation of microvascular channels by genetically deregulated, aggressive tumor cells

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was termed “vasculogenic mimicry” (VM) to emphasize their de novo generation without participation by endothelial cells. VM is thought to represent a vascular channel formation without the involvement of endothelial cells, in contrast to ▶ angiogenesis. Vasculogenic mimicry refers to a blood supply pathway in tumors that is formed by tumor cells and that is independent of endothelial cell-lined blood vessels. Three factors are thought to govern the formation of functional and patterned microcirculation channels by VM: (i) plasticity of highly malignant tumor cells, (ii) remodeling of the extracellular matrix (ECM), and (iii) connection of the VM channel with host blood vessels to acquire blood supply from the host tissue. Formation of VM in tumors may have substantial impact on clinical outcome of tumor patients. Tumor patients in the presence of VM have a poorer prognosis than those without VM, and VM-targeted therapy is a perspective for tumors showing VM. At this point, VM is a new concept originally described for ▶ melanoma that needs to be studied further in detail.

Characteristics Introduction Tumor angiogenesis is a key for tumor growth, invasions, and metastasis. Tumor growth is ▶ angiogenesis dependent, and angiogenic switch is an essential step for a small and noninvasive tumor to transit into a tumor with invasive and metastatic ability. Blood vessels are assembled by two processes: (i) vasculogenesis, the reorganization of randomly distributed cells into a blood vessel network, and (ii) angiogenesis, the sprouting of new vessels from preexisting vasculature in response to external chemical stimulation. Current Status of Studies on VM in Tumors It is believed that VM consists of tumor cells and PAS-positive ECM on the inner wall of channels. The constituents of PAS-positive ECM are laminin, collagens IV and VI, mucopolysaccharide, and heparin sulfate glucoprotein (HSPG). Initially, PAS-positive ECM was considered as an

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Vasculogenic Mimicry, Fig. 1 Vasculogenic mimicry (VM). Melanoma cells form VM channels, and red blood cells (RBC) flow into the channel. Necrosis and inflammatory cells are not observed in tumors undergoing VM

Vasculogenic Mimicry, Fig. 2 The connection of VM channel and endothelium-dependent vessel shows VM is a functional microcirculation

absolutely indispensable element. However, VM channels in the absence of PAS-positive ECM were observed in a melanoma mouse model. VM is an independent blood supply pattern in tumors (Figs. 1, 2, and 3). Compared with endothelium-dependent vessels, it has several characteristics as follows: (i) VM channels are lined by tumor cells but not endothelial cells. (ii) There is red blood cell (RBC) leakage into tumor tissue near to endothelium-dependent vessels, while leaking RBCs are lacking in tumors with VM. (iii) Necrosis and inflammatory cells are not observed in tumors undergoing VM.

Vasculogenic Mimicry

Vasculogenic Mimicry, Fig. 3 VM consists of tumor cells and PAS-positive ECM on the inner wall of channels. The PAS-positive patterns are lined by tumor cells, and there are red blood cells in the center of the pattern

Molecular Mechanisms Underlying VM Compared with less aggressive melanoma cells, highly aggressive melanoma cells express higher levels of ▶ matrix metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-14) and the 5g2 chain of laminin. This increases the expression of MMPs and the presence of the laminin receptor on the surface of tumor cells helps cells adhere to more laminin. The activated MMPs cleave laminin into several short chains and eventually promote the formation of VM. Phosphoinositide-3kinase modulates the function of MMP-14 (MT1-MMP), which activates MMP-2 with the help of tissue inhibitor of MMP-2 (TIMP2), and the activated MMP-2 then cleaves 5g2 chain into g20 and g2x chains. The two chains facilitate the formation of VM. The cleavage fragments of 5g2 can be secreted by highly malignant melanoma cells directly. VE-cadherin has been proved to be closely related to the formation of VM channels. Highly aggressive melanoma cells express VE-cadherin but less aggressive ones do not and inhibition of VM formation by downregulating the expression of the VE-cadherin gene. Dedifferentiation of Tumor Cells Is the Key to Formation of VM Channels Much information on VM has come from studies of highly malignant melanomas. Tumor cells having the ability of VM formation show an

Vasculogenic Mimicry

embryonic phenotype. A cDNA microarray study of 5,000 genes from a patient with poorly and highly aggressive melanoma cells revealed that there was a differential expression in 210 genes, including some genes associated with the phenotypes of endothelial and hematopoietic stem cells. Except for embryonic genotypes, cells of tumors with VM express various angiogenesisrelated cytokines. Flt-1 and Tie-2 are expressed by tumor cells of naked mice bearing human inflammatory breast carcinoma cells. Ovarian cancer cells with high aggressivity express the vascular endothelial growth factor (VEGF) and other angiogenesis-related cytokines (e.g., Ang-1 and Ang-2), whereas those with low aggressivity express VEGF only. The expression of tyrosine kinase (an enzyme that catalyzes the phosphorylation of several signal transduction proteins) is upregulated in highly aggressive melanoma cells than in less aggressive melanoma cells. Aggressive melanoma cells show an increased activity of tyrosine kinase around VM channels. Epithelial cell kinase (EphA2), a tyrosine kinase receptor, is specifically expressed in highly aggressive melanoma cells. Inhibitors of tyrosine kinase activity hinder VM channel formation, and a transient knockout of EphA2 shows reduced VM channel formation. Linearly Patterned Programmed Cell Necrosis and Three-Stage Phenomenon Linearly patterned programmed cell necroses (LPPCN) and three-stage phenomenon are thought to play essential roles in the blood supply for melanoma. At the early stage of tumor generation, endothelium-dependent vessels do not sprout into tumor center. Under the pressure of hypoxia, some tumor cells activate ▶ apoptosisassociated genes, and lacunas left by dissolving LPPCN cells connect with each other and form channel networks (Fig. 4). The channels coming from LPPCN cells face two opposite ends. If they connect with endothelium-dependent vessels, blood will flow into these channels lined by tumor cells and the channels will be a functional microcirculation. In contrast to this, the mass of

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Vasculogenic Mimicry, Fig. 4 LPPCN. Cells undergoing LPPCN have spindle-like figure and dark blue nuclei. They connect with each other as lines and some cells enter the wall of VM channels

tumor cells will undergo necrosis if these channels fail to link to endothelium-dependent vessels. Three microcirculation patterns – VM, mosaic vessels, and endothelium-dependent vessels – coexist in melanoma tissue. Angiogenesis requires the recruitment of normal endothelial cells, which may not be efficient and/or sufficient enough for sustaining aggressive tumor growth at the initial stage of rapid growth. Some tumor cells dedifferentiate, connect with other tumor cells or endothelium, and finally line the wall of tube. They are vasculogenic mimicry and mosaic vessels. Mosaic vessel may be a transition between VM channel and endothelium-dependent vessel. The three-stage phenomenon on tumor blood supply pattern assumes that there is a transformation among VM channels, mosaic vessels, and endothelium-dependent blood vessels (Fig. 5). In the stage of rapid tumor growth, endotheliumdependent vessels sprout from normal tissue but are insufficient to support the rapid tumor growth. VM occurs on the base of LPPCN and acts as the major blood supply pattern for tumor growth. As tumor size becomes bigger, endothelial cells from peripheral blood home, proliferate on the wall of VM, and cover some tumor cells forming VM. Mosaic vessels appear and become the major microcirculation pattern in tumors. Finally, endothelium-dependent vessels take over the dominant role in tumor blood supply.

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Vasculogenic Mimicry

LPPCN

Sprouting from host mitovessel VM

Mosaic vessel Tumor cell Red cell

Endothelium-dependent vessel

Endothelial cell Endothelial progenitor cell

Vasculogenic Mimicry, Fig. 5 Three-stage phenomenon on tumor blood supply pattern. During rapid tumor growth, endothelium-dependent vessels sprouting from normal tissue cannot satisfy the need for growth. VM occurs on the base of LPPCN and acts as the major blood supply pattern for tumor growth. As endothelial cells from

host microvessels migrate and endothelial progenitor cells from peripheral blood home into the wall of VM, endothelial cells cover some tumor cells forming VM. Mosaic vessels appear and become the major microcirculation pattern in tumors. Finally, endothelium-dependent vessels get the dominant role in tumor blood supply

The Effect of Local Tumor Microenvironment on the Formation of VM Channels Tumor growth and evolution are regulated by a good many factors, and tumor cells display distinguished blood supply pattern and biological behavior to adapt to different microenvironments. The environmental factors impacting VM channel formation include oxygen pressure, interstitial fluid pressure (IFP) in tumor tissue, pH, focal concentration of cytokines, and ECM. ▶ Hypoxia is a two-edge sword for tumor generation and development. Hypoxia and ischemia induce tumor cells to necrosis and tumor suppression, whereas hypoxia activates metastasis-related genes to promote tumor invasion. The hypoxic condition enhances the formation of VM channels, which exerts its function through HIF-1a and its downstream molecules. In the hypoxic environment, accumulated HIF-1a in tumor cells induces MMP-2, MMP-9, and VEGF expression and activation. MMP-2 and MMP-9 proteinases degrade the ECM components and facilitate VM formation, tumor invasion, and metastasis. VEGF secreted by tumor cells results in permeability of blood vessels, resulting in an increase and leaking out of many

serum proteins. Such a milieu provides a temporary matrix for VM channel formation. Under the stimuli of hypoxia, LPPCN-associated genes can be triggered and some tumor cells undergo LPPCN. Interspaces left by dissolving cells connect with each other as networks to provide the space basement for VM. Interstitial fluid pressure (IFP) is another important factor affecting tumor microcirculation patterns. Increased IFP is a characteristic of malignant tumors because of its rapid proliferation. It is similar to hypoxia and has double impact on tumor development. High IFP inhibits both endothelial cells of blood vessels and lymphatic vessels to migrate into the tumor center, with the result of tumor hypoxia. Elevated IFP stimulates tumor cells to secrete invasion-associated proteins. High IFP is a barrier for endothelium-dependent vessels sprouting into tumor tissue, but hypoxia induced by it is an inducer for VM. Moreover, the expressions of MMP-2, MMP-9, integrin, selectin, and kinesin increase significantly in tumor cells growing in the microenvironment with high IFP, which promote VM formation to provide sufficient nutrition and oxygen for tumor growth.

Vasculogenic Mimicry

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Vasculogenic Mimicry, Fig. 6 Comparison of overall survival time of patients with bidirectional differentiated malignant tumors. A Kaplan–Meier algorithm reveals that the survival time of melanoma, mesothelial sarcomas (MS),

alveolar rhabdomyosarcomas (AS), and synovial sarcomas (SS) without VM is significantly longer than that of patients with VM

Clinical Significance of VM VM has been observed in several human malignant tumor types, such as highly aggressive uveal melanomas, breast cancer, liver cancer, glioma, ▶ ovarian cancer, ▶ melanoma, prostate cancer, malignant ▶ astrocytoma, and bidirectional differentiated malignant tumors. Such tumors possess features of both endothelial cells and mesenchymal cells. Tumor cells lining on the inner surface of VM channels are directly exposed to blood flow, may move into the bloodstream, and metastasize to other organs. VM is associated with poor prognosis in patients. Tumors with VM have a higher rate of metastasis compared with tumors without VM, and the patients have a lower 5-year survival rate. Routine antiangiogenic drugs, such as

angiostatin and ▶ endostatin, which target endothelial cells, have not achieved a therapeutic effect on tumors that exhibit VM because of the absence of endothelium-dependent vessels (Fig. 6). Advances and Challenges VM and Lymphogenesis in Tumors

VM, a new pattern of blood supply to the tumors, has attracted the attention of many researchers, but many phenomena unique to VM channel formation remain to be elucidated. As a functional tumor microcirculation, VM channels need to be studied with regard to their connection with endothelium-dependent vessels, their relationship with lymphatic tubes, and their dual function as vessels and lymphatic tubes. Uveal melanoma

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cells have a specific vortex vein but no lymphatic tube. Highly aggressive melanoma expresses lymphatic-vessel endothelial hyaluronan receptor 1 (LYVE1) and VEGF-C, a lymphatic tuberelated growth factor. VM-Targeted Therapy

Given the important role of angiogenesis in tumor growth and metastasis, therapies aiming at endothelial cells represent promising antitumor strategies. As VM has a different structure from endotheliumdependent vessels, traditional antiangiogenic agents targeting at endothelial cells, such as anginex, TNP-470, and ▶ endostatin, have no remarkable effects on malignant tumors with VM. One of the distinguished features of tumors with VM is that cell adhesion molecules, tumor invasion-related proteinases and ECM synthesis, and secretion-associated proteins are overexpressed by tumor cells. These molecules represent potential targets for anti-VM strategies of highly aggressive and blood metastatic tumors with VM. Suppressing tyrosine kinase activity, using a knockout EphA2 gene, downregulating VE-cadherin, using antibodies against human McMPs and the laminin 5g2 chain, and using anti-PI3K therapy are strategies that have been employed to inhibit VM.

