Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy: Volume 5 [5, 1 ed.] 012817661X, 9780128176610

Table of contents :
Cover
BREAKING TOLERANCE
TO PANCREATIC
CANCER
UNRESPONSIVENESS
TO CHEMOTHERAPY
Copyright
Dedication
Contributors
About the Editor
About the Series Editor
Aims and Scope for Series ``Cancer Sensitizing Agents for Chemotherapy´´
Preface
1
Overview of Pancreatic Cancer Biology
Conflict of Interest
Introduction
Pathogenesis and Molecular Biology
Three Biological Stages of Cancer Development
Initiation
Clonal Expansion
Oncogenesis
KRAS
Raf/Mitogen-Activated Protein Kinase (MAPK) Cascade
Phosphoinositide 3-Kinase/ATK Cascade
Nuclear Factor Kappa B Signaling Cascade
Tumor Suppressor Genes
CDKN2A
TP53
SMAD4
Introduction to Local and Distant Microenvironments
Familial Pancreatic Cancers
Conclusions
References
Further Reading
2
Chemoresistance in Pancreatic Cancer: Emphasis on Age and Gender
Conflict of Interest
Introduction
Pancreatic Cancer Incidences and Prognosis: Association of Age and Gender
Chemoresistance: Association With Age and Gender
Mechanisms Associated With Chemoresistance
Factors Modulated in Various Age Groups
Conclusion
Acknowledgments
References
3
EMT Contributes to Chemoresistance in Pancreatic Cancer
Conflict of Interest
Introduction
Pancreatic Cancer Metastasis
EMT Regulation in Pancreatic Cancers
ZEB1
SNAIL
Twist
PRRX1 (Paired Related Homeobox 1)
Signaling Pathways Involved in the Regulation of EMT
TGF-β
Wnt/β-catenin
Notch
TNF-α
EMT in Chemoresistance in Pancreatic Cancer
5-Flurouracil Chemoresistance
Gemcitabine Chemoresistance
Role of EMT in Gemcitabine Resistance
Promising Ways to Improving Gemcitabine Efficacy
Conclusions and Future Perspectives
Acknowledgments
References
4
Pancreatic Cancer Resistance to Gemcitabine
Conflict of Interest
Introduction
Gemcitabine Structure and Mechanism
Gemcitabine Sensitivity
Gemcitabine and Chemoresistance
Role of Gemcitabine in Targeting Signaling Pathways
Conclusion
Acknowledgment
References
5
Pancreatic Cancer and Possible Therapeutic Options
Conflict of Interest
Introduction
Reasons Behind the Above Statistics?
Brief Outline of Treatment
Tumor-Node-Metastasis Staging and Clinical Classification
Modalities for Evaluating TNM Staging
Clinical Classification
Treatment Options for Pancreatic Adenocarcinoma
Localized Surgically Resectable Stage I and II Disease
Adjuvant Therapy
Gemcitabine and 5-Fluorouracil
Gemcitabine and S-1
Gemcitabine and Capecitabine
Adjuvant Chemoradiation
Gemcitabine Plus Nab-Paclitaxel
Neoadjuvant Therapy
Adjuvant Therapy in Borderline Removable Pancreatic Carcinoma
Evidence Supporting Neoadjuvant Therapy
FOLFIRINOX as Neoadjuvant Therapy
NAC-GSL (Neoadjuvant Chemotherapy-Gemcitabine, S-1, LV)
Metastatic Pancreatic Cancer: Treatment Options
Early Period
First-Line Therapies
FOLFIRINOX
PRODIGE-4/ACCORD-11 Trial
Outcomes of PRODIGE-4/ACCORD-11 Trial
Precautionary and Toxicity Reducing Strategies
Gemcitabine Plus Nab-Paclitaxel
The MPACT Trial
When to Use FOLFIRINOX Versus Gemcitabine/Nab-paclitaxel?
Clinical Parameters Indicating the Preferred Regimen
Regimen Selection for Poor Performance Status Patients
Second-Line Options
5-FU Plus Oxaliplatin-Based Combination
Nanoliposomal Irinotecan and 5-FU/Leucovorin Combination
The NAPOLI-1 Trial
Newer Treatment for Pancreatic Carcinoma
KRAS Inhibitors
RAF/MEK/ERK
HER-2 Inhibitors
Epidermal Growth Factor Receptor Inhibitors
JAK/STAT Inhibitors
Insulin-Like Growth Factor 1 Receptor Inhibitor
Vascular Endothelial Growth Factor Receptor Inhibitors
Tumor Stroma/Desmoplasia
Transforming Growth Factor-β
Platelet-Derived Growth Factor
Hyaluronic Acid
Matrix Metalloproteinase Inhibitors
Hedgehog Inhibitors
Conjugated Drugs
Nab-Paclitaxel
PEP02 (MM-398)
Endo Tag-1
Trastuzumab Emtansine
Tumor Hypoxia
Conclusions and Future Perspectives
References
6
Curcumin and Genistein Enhance the Sensitivity of Pancreatic Cancer to Chemotherapy
Conflict of Interest
Introduction
Chemotherapy for Pancreatic Cancer
Tumor Microenvironment Developing Chemoresistance
Curcumin Effecting as a chemo-sensitizer
Genistein Effecting as a Chemo Sensitizer
Signaling Cascades
EGFR
VEGF
STAT Pathway
Mitochondrial Pathway
MiRNAs
Notch Signaling Pathway
Epigenome Changes
Bioavailability
Conclusion
Acknowledgment
References
7
Terpenoids as Potential Targeted Therapeutics of Pancreatic Cancer: Current Advances and Future Directions
Conflict of Interest
Introduction
Pancreatic Cancer
Risk Factor of Pancreatic Cancer
Terpenoids
Molecular Targets of Terpenoids in Pancreatic Cancer
Terpenoids as NF-κB Signaling Inhibitors
Terpenoids in the Regulation of Caspase Activity
Terpenoids With Targeting DNA Damage
Terpenoids Targeting Apoptotic Proteins
Terpenoids With Differential Targets in Pancreatic Cancer
Conclusion
Acknowledgment
References
Further Reading
8
Small Molecules and Pancreatic Cancer Trials and Troubles
Conflict of Interest
Pancreatic Cancer: Causes and Treatments
Causes and Prevention
Therapeutic Treatments
Pancreatic Cancer Resistance
Causes of Treatment Resistance
Combatting the Various Chemotherapy Resistance Pathways
Pancreatic Cancer and Small Molecules
General Overview of Small Molecules
Heat Shock Proteins and HSP90 in Pancreatic Cancer
HSP90 Inhibitors in Clinical Trials
Acknowledgment
References
9
Targeting the Epigenome as a Therapeutic Strategy for Pancreatic Tumors: DNA and Histone Modifying Enzymes
Conflict of Interest
Introduction
DNA Methylation and Demethylation
Histone Deacetylation and Acetylation
Inactivating Histone Methylations
Activating Histone Methylations
Conclusions and Future Perspectives
Acknowledgments
References
10
Are Nanocarriers Effective for the Diagnosis and Treatment of Pancreatic Cancer?
Conflict of Interest
Current Status About Pancreatic Cancer
Commonly Used Therapeutic Agents
Chemotherapy
Radiation Therapy
Nanoscience and Nanotechnology
Nanocarriers
Polymeric Nanoparticle
Polymeric Micelle
Liposome
Nanogel
Mesoporous Inorganic Nanoparticle
Metal Oxide Nanoparticle
Inorganic Carbon Nanotubes
Nanoparticles Used in Pancreatic Cancer
References
11
Molecular Markers for Treatment Response and Toxicity of Gemcitabine
Conflict of Interest
Introduction
Gemcitabine Transport and Metabolism of Action
Dosing
Toxicities with Gemitabine
Candidate Genes Affecting Gemcitabine Therapy
Neucleoside Transporter Genes
SLC28 Family Gene Polymorphisms
SLC29 Family
Genes of Metabolizing Enzymes
Cytidine Deaminase
Deoxycytidine Kinase
Deoxycytidylate Deaminase
Cytidine Monophosphate Kinase 1
Thymidylate Synthase
Genes of Drug Targets
Ribonucleotide Reductases 1
Conclusions
References
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
R
S
T
U
V
W
X
Z
Back Cover