References Folberg R, Maniotis AJ (2004) Vasculogenic mimicry. APMIS 112(7–8):508–525 Hess AR, Gardner SE, Carles-Kinch LM et al (2001) Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer Res 61(8):3250–3255 Maniotis AJ, Hess FR, Seftor A et al (1999) Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol 155(3):739–752 Zhang S, Guo H, Zhang D et al (2006) Microcirculation patterns in different stages of melanoma growth. Oncol Rep 15(1):15–20

Vasoactive Intestinal Contractor ▶ Endothelins

Vasoactive Intestinal Contractor

Vasopressin Definition Antidiuretic hormone, also known commonly as vasopressin, is a nine-amino acid peptide secreted from the posterior pituitary. Within hypothalamic neurons, the hormone is packaged in secretory vesicles with a carrier protein called neurophysin, and both are released upon hormone secretion. Roughly 60% of the mass of the body is water, and despite wide variation in the amount of water taken in each day, body water content remains incredibly stable. Such precise control of body water and solute concentrations is a function of several hormones acting on both the kidneys and vascular system, but there is no doubt that antidiuretic hormone is a key player in this process. The single most important effect of antidiuretic hormone is to conserve body water by reducing the loss of water in urine. A diuretic is an agent that increases the rate of urine formation. Injection of small amounts of antidiuretic hormone into a person or animal results in antidiuresis or decreased formation of urine, and the hormone was named for this effect. Antidiuretic hormone binds to receptors on cells in the collecting ducts of the kidney and promotes reabsorption of water back into the circulation. In the absence of antidiuretic hormone, the collecting ducts are virtually impermeable to water, and it flows out as urine. Antidiuretic hormone stimulates water reabsorption by stimulating insertion of “water channels” or aquaporins into the membranes of kidney tubules. These channels transport solute-free water through tubular cells and back into the blood, leading to a decrease in plasma osmolarity and an increase osmolarity of urine.

See Also http://www.vivo.colostate.edu/hbooks/pathphys/endo crine/hypopit/adh.html

Viral Oncology Epigenetics

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VDAs

Villi

▶ Vascular Disrupting Agents

Isabelle Gross INSERM U1113, Université de Strasbourg, Strasbourg, France

VEGF ▶ Vascular Endothelial Growth Factor

VEGF Trap ▶ Aflibercept

Velcade ® ▶ Bortezomib

v-erb-B2 ▶ HER-2/neu

Definition Villi are small, slender, vascular projections that augment the surface area of a membrane. Although villi are present on the surface of the embryonic chorion and the brain arachnoid membrane, most of them are found in the gut. The typical velvety appearance of the intestinal wall is due to the presence of millions of such finger-shaped outgrowths pointing toward the lumen that considerably increase its absorptive area. They are most prevalent at the beginning of the small intestine and diminish in number toward the end of the tract. Each villus is covered by an epithelial lining and has a central core composed of one artery and one vein, a strand of muscle, a centrally located lymphatic capillary, and connective tissue that adds support to the structures.

Vesical Cancer

Villin 2

▶ Bladder Cancer Pathology

▶ ERM Proteins

VHL

Viral Oncology Epigenetics

▶ Von Hippel-Lindau Disease

VHL Tumor Suppressor Gene ▶ Von Hippel-Lindau Tumor Suppressor Gene

VIC ▶ Endothelins

James Flanagan Institute of Reproductive and Developmental Biology, Imperial College London, London, UK

V Definition Viral oncology epigenetics can represent the ▶ epigenetic alterations that occur within the host cell genome as a result of viral infection or virally induced ▶ carcinogenesis. Alternatively, viral

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oncology epigenetics can refer to epigenetic alterations of the viral genome during latent or lytic infection or during carcinogenesis.

Characteristics One of the emerging concepts in cancer biology is that epigenetic alterations are important in the initiation and early progression of the majority of human cancers. However, differentiation of the early cancer causing epigenetic alterations from later consequences is difficult. Oncogenic viruses are typically very small and with very few genes and yet can induce transformation. Therefore, investigations into the epigenetic alterations that viruses make to the host cell genome may provide an indication of which epigenetic alterations are critical for early carcinogenesis. Oncogenic viruses include any virus that has been identified as the causative agent for cancer. They can induce cellular alterations that enable transformed cells to evade host responses, including the immune system and apoptosis. In vitro, oncogenic viruses induce cellular transformation creating cells capable of unrestrained proliferation and are often tumorigenic in athymic mice. Oncogenic viruses in humans include DNA viruses, such as hepatitis B viruses (HBV), human papillomavirus (HPV), polyomaviruses (BKV, JCV, and ▶ SV40) and the gamma-herpesviruses, ▶ Kaposi sarcoma-associated herpesvirus (KSHV), and ▶ Epstein-Barr virus (EBV) as well as retroviruses, such as human T-cell lymphotropic viruses 1 and 2 (HTLV1/HTLV2), the RNA flavivirus, and ▶ hepatitis C virus (HCV). Epigenetics is a term used to describe the regulation of gene expression and genomic stability by heritable, but potentially reversible, changes in DNA methylation and chromatin structure. DNA ▶ methylation is controlled in the genome by various epigenetic regulators, including the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), methylated DNA-binding proteins (e.g., MECP2, MBD1-MBD4), and DNA demethylases (e.g., GADD45A, GADD45B). Chromatin structure is regulated by

Viral Oncology Epigenetics

posttranslational modifications to the tails of the core histones that make up the nucleosome including lysine acetylation, lysine and arginine methylation, lysine ubiquitination, serine phosphorylation, and proline isomerization. The enzymes that catalyze these modifications (histone deacetylases, histone acetyltransferases, histone demethylases, and histone methyltransferases) interact with other chromatin structure regulators, such as the polycomb group (repression) and trithorax group (activation) genes, ATPdependent chromatin remodeling complexes and microRNAs, and the RNAi machinery to coordinate together the regulation of gene expression. In normal cells transcriptionally active genes typically contain unmethylated promoter ▶ CpG islands, gene-wide histone hyperacetylation, and a number of specific histone modifications, such as H3 lysine 4 (H3K4) methylation, H3K79 methylation, H3 arginine methylation, H3S10 phosphorylation, and H2B ubiquitination. Transcriptionally repressed genes often contain methylated promoter CpG islands, histone hypoacetylation, and H3K27 and H3K9 methylation. In cancer cells, however, there are marked differences in the epigenetic landscape of the genome. Genome wide, there is an overall hypomethylation associated with repetitive DNA in cancer cells, as well as promoter hypermethylation of specific tumor suppressor genes and hypomethylation of oncogenes. In addition to altered DNA methylation, chromatin remodeling in cancers is also considered a common epigenetic alteration, including loss of H4K16 acetylation and H4K20 trimethylation and gains in H3K4 di- and tri-methylation, H3K79 methylation, and H3 and H4 hyperacetylation. Host Epigenetic Changes Due to Viruses and Virus-Associated Cancers Differentiating epigenetic causes of cancer from epigenetic consequences is one of the more challenging goals of current research in the epigenetics field. This conundrum can equally be applied to virally induced cancers. It is challenging to differentiate epigenetic changes that are directly due to viral infection, due to the antiviral response of the host, or due to downstream effects

Viral Oncology Epigenetics

of the virally induced transformation. Important early epigenetic changes in cancer may be distinguished from consequences via the identification of specific interactions between epigenetic regulators and the viral proteins effecting those changes and then determining the downstream effects of these interactions. Kaposi Sarcoma-Associated Herpesvirus

▶ Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus identified as the causative agent of the endothelial tumor Kaposi sarcoma (KS) and associated with the lymphoproliferative disorders primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other gamma-herpesviruses, KSHV has both a lytic and latent phase of its life cycle. In the majority of tumor cells, the more restricted set of latent genes are often expressed. Evidence suggests that KSHV can reprogram the cellular gene expression profiles of both blood and lymphatic vessel endothelial cells, although the mechanisms behind this reprogramming remain unclear. A direct link between the KSHV protein latency-associated nuclear antigen (LANA) and the de novo methyltransferase DNMT3a was examined in a study revealing recruitment of DNMT3a by LANA to the chromatin possibly via its interaction with histones H2A and H2B. This targeted repression of ~80 cellular genes, many of which are typical targets of epigenetic inactivation in numerous cancers. KSHV has been shown to hypermethylate the ▶ CDKN2A gene promoter in the majority of PEL cell lines harboring KSHV and in primary PEL samples. This is not unexpected, given that CDKN2A is probably the gene most commonly hypermethylated in cancers. This suggests that KSHV has the ability to invoke DNA methyltransferase activity, via the LANA protein, and may inactivate numerous cellular genes by promoter hypermethylation. In addition, LANA has numerous other roles in epigenetic gene regulation via interactions with a methylated DNA-binding protein MECP2, as well as the mSin3 repression complex and the SUV39H1 histone methyltransferase. Numerous studies

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have also proposed a direct interaction between the KSHV-encoded interferon regulatory factors (viral IRF 1, IRF2, and IRF3) and the histone acetyltransferase complex p300/CBP. The binding of CBP by the cellular IRF3 is thought to be a necessary interaction for transcriptional upregulation of the antiviral cytokine interferonb (IFN-b). In effect, the interactions of the viral IRFs with CBP inhibit histone acetyltransferase activity and promote histone hypoacetylation, altered chromatin structure, and reduction of cytokine gene expression. Presumably other genes that are activated by p300/CBP would also be downregulated. Epstein-Barr Virus

Epstein-Barr virus (EBV) is another gammaherpesvirus that has been identified as the causative agent of numerous lymphoproliferative diseases such as ▶ Burkitt lymphoma (BL), ▶ Hodgkin disease (HD), and posttransplant lymphoproliferative disease (PTLD). EBV is also involved in the carcinogenesis of epithelial origin tumors, particularly nasopharyngeal carcinomas (NPC) and some gastric cancers. The EBV latent membrane protein 1 (LMP1) increases DNA methyltransferase activity by upregulating the maintenance methyltransferase DNMT1 as well as both de novo methyltransferases, DNMT3b and DNMT3a. In EBV-related epithelial cancers, this causes increased promoter methylation and reduction of e-cadherin expression, resulting in increasing cell migration, an important step during carcinogenesis. Other cellular genes that are methylated by EBV are yet to be identified. The EBV latent nuclear antigens EBNA 2 and EBNA 3c appear to alter histone acetylation via their interactions either with the p300/CBP complex or with histone deacetylases, respectively. One of the interesting observations regarding the EBV proteins that coordinate epigenetic regulation (EBNA2, EBNA3c, and LMP1) is that they are all latent genes that are not expressed in most Burkitt lymphoma, EBV-associated gastric cancer, or nasopharyngeal carcinomas. This may lead one to conclude that the role of the virusinduced host epigenetic alterations may be limited

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in these cancers. However, in these cancers and in other virally induced cancers, it cannot be ruled out that the early cancer precursor cells may have been infected with the virus expressing these proteins that could alter the host epigenome. Epigenetic fingerprints such as histone modifications and DNA methylation are mitotically heritable, and therefore even if the progeny cancer cells no longer express these latent genes, the epigenetic history of the cell may remain. Hepatitis B Virus

The hepatitis B virus (HBV) is a DNA virus from the hepadnavirus family and is the causative agent of viral hepatitis, a chronic inflammation of the liver, which can develop into ▶ hepatocellular carcinoma. The hepatitis B virus oncogenic protein HBx has been shown to increase the activity of DNMT1, and similar to EBV-related cancers, this results in increased DNA methylation of e-cadherin (CDH1) and an increased cell migration. Whether this can be attributed directly to increased activity of the maintenance methyltransferase, DNMT1, is not clear as EBV also activates the de novo methyltransferases, DNMT3a and DNMT3b, which HBV does not. The tumor suppressor gene CDKN2A and the glutathione-S-transferase (GSTP1) genes are both commonly hypermethylated in hepatocellular carcinomas associated with HBV; however, there is currently no direct evidence that the virus causes the increased DNA methylation of these genes. Human Papillomavirus

The human papillomavirus (HPV) family is a large family of DNA viruses of which some subtypes (particularly HPV 16 and 18; ▶ early genes of human papillomaviruses) are associated with ▶ cervical cancer and rarer epithelial origin cancers. The two important HPVoncoproteins E6 and E7 have both been implicated in epigenetic alterations during carcinogenesis. HPV E7 protein increases the DNA methyltransferase enzymatic activity by direct interaction with DNMT1 and E6 can bind and inhibit the histone acetyltransferase activity of p300 and CBP similarly to KSHV. This

Viral Oncology Epigenetics

transcriptional repression by HPV is supported by E7 protein interaction with the Nurd ATP-dependent chromatin remodeling complex and histone deacetylase 1 which are both involved in transcriptional repression. Numerous cellular epigenetic alterations have been described in ▶ cervical cancers including hypermethylation of tumor suppressor genes RB, CDKN2A, MLH1, VHL, and CDH1. Whether the E7-mediated increase in DNA methyltransferase activity is responsible for methylation of these genes is yet to be established. Polyomaviruses (SV40, BK Virus, and JC Virus)

Polyomaviruses are very small DNA viruses (~5 kb) encoding only six genes. In humans the BK virus is associated with some brain tumors, and the JC virus has been associated with gliomas, medulloblastomas, and a minority of colorectal carcinomas; however, neither virus has been implicated as direct causative agents in these tumors. The simian virus SV40 is not a causative agent of any human tumors, but has been found in some mesotheliomas. These three polyomaviruses encode the T-antigen oncoprotein which is involved in inducing DNA methylation alterations. The SV40 virus upregulates the de novo methyltransferase DNMT3b which results in increased DNA methylation and increased tumorigenicity in normal human bronchial cells. Aberrant methylation of RASSF1A, HPP1, CCND2, DCR1, TMS1, CRBP1, HIC1, and RRAD has all been detected in SV40-associated malignant mesotheliomas or SV40-infected human mesothelial cells. The BK virus increases transcription of DNMT1 through the pRB/E2F pathway in cells that have pRB inactivated; however, this has not yet been linked to increased methylation of any specific genes. The JC virus T-antigen expression is associated with the methylator phenotype in colorectal cancer; however, this link with methyltransferase is still rather speculative. Adenovirus

The ▶ adenovirus protein E1A can induce transformation in vitro and can induce tumors in animal models, but the virus does not in itself cause

Viral Oncology Epigenetics

any human cancers. E1A interacts with the p300/ CBP complex and is likely to result in a loss of histone acetylation across the genome. It has been proposed that this interaction could be one of the key events in E1A-induced cellular transformation. Another important role could be its capability of increasing DNMT1 activity, although which cellular genes are hypermethylated as a result of this increased activity is yet to be determined. Human T-Cell Lymphotropic Viruses

Human T-cell lymphotropic viruses (HTLV1 and HTLV2) are single-stranded RNA retroviruses that are causative agents of adult T-cell leukemias. While there is little data on host DNA methylation alterations directly mediated by this virus, the HTLV1 Tax protein does interact with the p300/CBP complex to mediate transcriptional repression. Epigenetic Alterations in the Virus Due to the Host The opposite side of viral oncology epigenetics is the epigenetic alterations that occur on the viral genome during the lytic and latent stages of infection and during carcinogenesis. In addition to regulation of their own genome, DNA methylation of viral genes has also been proposed as a mechanism for silencing potentially highly immunogenic proteins that would otherwise elicit an immune response. Due to spontaneous deamination of methylated cytosines to thymine, there is a reduced rate of CpG dinucleotides (▶ CpG island) in the genomes of organisms that use DNA methylation as a mechanism of gene silencing. This mechanism, known as CpG suppression, is seen in the human genome with an observed CpG dinucleotide rate of ~1% where the statically expected rate would be 1/16 (6%) of the total bp. The genomes of many gamma-herpesviruses, including EBV, show CpG suppression suggesting that their genomes have been methylated similarly to the DNA of the host cells in which they reside. In this way the epigenetic state of the viral genome can be considered due to the host in which they reside. KSHV on the other hand only shows CpG suppression at the lytic switch promoter, ORF50,

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suggesting that the rest of the viral genome is not extensively methylated. KSHV in fact controls its entry into the lytic phase by employing both demethylation and chromatin remodeling of the lytic switch gene Rta (ORF50) promoter, and its latent cycle replication is controlled by hyperacetylation of the replication origin. The EBV C promoter which drives the expression of the latent gene transcripts is often methylated and silenced in most EBV-associated tumors. This prevents the expression of highly immunogenic Epstein-Barr nuclear antigen (EBNA) proteins that would elicit a cytotoxic T-cell response and provides a very good example of how viruses exploit epigenetic mechanisms to evade the immune system. Conclusions It is clear that oncogenic viruses increase the activity of DNA methyltransferases and have the ability to decrease histone acetylation via p300/ CBP. These are both likely to be essential for the inactivation of tumor suppressor genes. That both of these events occur in many nonviral cancers as well suggests that they may be some of the earliest epigenetic alterations in carcinogenesis. The common pathways utilized by viruses to effect these epigenetic changes suggest that perhaps very few changes are required to initiate epigenetic misregulation in cancer. For example, the BK virus increases transcription of DNMT1 through the pRB/E2F pathway in cells that have pRB inactivated: however, this has not yet been linked to increased methylation of any specific genes. The HPV E7 protein, KSHV LANA, polyomavirus T antigen, and adenovirus E1A are all known to inactivate pRB as well as increase DNA methyltransferase activity, suggesting that inactivation of pRB may be a critical step toward epigenetic alterations in carcinogenesis. It is also interesting to note that many of the viral proteins described here, LMP1, E6 and E7, LANA, E1A, large T antigen, and Tax, are all often portrayed as the viral “oncoproteins” as they are often either essential components of viral transformation or oncogenic on their own. This is often used as evidence that the functions of these proteins, in this case the epigenetic interactions, are indeed essential for carcinogenesis induced by these viruses.

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Cross-References ▶ Adenovirus ▶ Amplified in Breast Cancer 1 ▶ Burkitt Lymphoma ▶ Carcinogenesis ▶ CDKN2A ▶ Cervical Cancers ▶ Chromatin Remodeling ▶ CpG Islands ▶ Early Genes of Human Papillomaviruses ▶ Epigenetic ▶ Epstein-Barr Virus ▶ Hepatitis B Virus ▶ Hepatitis C Virus ▶ Hepatocellular Carcinoma ▶ Hodgkin Disease ▶ Human T-Lymphotropic Virus ▶ Kaposi Sarcoma ▶ Methylation ▶ MicroRNA ▶ Nasopharyngeal Carcinoma ▶ Oncogene ▶ SV40

References Fields BN, Knipe DM, Howley PM (2001) Fields’ virology, vol 2, 4th edn. Lippincott Williams & Wilkins, Philadelphia, p xix, 3087, I–72 Flanagan JM (2007) Host epigenetic modifications by oncogenic viruses. Br J Cancer 96(2):183–188 Kouzarides T (2007) Chromatin modifications and their function. Cell 128(4):693–705, This Cell Special Review Issue has many excellent reviews on epigenetic control of transcription and chromatin organisation

See Also (2012) DNA Methylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1140. doi:10.1007/978-3-642-16483-5_1682 (2012) Histone Acetylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1697. doi:10.1007/978-3-642-16483-5_2750 (2012) HTLV-1. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1752. doi:10.1007/978-3-642-16483-5_2844 (2012) Hypomethylation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1791. doi:10.1007/978-3-642-16483-5_2922

Viral Vector-Mediated Gene Transfer (2012) LMP2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2068. doi:10.1007/978-3-642-16483-5_3401 (2012) Retrovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3296–3297. doi:10.1007/978-3-642-16483-5_5084

Viral Vector-Mediated Gene Transfer Yuanan Lu1 and Lynn F. Gottfried2 1 Department of Public Health Science, University of Hawaii, Honolulu, HI, USA 2 LeClairRyan, Rochester, NY, USA

Synonyms Oncolytic virus; Virus vector; Virus vectormediated gene transfer

Definition Refers to the process by which virus vectors are used to deliver functional genes of interest into target cells and tissues, either in vitro or in vivo.

Characteristics Viruses have evolved natural mechanisms to efficiently transport their own genetic materials into host cells while also commandeering host cell machinery for replication. Therefore, the use of viruses for the introduction of therapeutic genes and stimulation of the immune system has become an attractive and promising method for the treatment of a variety of diseases, including cancer. The genomes of a wide array of viruses can be modified and used as a tool for the efficient transfer of exogenous genes into living cells or organisms. In broad terms, two types of virus vectors exist: replication competent and replication defective. Replication-competent vectors are exemplified by live-attenuated strains of many common viruses, including both DNA viruses (such as ▶ adenovirus, herpes simplex virus type-1

Viral Vector-Mediated Gene Transfer

(HSV-1), and vaccinia virus) and RNA viruses (such as recombinant derivatives of vaccine strains of measles virus and poliovirus and vesicular stomatitis virus (VSV)). Replication-defective vectors have an intrinsically more favorable safety profile than replication-competent vectors, but can be more difficult to engineer and manufacture. Commonly used replication-defective vectors include both small (adeno-associated virus (AAV)) and large (adenovirus, HSV-1) DNA viruses, as well as RNA viruses (including alphaviruses such as Venezuelan equine encephalitis virus (VEE), lentiviruses, and other retroviruses). In all cases, the vectors are designed such that one or more genes essential for viral replication or assembly have been deleted or otherwise rendered defective. Some viral vectors combine aspects of both replication-competent and replication-defective vectors. These include vectors that are fully competent for replication in cells of one type or species, but not in cells of a different type, or from a different host species. Widely used examples include baculovirus vectors, which are able to transduce mammalian cells but which can replicate only in insect cells, and certain poxviruses such as the modified vaccinia Ankara (MVA) strain of vaccinia virus, which replicates efficiently in chick embryo fibroblasts, but undergoes abortive infection in human cells. Conditionally replicating virus vectors, such as oncolytic viruses, are another example and are especially attractive as potential therapeutic agents for cancer treatment, as will be discussed later in this entry. Finally, there is considerable interest in the development of artificial viruslike vectors, including viruslike particles (VLPs). VLPs offer potential advantages for vaccine delivery or display of vaccine antigens due to the densely repetitive nature of the surface of many VLPs. These properties explain, in part, the tremendous effectiveness of the licensed VLP-based vaccines for oncogenic human papillomavirus (HPV) subtypes. Completely synthetic gene transfer vectors have particular appeal because of the potential to mass-produce such agents using conventional chemical technologies, rather than the more

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complex and cumbersome biological production methods required to manufacture conventional virus vectors. However, the efficiency of gene transfer by completely synthetic vectors currently remains far inferior to that of conventional virus vectors. Detection of Viral Vector-Mediated Gene Expression and Vector Distribution. In order to analyze the success of viral vector-mediated gene transfer, it is important to be able to monitor both the distribution of the vector and the effectiveness of vector-mediated gene expression. This can be achieved by subcloning a reporter gene into the viral vector backbone. Several reporter genes are commonly used for this purpose including fluorescent proteins of various colors (including green fluorescent protein, GFP), E. coli b-galactosidase (LacZ), and various forms of luciferase (Luc). It is simple to detect and quantitate cells expressing these marker genes in vitro, using FACS analysis or fluorescence microscopy. Detection of vectortransduced cells in vivo often requires a different approach. Modified luciferase reporters such as the Gaussia luciferase can generate a very bright light emission that can be detected and localized to specific tissues by using highly sensitive light detection methods in combination with 3D tomography. For in vivo imaging applications, a number of marker genes are compatible with the use of currently available gamma cameras or ▶ positron emission tomography (PET) instruments. These include the sodium-iodide symporter (NIS), which has been used to target radioisotopes of iodine to cancer cells. This allows in vivo imaging of the distribution of the vector, quantitation of gene expression, and also delivery of therapeutic radiation to cancer cells. The HSV-1 thymidine kinase (TK) gene has also been a successful tool for imaging when coupled with PET tracers. Furthermore, in the case of cancer therapy, the TK gene can be combined with prodrugs, such as ganciclovir (GCV), which become activated by TK and exert cytotoxic effects on rapidly dividing cancer cells. Viral Vectors for Cancer ▶ Gene Therapy. A number of vectors, including AAV, adenovirus, HSV, measles virus, and retroviruses, have been

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developed to promote the selective elimination of tumor cells. Cancer gene therapy approaches include immunotherapy and suicide gene therapy. In addition, naturally oncolytic viruses such as reovirus (exemplified by Reolysin) and oncolytic viral vectors that result in the destruction of transduced tumor cells are also being pursued. For reasons of space, we will focus the rest of this review on the latter. Virus platforms that are being used to develop oncolytic vectors include adenovirus, HSV-1, measles virus, and vaccinia virus. The field of ▶ oncolytic virotherapy has made rapid progress in the past decade, resulting in human clinical trials of multiple agents based on all of the above vector platforms. Clinical trials have included phase III studies, and, in 2005, the field received a major boost when the Chinese government approved the first oncolytic virus therapy for cancer treatment. This is a significant landmark that likely presages the approval of other oncolytic virus therapies elsewhere in the world. There are several approaches that can be taken in the use of oncolytic vectors for cancer therapy. One approach simply utilizes the replicating virus itself as therapy. As the virus replicates within the tumor cells, the cells are lysed and destroyed. Considerable efforts are being made to improve tumor selectivity and to ensure that the ▶ virotherapy spares surrounding healthy tissue. This can be accomplished by (i) placing viral genes under the control of tumor-specific promoters (such as the PSA promoter in ▶ Prostate Cancer), (ii) altering the receptor-binding properties of the vector in order to target tumors, and (iii) utilizing the oncolytic properties of the vector in conjunction with “arming” of the virus with therapeutic genes. The use of “armed” vectors is attractive due to the fact that it draws on multiple mechanisms to achieve the desired effect. For example, engineering oncolytic HSV vectors to deliver a therapeutic gene, such as an antiangiogenic factor, has been shown to enhance the therapeutic efficacy of the vector in small animal model systems. It is also possible to introduce a tumor suppressor gene, such as ▶ p53, into cancer cells via an oncolytic vector in order to exert control over the cell cycle of the tumor

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cells while also subjecting the cells to the oncolytic activity of the virus itself. The success of oncolytic vectors as anticancer therapies can be further enhanced by combining the virotherapy with either chemotherapy or radiation treatment. Not only is it possible to use radiation-inducible promoters to direct the expression of essential viral genes but also it has been shown that radiation and chemotherapy are able to enhance the replication of certain oncolytic vectors. For example, in the case of oncolytic HSV-1 vectors, the induction of DNA repair genes by chemotherapy actually augments viral replication. Also, the use of a reporter gene, which has therapeutic effects when combined with radiation, such as NIS, offers the promise of an effective synergistic approach to tumor treatment. Overall, the field of cancer gene therapy has made important advances within the past few years, and while oncolytic virotherapy may not offer a simple “magic bullet,” it has considerable potential to synergize with, and enhance the effectiveness of, other established therapies such as chemo- and radiotherapy. Viral Vector Cancer Gene Therapy – Pitfalls. While important advances have been made, concerns remain. First, oncolytic virotherapies may not be sufficiently effective to provide optimal therapeutic benefit as a stand-alone treatment. Therefore, the effectiveness of the vectors needs to be increased. In addition, the issue of pre-existing immunity to common viruses such as HSV-1 and adenovirus serotype 5 may need to be overcome, in order to make most effective use of oncolytic viruses based on these agents. Second, biosafety questions must be addressed. These include the potential for vector replication in normal tissues, such as rapidly dividing stem cells, and the inherent genetic instability of certain viruses. For example, it was found that an oncolytic HSV-1 vector being used in human clinical trials contained a previously unrecognized mutation (a truncation in the UL3 open reading frame). In conclusion, virus vector-mediated gene transfer holds significant promise as a potential therapeutic approach for many human diseases,

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including cancer. The approval of the world’s first oncolytic virus for human tumor therapy is indicative of the rapid and exciting progress in the field. Moreover, evolving success in combining oncolytic virotherapies with sophisticated tumortargeting and gene transfer approaches, as well as conventional chemo- and radiotherapy, provide a compelling reason to anticipate new cancer treatments that more effectively exploit the potential of viral vector systems.

References Advani SJ, Mezhir JJ, Roizman B et al (2006) ReVOLT: radiation-enhanced viral oncolytic therapy. Int J Radiat Oncol Biol Phys 66:637–646 Dambach MJ, Trecki J, Martin N et al (2006) Oncolytic viruses derived from the g34.5-deleted herpes simplex virus recombinant R3616 encode a truncated UL3 protein. Mol Ther 13:891–898 Kirn DH (2006) The end of the beginning: oncolytic achieves clinical proof-of-concept. Mol Ther 13:237–238 Liu TC, Galanis E, Kirn D (2007) Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol 4(2):101–117 Young LS, Searle PF, Onion D et al (2006) Viral gene therapy strategies: from basic science to clinical application. J Pathol 208:299–318

Virology Paul G. Murray CRUK Institute for Cancer Studies, Molecular Pharmacology, Medical School, University of Birmingham, Birmingham, UK

Definition Virology addresses the molecular nature of viruses, their genetic content, the pathway by which they enter cells and multiply using the molecular machinery of the host cell, and the mechanisms by which they elicit diseases. Tumor virology is a specialized discipline analyzing the association of particular virus types with cancers in animals and humans.

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Characteristics In 1964 the first human tumor virus, the ▶ Epstein–Barr virus (EBV), was isolated from tumor samples of a patient with African ▶ Burkitt lymphoma. Subsequently, EBV was linked to the development of other forms of cancer. In the 44 years since the discovery of EBV, other human tumor viruses have been identified (Table 1). Tumor Virus Epidemiology The development of cancer is an infrequent consequence of viral infection and often occurs many years after any initial infection. Therefore, tumor viruses often infect individuals without adverse effects. For example, approximately 95% of the world’s adult population are infected with EBV. The majority of these individuals carries the virus asymptomatically and will not develop cancer as a consequence of EBV infection. Likewise, tumors associated with the human T-lymphotropic virus 1 (HTLV-1) arise infrequently in populations where the virus is endemic. Thus, presumably alone tumor viruses are usually insufficient to cause malignancy; virus infection is only one step in the multistep process leading to a cancer. Although virus infection may be linked to a particular cancer type, in order to establish a clear association between the virus and the development of a cancer, it is usually necessary to detect the virus within the tumor cells. Immunosurveillance and Viral Oncogenesis Virus-associated cancers occur in both immunocompetent and immunodeficient patients. However, the latter group has a particularly high risk for the development of these tumors, suggesting that the immune system can prevent the development of virus-associated cancers. ▶ Cytotoxic T cells (CTLs) are particularly important in the recognition and elimination of virus-infected tumor cells. CTLs recognize virus-derived peptides that are presented by the infected cell in association with MHC class I. The development of virus-associated cancers in immunocompetent individuals suggests that the virus-infected tumor cell or its progenitor has developed mechanisms

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Virology

Virology, Table 1 Major oncogenic viruses Natural host Man

Host in which virus is oncogenic Hamster, rat

Tumor Various

Mouse

Mouse

Various

SV40

Monkey

Hamster, rat

Various

Papillomaviridae

HPV 16,18

Man

Man

Herpesviridae

EBV

Man

Man

KSHV

Man

Man

Hepadnaviridae

Hepatitis B virus

Man

Man

Skin cancer, cervical cancer, anal cancer Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin lymphoma Kaposi sarcoma, multicentric Castleman disease, primary effusion lymphoma Hepatocellular carcinoma

Flaviviridae

Hepatitis C virus

Man

Man

Hepatocellular carcinoma

Retroviridae

HTLV1

Man

Man

Adult T-cell leukemia

Virus family Adenoviridae

Polyomaviridae

Virus Human adenoviruses A, B, D Polyoma

to escape immune recognition. In some cases virus-specific CTLs have been used to treat virus-associated cancers (▶ Adoptive Immunotherapy). In some cases gene therapy may be used to modify CTL function (CTL therapy). Tumor Viruses There are a number of different viruses that have been associated with the development of cancer. The acutely transforming retroviruses cause cancer in animals but to date none have been associated with the development of human tumors. The following sections consider some of the important human tumor viruses. Herpesviridae The major oncogenic herpesviruses are the Epstein–Barr virus (EBV) and the Kaposi sarcoma herpesvirus (KSHV). Epstein–Barr Virus EBV (Epstein–Barr virus) is a double-stranded DNA virus of the gamma herpesvirus family.

Major oncogenic protein(s) E1A, E1B

Middle T antigen, large T antigen Large T antigen E6, E7 LMP1

Hepatitis B x antigen HCV core, NS3, NS4B, NS5A Tax

EBV infects the majority of the world’s adult population, and following primary infection, the individual remains a lifelong carrier of the virus. In poorly developed countries, primary infection with EBV usually occurs during the first few years of life and is often either asymptomatic or produces only a mild febrile illness. However, in developed populations, primary infection is frequently delayed until adolescence or adulthood, in many cases producing the characteristic clinical features of infectious mononucleosis (also known as glandular fever) including sore throat, fever, malaise, lymphadenopathy, and mild hepatitis. EBV is often transmitted from one individual to another in saliva, and the oropharynx is believed to be both the primary site of infection and where virus replication occurs. Virus replication in the oropharynx ensures the production of new virions for transfer in saliva to other susceptible hosts. Because EBV is transmitted in this way, infectious mononucleosis is often referred to as the “kissing disease.” Soon after primary infection, EBV infects B-lymphocytes through an interaction of the

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Virology, Table 2 Three major forms of viral latency are associated with EBV-positive malignancies Latency I II

III

Viral genes expressed EBERs, BARTs EBNA1 EBERs, BARTs EBNA1, LMP1, LMP2A, and LMP2B EBERs, BARTs EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA leader protein (LP) LMP1, LMP2A, and LMP2B

viral envelope glycoproteins, gp350/220, with the cellular EBV receptor, CD21. EBV does not usually replicate in B-lymphocytes but instead establishes a latent infection during which no new virions are produced and only a subset of viral genes are expressed. As a consequence of the host immune response, the number of latently infected B-lymphocytes in the peripheral blood falls to approximately 1 in 106 during the months following primary EBV infection. This low number is maintained in healthy carriers by the continual elimination of proliferating EBV-transformed B-lymphocytes by virus-specific CTLs. The EBV genome usually exists in cells as extrachromosomal pieces of circular DNA, known as episomes. During latency only a limited number of viral genes are expressed; these include six nuclear proteins, referred to as the Epstein–Barr nuclear antigens (EBNAs), two latent membrane proteins (LMPs), two non-translated RNA molecules known as the Epstein–Barr-encoded RNAs (EBERs 1 and 2), and transcripts from the BamH1A region of the viral genome (BamH1A rightward transcripts; BARTs). Three major forms of viral latency exist in tumors (Table 2). The restricted forms of latency have evolved partly to prevent expression of the immunodominant EBNA proteins and are controlled by differential ▶ methylation of viral promoters. Demethylating agents can induce expression of these EBNAs and in some cases can also induce lytic cycle; this has been proposed as an alternative treatment for some EBV-associated cancers (▶ Epigenetic Therapy).

During EBV latency, only a limited number of viral genes are expressed; these include six nuclear proteins, referred to as the Epstein–Barr nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and EBNA-LP), two proteins found in the cell membrane of infected cells known as the latent membrane proteins (LMPs), two non-translated RNA molecules known as the Epstein–Barr virusencoded RNAs (EBERs), and the BARTs. Many viruses, including EBV, are able to subvert the cellular epigenetic machinery, not only to regulate virus gene expression but also potentially to influence cell growth by silencing ▶ tumor suppressor genes). LMP1 is the major transforming protein of EBV; it is a transmembrane protein which mimics a constitutively activated tumor necrosis factor receptor (TNFR). In normal cells, binding of the appropriate ligand to these receptors causes intracellular signaling and a number of effects including either proliferation or ▶ apoptosis, depending upon the nature of the signal and the cell type involved. In contrast, LMP1 requires no ligand for its activation and delivers constitutive intracellular signals leading to cell proliferation and protection from apoptosis, which in turn accounts for the transforming properties of this protein. Signaling pathways activated by LMP1 include ▶ NF-kB, JNK/AP-1, p38/MAPK, and JAK/STAT (▶ Signal Transducers and Activators of Transcription). Several of these pathways are aberrantly activated in EBV-associated tumors, such as Hodgkin lymphoma. LMP1 can decrease ATM expression and may represent one of a number of ways in which EBV can disable DNA repair. The malignant diseases associated with EBV include Burkitt lymphoma, ▶ nasopharyngeal carcinoma, Hodgkin lymphoma, and a variety of other cancer types. EBV-Encoded microRNAs ▶ MicroRNAs (miRNAs) are a class of small RNAs that are probably major regulators of gene expression. miRNAs are complementary to their cognate mRNA sequences; their interaction in the RNA-induced silencing complex (RISC) results in the cleavage of the target mRNA or in some cases inhibits its translation. The first

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virus-encoded miRNAs that were discovered were those of EBV, from which five miRNAs were cloned. EBV miRNAs are clustered in two distinct regions; the first is located within the 50 and 30 UTR of the BHRF1 transcript and the other within the BART region. The precise functions of these and other viral miRNAs are yet to be defined but are likely to play key roles in the regulation of virus and cellular gene expression, with potential involvement in oncogenesis. Burkitt Lymphoma ▶ Burkitt lymphoma (BL) is an aggressive tumor of B-lymphocytes. There are two main types of BL. The endemic (African) type occurs with high frequency (5–20 cases/100,000 children/year) in equatorial Africa and Papua New Guinea and with a distribution that matches that of holoendemic malaria. Almost all cases of endemic BL are EBV positive. On the other hand, sporadic BL occurs worldwide with a much lower incidence, and only around 15% of cases are EBV positive. A third form of BL occurs in AIDS patients and approximately one-third of these tumors harbor EBV. The cells of EBV-infected BL tumors usually display a latency I phenotype – that is, they only express one viral protein, EBNA1. Variant forms of BL have been described in which expression of some of the other EBNA genes is observed. These are known as Wp using BL since they use a distinct viral promoter (Wp) to drive EBNA expression. Although infected BL tumor cells could process peptides from the EBNA1 protein and present them to specific CTLs, the processing of endogenous EBNA1 through the class I pathway is inhibited. This, together with the downregulation of MHC class I and ▶ adhesion molecules, that is, a feature of BL cells, contributes to their ability to evade immunodetection. BL is characterized by reciprocal translocations that result in deregulation of the myc gene. In endemic BL, EBV-driven proliferation of B-lymphocytes, together with a general polyclonal stimulation of B cells induced by malaria infection, is thought to increase the chances of one of these specific translocations occurring in the B-lymphocytes that will eventually give rise to BL.

Virology

Nasopharyngeal Carcinoma ▶ Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the nasopharynx that is rare in the West but endemic in China, Southeast Asia, and North Africa. There are three main types of NPC: undifferentiated, nonkeratinizing, and squamous NPC. EBV is strongly associated with the undifferentiated type (UNPC). UNPC is an aggressive tumor with ▶ metastasis early to the bone, liver, and lung and lymph nodes of the neck. The exact contribution of EBV to the pathogenesis of NPC has yet to be established, although studies suggest that EBV infection is preceded by genetic changes which include deletions of chromosome regions rich in tumor suppressor genes. Dietary factors, including nitrosamines from preserved fish, as well as EBV, are important risk factors for UNPC. Hodgkin Lymphoma Hodgkin lymphoma (HL) is characterized by relatively low numbers of malignant, so-called ▶ Hodgkin/Reed–Sternberg cells (HRS cells) surrounded by a mass of “reactive” nonmalignant cells. HL is classified into four major subtypes on the basis of the relative proportions and morphology of the HRS cells, the nature of the reactive component, and the degree of fibrosis. In most cases the HRS cell is believed to derive from germinal center (GC), or post-GC B-lymphocytes. The frequency of the EBV association in HL is dependent upon a number of factors. EBV is most often associated with the mixed cellularity form and less commonly with the other subtypes. In North America and Europe, fewer (20–40%) tumors are EBV associated, compared to developing countries where the association approaches 100%. Males also seem to be more at risk than females for EBV-positive HL. In EBV-positive cases, HRS cells express EBNA1, LMP1, and LMP2 (latency II pattern, Table 2). Epitopes from both LMP1 and LMP2 can be processed and presented to CTLs by infected cells. Thus, EBV-infected HRS cells should be recognized by EBV-specific CTLs; however, their survival in immunocompetent patients suggests that they, like BL cells, can escape the immune response.

Virology

EBV-infected HRS cells express interleukin-10 (IL-10) which can inhibit EBV-specific CTL responses. Regulatory T cells are also detected in the microenvironment of HL. It is likely that EBV and other persistent virus are able to recruit regulatory T cells to inhibit virus-specific T-cell responses. Lymphoproliferative Disease in Immunosuppressed Patients In immunosuppressed patients, the lack of EBV-specific CTLs can lead to an increase in the numbers of EBV-infected B-lymphocytes. In persistent ▶ immunosuppression states, such as in posttransplant patients or in AIDS sufferers, EBV-infected B-lymphocytes can proliferate to produce tumorlike masses. Later, the acquisition of other genomic changes, such as those that affect p53, MYC, or BCL-6, can lead to the formation of a classic lymphoma. Therefore, these lymphoproliferative diseases constitute a spectrum of disorders ranging from relatively benign atypical lymphoproliferations which will regress if the immunosuppressive therapy is withdrawn through to highly aggressive lymphomas which do not respond to immune reconstitution. Other Tumors EBV is associated with a number of other tumors, including some T-cell lymphomas, such as nasal T/natural killer cell lymphomas (NK cell lymphomas). EBV is also associated with some gastric cancers and with smooth muscle tumors that arise in some immunodeficient patients. Thus, EBV is apparently able to infect and contribute to neoplastic growth in a number of different cell types. Kaposi Sarcoma Herpesvirus

▶ Kaposi sarcoma (KS), originally described in 1872, is a malignancy of endothelial cells that usually presents as a brown/purple skin tumor with more aggressive forms involving the lungs, lymph nodes, and gastrointestinal tract. Until the advent of the AIDS epidemic, it was a relatively rare disease whose etiology remained obscure. KS occurs frequently in HIV-positive individuals, particularly in homosexual/bisexual males.

4819 Virology, Table 3 KSHV encodes many homologues of cellular proteins; some of these viral proteins and their effects upon the infected cell are summarized Cellular gene Cyclin gene (cyclin D2)

KSHV gene v-Cyclin D

IL-6

v-IL-6

IL-8R

v-GPCR

FLICE inhibitor protein (FLIP) Interferon regulatory proteins (IRFs) Bcl-2

v-FLIP

Effect of virus gene expression Phosphorylates Rb and releases cell from cell cycle arrest Autocrine stimulation of cell growth Constitutive activation of phosphatidylinositol pathway leading to cell growth Inhibits CD95mediated apoptosis

v-IRFs 1–4

Inhibits interferon signaling

v-Bcl-2

Protects infected cell from apoptosis

Suspicions that KS might be due to an infectious agent were confirmed when a new human herpesvirus, known as Kaposi sarcoma herpesvirus or KSHV (also referred to as human herpesvirus 8, HHV8), was discovered in KS tumors from AIDS patients. In fact, viral sequences are present in all types of KS including KS that arises in HIV-negative individuals. Serological assays to detect antibodies to KSHV were developed and showed a higher prevalence of infection in those groups at high risk for the development of KS. KSHV is also associated with primary effusion lymphomas and a rare lymphoproliferative disease known as multicentric Castleman disease. KSHV is a double-stranded DNA virus that is closely related to EBV. Its genome encodes many genes with homology to human genes (Table 3). Some of these are involved in the regulation of both innate and adaptive immune responses; many of them function to inhibit the immune responses to viral infection. For example, several virus-encoded interferon (IFN)-regulatory factors (IRFs) negatively influence antiviral interferon responses, while others protect cells from apoptosis (e.g., v-FLIP, v-▶ BCL-2).

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Polyomaviridae

Polyomaviruses are DNA viruses with small circular genomes encoding only six proteins. They include the mouse polyomavirus, the simian virus 40 (SV40), and the human viruses, BK virus and JC virus. With the exception of the mouse polyomavirus, these viruses do not cause cancer in their natural hosts but do induce tumors in newborn animals, including hamsters and rats. Polyomaviruses do not themselves encode replication proteins and must drive cells into S-phase where host DNA replication proteins can be utilized for virus replication. The large T (tumor) antigen (T-Ag) and the small t antigen (t-Ag) are major effectors of this. T-Ags bind to pRb encoded by the RB gene and displace E2F thereby promoting cell cycle progression; this is a major mechanism by which T-Ag promotes the inappropriate cell proliferation leading to oncogenic transformation. The release of E2F from pRb activates p14ARF which can stabilize p53. However, SV40, JCV, and BKV T-Ags can bind to and inactivate p53 and thus prevent inhibition of the cell cycle or apoptosis. Apart from their well-established role in the binding and inactivation of p53 and pRb, T-Ag can influence other pathways leading to oncogenesis. Thus, JCV T-Ag can interact with insulin receptor substrate-1 (IRS-1); this is associated with transformation and might be involved in the development of childhood medulloblastomas. JCV T-Ag can also bind b-catenin, causing it to translocate to the nucleus where it can stimulate the expression of genes such as c-myc and cyclin D1. SV40 t-Ag binds and inhibits protein phosphatase 2A (PP2A), a major serine/threonine specific protein phosphatase; this leads to the activation of several pathways that promote cell proliferation, including the MAPK pathway. Although the polyomaviruses can transform cells in culture and under certain conditions are oncogenic in laboratory animals, their association with clinical human tumors has yet to be definitely proven (association of polyomaviruses with human cancers).

Virology

tissues. At present there are over 100 known subtypes of HPV, and the majority are responsible for benign lesions of the genital, upper respiratory, and digestive tracts. However, some HPV subtypes are associated with malignant diseases of the skin and cervix (▶ Cervical Cancers). Cutaneous HPV infection normally results in the appearance of benign warts; however, in the rare but lifelong skin disease, epidermodysplasia verruciformis (EV), these multiple benign warts can progress to malignant squamous carcinoma when exposed to ultraviolet light. The tumor cells often contain HPV 5 or 8. These two HPVs are also associated with the skin carcinoma observed in long-term immunosuppressed renal transplant patients. Virtually all squamous cancers of the cervix are HPV positive; HPV16, followed by HPV18, is the commonest subtype found in this disease. HPV18 is the type most strongly associated with adenocarcinoma of the cervix. HPV is sexually transmitted and the association of HPV with cervical cancer explains many of the risk factors for this disease including early age at first sexual encounter and multiple sexual partners. Although most women will have been infected with HPV at some time, very few will develop invasive cancer. In cancers, integration of HPV18 is almost universally observed, whereas integration of HPV16 is less common. Integration usually disrupts the E1 or E2 viral genes (▶ Early Genes of Human Papillomaviruses); this results in the loss of negative feedback control of E6 and E7 expression by the viral regulatory E2 protein. Overexpression of E6 and E7 proteins is important in oncogenesis; E6 binds to and inactivates p53 and E7 binds to Rb. A bivalent HPV16/18 and a quadrivalent HPV 6/11/16/18 vaccine are being evaluated in phase III clinical trials. Preliminary data suggest that these prophylactic HPV viruslike particle vaccines are effective in preventing infections and also in reducing epithelial abnormalities (Human Papillomavirus Vaccines). Hepadnaviridae

Papillomaviridae

Hepatitis B Virus

▶ Human papillomaviruses (HPVs) are small DNA viruses that commonly infect epithelial

▶ Hepatitis B virus (HBV) is associated with the development of ▶ hepatocellular carcinoma

Virology

(HCC) where integrated HBV DNA can been detected in the majority of tumors. The exact role of HBV in the development of HCC is yet to be established. However, the HBx protein is likely to be important since it can induce a number of cellular changes that contribute to transformation, including the activation of several intracellular signaling pathways, including NF-kB. Other factors in addition to HBV status contribute to the risk of developing HCC; these include smoking, dietary components such as ▶ aflatoxin, and exposure to other hepatotoxic agents, including ▶ hepatitis C virus. Flaviviridae Hepatitis C Virus

▶ Hepatitis C virus is a blood-borne RNA virus that can cause chronic hepatitis and later cirrhosis and hepatocellular carcinoma. HCV is spread by blood-to-blood contact. Many people with HCV infection have no symptoms and are unaware of the need to seek treatment. An estimated 150–200 million people worldwide are infected with HCV. At least four HCV gene products, namely, HCV core, NS3, NS4B, and NS5A, have been shown to exhibit transformation potential in tissue culture. Both HCV core and NS5A induce the accumulation of wild-type b-catenin. Retroviridae Human T-Lymphotropic Virus 1

Human T-lymphotropic virus 1 (HTLV1) (▶ human T-lymphotropic virus) infects CD4-positive T-lymphocytes and is associated with the development of adult T-cell leukemia/lymphoma (ATLL). Like other retroviruses, the HTLV1 genome consists of gag, pol, and env genes, which encode important structural and functional proteins, flanked by long terminal redundancies (LTRs). HTLV1 has an additional 3Υ region which encodes several proteins implicated in transformation; these include Tax, Rex, p12, p13, p30, and HBZ. Tax has been shown to be necessary and sufficient for transformation by HTLV-1. Tax activates both the canonical and noncanonical NF-kB pathways,

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in turn leading to increased expression of many ▶ cytokines and their receptors, including interleukin-2 and the interleukin-2 receptor, which leads to polyclonal proliferation of HTLV-1-infected cells by autocrine and paracrine mechanisms. Additional potentially transforming effects are contributed by some of the other HTLV1 genes.

Cross-References ▶ Adhesion ▶ Adoptive Immunotherapy ▶ Aflatoxins ▶ Apoptosis ▶ Bcl2 ▶ Burkitt Lymphoma ▶ Cervical Cancers ▶ Cytokine ▶ Cytotoxic T Cells ▶ Early Genes of Human Papillomaviruses ▶ Epigenetic Therapy ▶ Epstein-Barr Virus ▶ Hepatitis B Virus ▶ Hepatitis C Virus ▶ Hepatocellular Carcinoma ▶ Hodgkin and Reed/Sternberg Cell ▶ Immunosuppression and Cancer ▶ Kaposi Sarcoma ▶ Metastasis ▶ Methylation ▶ MicroRNA ▶ MYC Oncogene ▶ Nasopharyngeal Carcinoma ▶ Nuclear Factor-kB ▶ Signal Transducers and Activators Transcription in Oncogenesis ▶ Tumor Necrosis Factor ▶ Tumor Suppressor Genes

of

References Cullen BR (2006) Viruses and microRNAs. Nat Genet 38(Suppl):S25–S30 Khan G (2006) Epstein-Barr virus, cytokines, and inflammation: a cocktail for the pathogenesis of Hodgkin’s lymphoma? Exp Hematol 34:399–406

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4822 Poulin DL, DeCaprio JA (2006) Is there a role for SV40 in human cancer? J Clin Oncol 24:4356–4365 Stanley MA (2006) Human papillomavirus vaccines. Rev Med Virol 16:139–149 Tao Q, Young LS, Woodman CB et al (2006) Epstein-Barr virus (EBV) and its associated human cancers–genetics, epigenetics, pathobiology and novel therapeutics. Front Biosci 11:2672–2713

See Also (2012) Humanized antibodies. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 1760. doi:10.1007/978-3-642-164835_2863 (2012) Monoclonal antibody. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2367. doi:10.1007/978-3-642-16483-5_6842 (2012) Monoclonal antibody therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 2367–2368. doi:10.1007/978-3-64216483-5_3823 (2012) P53. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331

Virotherapy Zeng B. Zhu1, Bruce F. Smith2, Gene P. Siegal3 and David T. Curiel4 1 Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology and the Gene Therapy Center, Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, USA 2 Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL, USA 3 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA 4 Division of Cancer Biology, Washington University, St. Louis, MO, USA

Synonyms Oncolytic virotherapy

Zeng B. Zhu has retired.

Virotherapy

Definition Virotherapy utilizes ▶ oncolytic viruses, which may occur naturally or more commonly be engineered, such as conditionally replicative ▶ adenoviruses (CRAds), to selectively infect tumor cells and replicate within them, thus causing their demise while sparing surrounding normal cells in the host. Origins of replication result from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells whereby normal host cells are spared.

Characteristics Virotherapy represents an exciting and novel interventional strategy for a range of neoplastic disorders. In this strategy a virus is rendered conditionally replicative for tumor cells whereby direct oncolytic target killing is achieved. A variety of viral species have been adapted as virotherapy agents with the majority of human clinical trials exploiting conditionally replicative herpes simplex virus (HSV) and adenovirus. Of these, adenovirus has emerged as a promising model oncolytic vector to target tumor cells. Over 40 different human serotypes of adenoviruses have been identified with types 2 and 5 being the most extensively used in developing oncolytic constructs. Oncolytic Adenoviruses and Cancer Gene Therapy The concept of using conditionally replicative adenoviruses (CRAds) for the treatment of cancer, also known as oncolytic virus therapeutics, originated in the 1950s. The knowledge that adenoviruses could eliminate cancer cells in vitro, as a consequence of their reproductive cycle leading to cell lysis (“oncolysis”), resulted in clinical studies in which various wild-type adenoviral serotypes were examined for their effect on ▶ cervical cancer patients. In these studies, no significant toxicity was reported after intratumoral injection (i.p.) or intravenous (i.v.) administration and a

Virotherapy

moderate tumor response was observed. The most studied CRAd is the one originally generated (dl1520) by Arnold Berk and used initially by Frank McCormick as a selective vector, named ONYX-015. This viral vector originally was believed to only replicate in p53-defective cells (present in 50% of human tumors). It is now recognized that these agents have beneficial properties for cancer ▶ gene therapy when compared with their nonreplicating counterparts, although this mechanism has subsequently been questioned. The adenovirus-based vectors have maintained their positions as the leading candidates for in vivo oncolytic virotherapy because they are safe, produced in high titers, do not integrate into the host chromosome, and have a wide tropism in neoplastic cells. While adenoviral transfection is efficient, the expression of the transferred gene is transient, as the viral genome remains episomal. To maximize CRAd-mediated cell killing, one needs to achieve an amplification effect for transduction, via replication of the delivered viral vector postinfection, resulting in lateral spread of the progeny vector and cell killing via viral oncolysis. In addition, viral proteins expressed late in the course of infection are indirectly cytotoxic, including the E3 11.6 kDa adenovirus death protein, E4ORF4. CRAds in Human Clinical Trials and the Limitations Significant antitumor activity has been demonstrated using ONYX-015 both in vitro and in vivo using murine models. The preclinical potential of CRAds led to their rapid translation into human clinical trials, including those targeting recurrent head and neck, pancreatic, colorectal, ovarian, and hepatobiliary cancer. The overall conclusion was that adenoviral virotherapy is a safe method when applied via various routes, thus validating the concept in vivo. In clinical practice, however, employing CRAd as a single agent has demonstrated limited efficacy. This is due, in part, to relatively inefficient gene transfer to tumor cells. The replicative Ad system of ONYX-015, although demonstrating promise in preclinical studies, yielded no clinical effect in 16 patients with ▶ ovarian cancer

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treated intraperitoneally with up to 1  1011 plaque-forming units of adenovirus daily for 5 days. Important in this regard, cancer cells have been specifically shown to be profoundly resistant to Ad infection. The results obtained in these studies have been helpful in determining the limitations of the current generation of CRAds. The critical problems that have been encountered involve two main limitations: (i) poor infectivity of cancer cells by adenovirus at the transductional level due to the lack of native adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR), on the surfaces of tumor cells and (ii) poor tumor selectivity of CRAd agents at the transcriptional level due to the lack of tumor-specific promoters to selectively drive the viral replication in tumor cells. CAR Deficiency on Tumor Cells Results in the Poor Infectivity of Ad Agents Cancer cells have been specifically shown to be profoundly resistant to Ad infection because of their lack of the primary receptor for viral entry, the coxsackievirus-adenovirus receptor (CAR). As these primary tumor cells often express relatively low levels of the CAR, this results in the poor infectivity of CRAd agents and the difficulty of lateral dispersion of virus in tumor tissue. This has been demonstrated by the fact that low CAR levels strongly reduced viral replication and oncolysis in monolayer cultures and murine models. On this basis, it was proposed that delivery be achieved via a heterologous entry pathway or CAR-independent pathway to circumvent this key aspect of tumor biology. Infectivity-Enhanced CRAd Agents Retargeting CRAds to Tumor Cells Native adenoviral tropism is mediated by two capsid proteins, fiber and penton base. These proteins bind to the primary, high-affinity cellular receptor, CAR, and the integrins avb3 and avb5, respectively. Manipulation of these molecular interactions has been carried out to modify adenovirus tropism by routing entry through either a heterologous entry pathway or a CARindependent pathway. Many approaches have been described to enhance the viral infectivity.

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These include: (i) Ad capsid modification for circumventing tumor cell CAR deficiency. Specifically, Dmitriev et al. (1998) reported that construction of modified adenoviral vectors containing the RGD peptide in the HI loop region, which targets integrins avb3 and avb5 instead of CAR, increased gene transfer to ovarian cancer cell lines (30–600-fold) and to primary ovarian cancer cells obtained from patients (two to threefold). Many other approaches have been reported which include targeting Ad to the serotype 3 receptor with a chimeric fiber protein, such as F5/3, targeting Ad to tumor cells with the nonhuman canine Ad type 2 knob, targeting Ad to a heparin sulfate-containing receptor with an Ad fiber incorporating polylysine (pK7), and targeting Ad to the junction adhesion molecule 1 (JAM1) with an Ad fiber incorporating reovirus sigma 1 fiber. All these fiber modifications enhanced the viral infectivity of Ad vectors but with different levels of success dependent upon the tumor types. (ii) Transductional retargeting using heterologous targeting adapters. In adapter-mediated targeting, the tropism of the virus is modified by an extraneous targeting moiety, the ligand, which associates with an Ad virion either covalently or non-covalently. Zhu et al. described in 2004 that an adenoviral vector was successfully induced to transport itself across polarized epithelial monolayers by use of a fusion protein (sCAR-transferrin), a bifunctional adapter, which bound to the knob of Ad through a sCAR (secretory CAR) domain and to the transferrin receptor on cell surfaces through a transferrin domain. In addition, sCAR-EGF, sCAR-SCF, and sCAR-scFv have all been reported as having successfully targeted EGF receptor-positive cells, c-Kit(+) and CAR(–) hematopoietic cells and ovarian carcinoma cells, respectively. The genetic fusion proteins of single-chain variable fragment (scFv) antibodies directed against the fiber knob and receptor on the surfaces of tumor cells have also been used in virotherapy. In this schema, an anti-fiber-knob Ab has been employed to attach to the cell recognition motif of the fiber knob and, importantly, ablate the native tropism determinants. An Ab, or Fab derivative, was then conjugated to a second moiety, which provided

Virotherapy

targeting specificity. To that end, receptor ligands such as folate and basic fibroblast growth factor-2 (FGF-2) have been used to successfully target tumor cells. Thus, the strategy of tropism modification either using bispecific conjugates or scFv molecules allowed dramatic augmentation in gene delivery to tumor targets, with a specificity that would predict an improved therapeutic index. Tumor-Specific CRAd Agents: Using a TumorSpecific Promoter to Drive Specific Viral Replication Although viral replicative specificity is not the main limit of CRAd efficacy, our work and those of others have also sought to address this aspect to improve the overall therapeutic index. The method used to direct CRAd vector specificity is by regulation of viral replication via cellular promoters that are overexpressed or reactivated in selected tumor cells, sometimes referred to as “tumor-specific” promoters. A number of CRAd agents have been developed, which harbor the essential Ad E1A gene under the control of such promoters. They include the alpha-fetoprotein promoter, Cox-2 promoter, DF3/MUC1 promoter, midkine promoter, PSA/PSMA enhancer, secretory leukocyte protease inhibitor (SLPI) promoter, CXCR4 and survivin promoters, human telomerase reverse transcriptase (hTERT) promoter, and the tyrosinase promoter/enhancer. Most of these agents have demonstrated remarkable preclinical results in eradicating tumors in xenograft mouse models. A Double-Targeted CRAd for Ovarian Cancer Based on the foregoing considerations, we have developed a CRAd agent which addressed the limits of the current systems. As previously discussed, CAR deficiency in the context of the target tumor would clearly confound this process, thus undermining the overall efficacy of CRAd agents. Previous studies have demonstrated that incorporation of an Arg-Gly-Asp (RGD)containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. In the context of ovarian cancer, the RGD-modified nonreplicative adenovirus

Virotherapy

mediates enhanced gene transfer in vitro in both established cell lines and freshly cultured ▶ ovarian cancer cells. It exhibits preferential gene transfer to primary ovarian cancer cells when compared to non-transformed human mesothelial tissue, thus, indicating a degree of specificity for tumor cells. In addition, the RGD-modified nonreplicative adenoviral vector demonstrated enhanced infectivity of primary ovarian tumor cells where infectivity was known to be inhibited by preformed neutralizing antibodies against adenovirus found in the ascites fluid in which the tumor cells float. Investigators at the University of Alabama at Birmingham (UAB) and the University of Texas MD Anderson Cancer Center have constructed a novel infectivity-enhanced CRAd, designated Ad5-D24RGD, that has a 24 bp deletion in the E1A gene and that incorporates the RGD modification in its fiber. The deletion in the E1A gene is from Ad5bp923 to 946 which corresponds to the amino acid sequence L122TCHEAGF129 of the E1A protein known to be necessary for host cell Rb protein binding. Adenoviruses having this partial deletion cannot induce resting cells to pass the G2/M checkpoint and progress to mitosis and lysis. In contrast, most human tumor cells bypass the Rb/p16 pathway, thus allowing for selective replication of adenoviruses with this deletion. Preliminary experiments demonstrated that Ad5-D24RGD propagated more efficiently than Ad5-D24 in A549 cells. Specifically, A549 cells transfected with Ad5-D24RGD yielded 43-fold times increase in viral progeny (3.75  109 pfu/ ml) when compared to Ad5-D24-infected cells (8.74  107 pfu/ml). Cell lysis in Ad5-D24RGDinfected A549 and LNCap cells was 7 and 3.5 times higher, respectively, compared to that achieved with Ad5-D24. Validation of the potential benefits of incorporating infectivity enhancements into a CRAd provided the rational to translate this into clinical trials which has been approved by FDA. Future Directions for Improvements CRAd human trials were historically limited by lack of useful information regarding the biologic basis of adenovirus efficacy barriers. Absent

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“surrogate endpoints” imaging could potentially provide this information. We have thus developed an imaging system which addresses this problem, based upon fluorescently labeled adenovirus carrying EGFP (enhanced green fluorescent protein) on the pIX minor surface protein of Ad for vector detection. Furthermore, positron emission tomography (PET) scanning has been used to detect herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) which was fused to this same Ad protein IX (pIX). Other conventional imaging systems for virotherapy have been designed for the detection of transgene expression of reporters such as sodium iodide symporter, somatostatin receptor type 2 (SSTR-2), and luciferase. The combination of a noninvasive imaging modality with a genetic adenoviral labeling system for detection of viral replication and progeny localization has begun to provide a powerful means of real-time monitoring of CRAd function in vivo.

Cross-References ▶ Adenovirus ▶ Cervical Cancers ▶ Gene Therapy ▶ Oncolytic Virus ▶ Ovarian Cancer ▶ Raf Kinase

References Dmitriev I, Kransuyky V, Miller CR et al (1998) An adenovirus vector with genetically modified fibres demonstrates expanded tropism via utilization of a coxsackie virus and adenovirus receptor-independent cell entry mechanism. J Virol 72:9706–9713 Heise C, Sampson-Johannes A, Williams A et al (1997) ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 3:639–645 Kruyt FA, Curiel DT (2002) Toward a new generation of conditionally replicating adenoviruses: pairing tumor selectivity with maximal oncolysis. Hum Gene Ther 13:485–495 Stojdl DF, Lichty B, Knowles S et al (2000) Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 6:821–825

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4826 Yu DC, Chen Y, Dilley J et al (2001) Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel. Cancer Res 61:517–525 Zhu ZB, Makhija SK, LU B et al (2004) Transport across a polarized monolayer of caco-2 cells by transferrin receptor-mediated adenovirus transcytosis. Virology 325:116–128

See Also (2012) G2/M checkpoint. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1481. doi:10.1007/978-3-642-16483-5_2466 (2012) Herpes simplex virus. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1684. doi:10.1007/978-3-642-16483-5_2688 (2012) Origin of replication. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2655. doi:10.1007/978-3-642-16483-5_4255 (2012) RGD. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3297. doi:10.1007/978-3-642-16483-5_5090

Virus Vector ▶ Viral Vector-Mediated Gene Transfer

Virus Vector

Characteristics 1a,25-dihydroxy-vitamin D3 (1,25D), or calcitriol, the biologically most active form of vitamin D, is essential to bone and mineral metabolism. 1,25D deficiency has been traditionally associated with bone diseases characterized by decreased bone mass and reduced mineralization, such as rickets, osteomalacia, and osteoporosis. In addition to its well-known actions on bone and calcium homeostasis, this secosteroid hormone exerts a number of effects on many different target organs and systems. 1,25D actions include regulation of cell cycle, cell proliferation, and differentiation in breast, colon, and prostate, among others. 1,25D binds to the nuclear vitamin D receptor (VDR), which acts as a transcription factor for the regulation of gene expression. In addition, 1,25D stimulates signal transduction cascades implicated in non-genomic responses of target cells such as modulation of the electrical state of the plasma membrane, secretory activities, cell survival, and establishment of the apoptotic phenotype. Mechanisms

Virus Vector-Mediated Gene Transfer Natural compounds and synthetic analogs of vita▶ Viral Vector-Mediated Gene Transfer

Vitamin D Laura P. Zanello Department of Biochemistry, University of California-Riverside, Riverside, CA, USA

Definition The biologically most active form of vitamin D, 1a,25-dihydroxy-vitamin D3, and many synthetic analogs exert antiproliferative actions on different cancer cell types, most typically breast, colon, and prostate. Clinical studies indicate a potentially important role for vitamin D in the prevention and treatment of cancer.

min D have proved to have significant effects on cell cycle, differentiation, survival, and programmed cell death. More specifically, 1,25D has been shown to act as a powerful antiproliferative agent on certain cancer cell types expressing the VDR, including breast, colon, and prostate, both in vitro and in vivo. Typically, cell cycle arrest promoted by physiological doses of 1,25D is linked to cell differentiation and therefore reduction in the probability of cells to become cancerous. Human trials on anticancer properties of calcitriol and synthetic analogs have increased, with ▶ prostate cancer being the most studied one. Calcitriol and numerous analogs have shown potential to become therapeutic agents for the treatment of certain cancer types in the future. However, the molecular mechanisms by which 1,25D inhibits cancer cell proliferation remain only partially understood. To date, more than 1,000 vitamin D analogs have been synthesized,

Vitamin D

and many of these analogs have shown to have significant antiproliferative effects. The earliest evidences on a relationship between vitamin D and cancer come from epidemiological studies conducted in the 1990s. An inverse relationship was discovered between sunlight exposure – which is lower as latitude increases – and ▶ prostate cancer cases in the United States over 45 years of data (1950–1994). A main source of vitamin D in our bodies is by means of production of vitamin D3 or cholecalciferol, a hormone precursor, in the skin by ▶ UV radiation. The second main source is vitamin D2 or ergocalciferol in the diet. However, these products are biologically inactive. They require two sequential hydroxylations, first in the liver (to produce 25-hydroxy-vitamin D3 or 25D) and then in the kidney (to render 1a,25-dihydroxyvitamin D3). The major circulating source of the secosteroid hormone is 25D; therefore, measurement of circulating concentrations is a good indicator of the overall vitamin D3 status of the individual. In addition, many tissues express 25-hydroxy-vitamin D3 1a-hydroxylase, the enzyme that transforms 25D into 1,25D, which indicates that local production of the biologically active form may play an important role in autocrine and paracrine functions. Early epidemiological studies suggested that elevated circulating levels of 25D main protect against cancer and that ingestion of vitamin D supplements may be helpful in the treatment of tumors. In addition to case studies, extensive in vitro work has demonstrated that high concentrations of 1,25D (109–107 M) inhibit the growth and induce differentiation of tumor cells, including malignant melanoma, myeloid leukemia, ▶ prostate cancer, ▶ breast cancer, glioma cancer, and colon cancer. Clinical trials and treatment of cancer patients with high doses of 1,25D or synthetic analogs have been limited due to the fact that, at these high vitamin D3 doses, there is a significant risk to develop hypercalcemia with fatal side effects. This has encouraged the pharmaceutical industry to develop new synthetic analogs with high potency for cell growth inhibition and low hypercalcemic effects (e.g., analog EB1089).

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The highly conformationally flexible 1,25D molecule offers a broad source of possibilities for the design of chemical compounds with high affinity for the VDR. These are ideally capable of potentiating the genomic regulation of the cell cycle while inducing only low calcium effects more likely to develop via non-genomic mechanisms. An alternative binding site for non-genomic analogs has been proposed on the basis of molecular modeling of the crystal structure of the VDR. This new hypothesis for the binding of ligands to the VDR brings an even higher potential for the design of analogs with more specific antiproliferative actions and reduced hypercalcemic side effects. Additional aspects that contribute to the tumorsuppressive activity of vitamin D compounds include the ability of 1,25D and analogs to reduce ▶ angiogenesis and ▶ metastasis. While use of 1,25D and analogs with low calcemic effects for the treatment of cancer offers high promise, combination therapy with other antitumorigenic drugs may signify even higher beneficial effects. In vitro studies have demonstrated, for example, that 1,25D in combination with the antiestrogen ▶ tamoxifen has greater efficacy in the inhibition of breast cancer cell lines. Different combinations of vitamin D3 and ▶ retinoids, ▶ cytokines, and a number of cytotoxic compounds including ▶ adriamycin, carboplatin, ▶ cisplatin have proven to act synergistically on the suppression of cancer cell growth. The anticancer effects of vitamin D compounds and analogs can be explained on the basis of different aspects of cancer cell biology. More specifically, 1,25D effects on tumor cells include: 1. Regulation of the cell cycle 2. Induction of ▶ apoptosis or programmed cell death 3. Modulation of the expression of ▶ oncogenes and ▶ tumor suppressor genes 4. Induction of cell differentiation Studies performed with 1,25D and analogs on different cancer cell lines in vitro have shown that cells grown in the presence of the steroid significantly reduce their growth rate mostly by

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Osteosarcoma cell Vitamin D, Fig. 1 1,25D reduction of human osteosarcoma cell proliferation occurs via sustained activation of mitogen-activated protein kinases JNK and MEK1/MEK2 downstream of non-genomic VDR signaling, leading to upregulation of a c-Jun/c-Fos (AP-1) transcription factor

complex, which in turn modulates p21(waf1) gene expression. Transient refers to 1,25D treatment for 15 min; sustained implicates a treatment with hormone for 3 days. RXR ▶ retinoic acid receptor, VDRE vitamin D responsive element, p indicates phosphorylation of the protein

blockade of the transition from the G0/G1 phase (differentiated, nondividing cells) to the S phase (DNA synthesis). In addition to promoting cell cycle arrest and inhibition of cell division, 1,25D and related compounds have proved to induce apoptosis in glioma, breast, leukemia, and colon cancer cells. It is not clear, however, how a cell follows its path to either cell cycle progression or apoptosis once it reaches early G1 phase. Vitamin D3 compounds appear to act at this point through different molecular mechanisms, depending on whether they induce cell death or stop cells from dividing. In human ▶ osteosarcoma, for example, 1,25D treatment reduces cell proliferation in vitro by approximately 25% after 3 days. The mechanisms involve sustained activation of ▶ MAP kinase/▶ AP-1/p21(waf1) pathways. Upregulation of p21 gene expression led to control over the cell cycle and subsequent cell cycle arrest (Fig. 1).

The modulation of the expression of certain oncogenes and tumor suppressor genes has been also shown to be affected by 1,25D treatment of cancer cell lines. Among genes that modulate cell growth and apoptosis, the ▶ bcl-2, c-myc, and retinoblastoma gene products are the most widely studied. The bcl-2 oncogene product protects against apoptosis, therefore inducing cell survival. In MCF-7 breast cancer cells, 1,25D and analogs KH1060 and EB1089 decreased bcl-2 expression, and cells progressed through apoptosis. The expression of the oncogene c-myc has also been shown to be reduced by 1,25D treatment of breast cancer cells and resulted in reduction of cell proliferation. Finally, induction of cell differentiation by vitamin D3 compounds has been widely described in combination with its antiproliferative effects. Typically, 1,25D inhibits cell proliferation and induces differentiation in the hematopoietic system, osteoblasts, and keratinocytes.

Vitamin D

Currently, the safe upper limits for vitamin D intake as supplements have been set in the USA by The Institute of Medicine to be 1,000 IU/day for children under 1 year of age and 2,000 IU/day for adults. Although rare, vitamin D intoxication may develop if intake of higher doses happens over significantly prolonged periods of time and lead to hypercalcemia, hyperphosphatemia, and hypercalciuria. A clinical trial conducted on patients with premalignant colon and rectal tissues showed that a treatment with 1,500 mg of calcium carbonate and 400 IU of vitamin D3 per day significantly decreased the levels of expression of markers of cell proliferation in polyp tissues, which is an indicator of tumor size reduction. In Europe, fortification of milk and other food products with vitamin D has been prohibited because of an increase in hypercalcemia cases in infants after World War II. However, in consideration that vitamin D deficiency is widely recognized as a cause of impaired bone formation in children and that there are clear beneficial effects of the hormone in cases of cancer and osteoporosis, many European countries currently fortify margarine and cereals with vitamin D.

Conclusions In vitro and in vivo studies on anticancer effects of 1,25D and synthetic analogs have indicated that the steroid has high potential for therapeutic applications in a wide range of cancers. Of special interest is the development of future combination therapies with other antitumor drugs with synergistic effects on cell growth. However, it is imperative that the precise molecular mechanisms by which 1,25D modulates cell proliferation and death be investigated. In addition, more clinical studies are needed in order to establish whether 1,25D and analogs can be used as an effective treatment of cancer.

Cross-References ▶ Adriamycin ▶ Angiogenesis ▶ AP-1

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▶ Apoptosis ▶ Bcl2 ▶ Breast Cancer ▶ Cisplatin ▶ Cytokine ▶ MAP Kinase ▶ Metastasis ▶ Oncogene ▶ Osteosarcoma ▶ Prostate Cancer ▶ Retinoic Acid ▶ Retinoids ▶ Tamoxifen ▶ Tumor Suppressor Genes ▶ UV Radiation

References Holt RR, Bresalier RS, Ma CK et al (2006) Calcium plus vitamin D alters preneoplastic features of colorectal adenomas and rectal mucosa. Cancer 106:287–296 Schwartz GG, Hanchette CL (2006) UV, latitude, and spatial trends in prostate cancer mortality: all sunlight is not the same (United States). Cancer Causes Control 17:1091–1101 Schwartz GG, Skinner HG (2007) Vitamin D status and cancer: new insights. Curr Opin Clin Nutr Metab Care 10:6–11 Van Leeuwen JPTM, Pols HAP (1997) Vitamin D: anticancer and differentiation. In: Feldman D, Glorieux FH, Pike JW (eds) Vitamin D. Academic, New York, pp 1089–1105 Wu W, Zhang X, Zanello LP (2007) 1a,25-dihydroxyvitamin D antiproliferative actions involve vitamin D receptor-mediated activation of MAPK pathways and AP-1/p21(waf1) upregulation in human osteosarcoma. Cancer Lett 254:75–86

See Also (2012) Autocrine. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 311. doi:10.1007/978-3-642-16483-5_468 (2012) Carboplatin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 641. doi:10.1007/978-3-642-16483-5_833 (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) C-Myc. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 886. doi:10.1007/978-3-642-16483-5_1232 (2012) Glioma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1557. doi:10.1007/978-3-642-16483-5_2423

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4830 (2012) Hypercalcemia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1780. doi:10.1007/978-3-642-16483-5_2903 (2012) Paracrine. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2783. doi:10.1007/978-3-642-16483-5_4380 (2012) Transcription Factor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3752. doi:10.1007/978-3-642-16483-5_5901 (2012) Vitamin D Receptor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3928. doi:10.1007/978-3-642-16483-5_6208

mutations are spread in all three exons. Missense mutations usually confer better prognosis and are more frequently detected in patients presenting with pheochromocytoma. About 20–30% of VHL type 2 patients develop a pheochromocytoma. The age at diagnosis is younger than in sporadic cases. They are frequently multiple, bilateral adrenal, and multifocal extra-adrenal. Rarely, they are malignant.

Cross-References

v-Ki-ras2 ▶ KRAS

Vomeroglandin (Mouse) ▶ Deleted in Malignant Brain Tumors 1

▶ Von Hippel-Lindau Tumor Suppressor Gene

See Also (2012) Hemangioblastoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1640. doi:10.1007/978-3-642-16483-5_2611 (2012) Missense mutation. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2330. doi:10.1007/978-3-642-16483-5_3761 (2012) VHL. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3907. doi:10.1007/978-3-642-16483-5_6183

Von Hippel-Lindau Disease Synonyms

Von Hippel-Lindau Tumor Suppressor Gene

VHL

Definition There are two major subgroups of the VHL disease: VHL type 1 mainly without and VHL type 2 mainly with pheochromocytoma presentation. The clinical features of the VHL syndrome include retinal (von Hippel) and cerebellar (Lindau) hemangioblastoma, as well as brain stem and spinal hemangioblastoma. They also include the presence of renal cysts and renal cell carcinoma, pancreatic cysts and islet cell tumors, endolymphatic sac tumors, and cysts and cystadenomas of the epididymis and broad ligament. The ▶ Von HippelLindau tumor suppressor gene lies on the short arm of chromosome 3 (3p25), with three exons coding for two isoforms of the protein. The

Jochen Decker1 and Hiltrud Brauch2 1 Hematology Oncology Medical School Clinic III, University of Mainz, Mainz, Germany 2 Breast Cancer Susceptibility and Pharmacogenomics, Dr. Margarete FischerBosch-Institute of Clinical Pharmacology, University of Tübingen, Stuttgart, Germany

Synonyms VHL tumor suppressor gene

Definition The von Hippel-Lindau ▶ tumor suppressor gene (VHL gene) is a cellular gene that is required for

Von Hippel-Lindau Tumor Suppressor Gene

normal development, differentiation, and cellular stress response (hypoxia, lack of glucose). VHL was discovered in families with the hereditary von Hippel-Lindau (VHL) syndrome by virtue of its two-hit mechanism of inactivation and identified in 1993 following a positional cloning strategy. The VHL gene may be subject to mutation either in the germline giving rise to VHL disease or in somatic renal epithelial cells giving rise to sporadic renal conventional (clear cell) carcinoma (CCRCC) functioning as a gatekeeper, which is a type of ▶ tumor suppressor gene that refers to a subgroup of gene products, whose major cellular function is in the control of cell division, death, or life span. Inactivation of gatekeeper genes favors, through a variety of mechanisms, the unrestrained growth typical of cancer cells. Multiple gatekeeper mutations may be required for the neoplastic transformation of a cell. Gatekeeper gene products often participate in the control of cell cycle (e.g., the Rb gene product [▶ retinoblastoma protein, cellular biochemistry]) or in the signals that regulate proliferation (e.g., the APC gene product). It is possible that large proteins with several functional domains (pleiotropic genes), such as BRCA1 and BRCA2 proteins originally described as gatekeepers, may also have caretaker functions. VHL mutation patterns suggest two functional domains within the protein pVHL. Current knowledge indicates similarities to the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complex that targets proteins for degradation. Therefore, pVHL may function as a molecular adaptor in a similar proteolytic pathway. Today’s best understood function of pVHL is its role in the VHL/HIF (hypoxia-inducible factor 1-a) pathway.

Characteristics Molecular Features • Gene located at 3p25.3; single copy locus, NCBI GenBank; range, bp 10.158.319–10.168.762, as published in Nature 431(7011):931–945 (2004) (Fig. 1). • 639 Nucleotides in three exons (originally reported sequence contained 852 nucleotides with 213 untranslated base pairs at the 50 end).

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• Exon 1 is a CpG island (G + C content is 70%, CpG/GpC > 1). • TATA-less and CCAAT-less promoter. • Two transcription initiation codons (amino acid 1 and amino acid 54). • Follows a two-hit mechanism of inactivation characteristic for tumor suppressor genes. • Evolutionarily highly conserved. • No homologies known. • The full-length protein pVHL contains 213 amino acids. • Known isoforms result from tissue-specific and developmentally selective alternative splicing (skipping of exon 2). • Differentially phosphorylated at serin 68 by casein kinase 2 or glycogen synthase kinase 3. • Expressed in a variety of adult and fetal tissues including those of VHL target organs. • There are two protein-binding domains that allow pVHL to function as an adaptor molecule in a proteolytic pathway. • The gene may be subject to mutations at almost any nucleotide of the 470bp COOH terminal sequence. • Phenotypic variation may result from confounding effects of modifier genes. • No imprinting reported. • Posttranslational negative regulation by E2-EPF UCP (E2-EPF ubiquitin carrier protein).

Structure of the von Hippel-Lindau gene (VHL), and protein, indicating the functional domains: The pattern of germline mutations published so far reflects the functional importance of the binding sites to the target as well as to the multimeric protein complex.

Role in Diseases: Clinical, Molecular, and Cellular Characteristics Von Hippel-Lindau (VHL) disease (OMIM 193300) is an inherited tumor susceptibility syndrome predisposing gene carriers to a variety of benign and malignant tumors. VHL segregates in

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Von Hippel-Lindau Tumor Suppressor Gene, Fig. 1 Structure, function, and germline mutations of the VHL gene

affected families as an autosomal dominant inherited trait. Phenotypic expression is highly variable.

Clinical and Molecular Features Lesions Associated with von Hippel-Lindau Disease (Fig. 2). VHL patients may present with a variety of tumors affecting eye, central nervous system, inner ear, adrenal gland, kidney, pancreas, and epididymis. Most frequent tumors include retinal angiomas and hemangioblastoma of the cerebellum and of the spinal cord which are usually benign. Other benign lesions include ▶ pheochromocytoma and renal and pancreatic cysts. Renal clear cell carcinomas are malignant (Table 1):

• Tumors and cysts are frequently bilateral and/or multiple in origin. • All ethnic groups are involved; there is no sex bias. • Birth incidence is estimated from 1/39,000 (Germany) to 1/53,000 (East Anglia). • Prevalence is 1/85,000–1/31,000. • Incidence of de novo mutations is about 5% (up to 20%). • Mean age at diagnosis is 26 years. • Penetrance by age of 65 years is more than 90%. • Most severe complications are hemangioblastomas due to unrestricted growth in the confined space of skull or vertebral canal and CCRCC due to metastasis. • Major cause of death is hemangioblastomas.

Von Hippel-Lindau Tumor Suppressor Gene Von Hippel-Lindau Tumor Suppressor Gene, Fig. 2 Lesions associated with von Hippel-Lindau disease

Retinal angiomas

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Cerebellar and spinal hemangioblastomas Endolymphatic sac tumors

Pheochromocytomas

Pancreatic cysts islet cell tumors

Conventional clear cell renal cell carcinomas renal cysts Epididymal cystadenomas

Von Hippel-Lindau Tumor Suppressor Gene, Table 1 Lesions associated with von Hippel-Lindau disease Affected organs Eyes Central nervous system Adrenal glands Kidneys

Pancreas

Inner ear Epididymis Broad ligament Liver

Clinical symptoms Angiomatosis retinae Cerebellar hemangioblastomas Spinal hemangioblastomas Pheochromocytomas Renal cysts Conventional (clear cell) renal cell carcinomas (CCRCC) Pancreatic cysts Serous cystadenomas Islet cell tumors usually asymptomatic Endolymphatic sac tumors Cystadenomas Benign adnexal papillary tumors Cysts

Frequency 50–57% 55–59% 13–14% 7–19% 76% 24–52%

22% Occasionally Infrequent