Citation preview

Cancer Sensitizing Agents for Chemotherapy

BREAKING TOLERANCE TO PANCREATIC CANCER UNRESPONSIVENESS TO CHEMOTHERAPY VOLUME 5

Cancer Sensitizing Agents for Chemotherapy

BREAKING TOLERANCE TO PANCREATIC CANCER UNRESPONSIVENESS TO CHEMOTHERAPY VOLUME 5 Edited by

GANJI PURNACHANDRA NAGARAJU Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom # 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISSN: 2468-3183 ISBN: 978-0-12-817661-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisition Editor: Rafael Teixeira Editorial Project Manager: Sandra Harron Production Project Manager: Punithavathy Govindaradjane Cover Designer: Greg Harris Typeset by SPi Global, India

Dedication This book is dedicated to my family, teachers, students, and friends.

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Contributors Sarfraz Ahmad Florida State University, Tallahassee; UCF Colleges of Medicine, Orlando; Gynecologic Oncology, Florida Hospital, Orlando, FL, United States

Cancer Institute, Emory University, Atlanta, GA, United States Shailender Guganavath Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India

Afroz Alam Department of Bioscience and Biotechnology, Banasthali University, Banasthali, India Saeed Ali Department of Internal Medicine, Florida Hospital, Orlando, FL, United States

Myrna Hurtado University of North Texas Health Science Center, Fort Worth, TX, United States

Sheik Aliya Department of Biotechnology, Jawaharlal Nehru Technical University, Hyderabad, India

Ishtiaq Hussain Department of Internal Medicine, Khyber Teaching Hospital, Peshawar, Pakistan

Riyaz Basha University of North Texas Health Science Center, Fort Worth, TX, United States

Akriti Gupta Jain Department of Internal Medicine, Florida Hospital, Orlando, FL, United States

Shipra Bethi Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States

Sundas Jehanzeb Department of Internal Medicine, Khyber Teaching Hospital, Peshawar, Pakistan

Suresh Chava Laboratory of Cell Signaling, Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India

Deepak K.G.K. Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India Prameswari Kasa Dr. LV Prasad Diagnostics and Research Laboratory, Hyderabad, India

Begum Dariya Department of Bioscience and Biotechnology, Banasthali University, Banasthali, India

Neelam Khetpal Department of Internal Medicine, Florida Hospital, Orlando, FL, United States

Batoul Farran Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States

Ranjeet Kumar Department of Internal Medicine, Florida Hospital, Orlando, FL, United States

Murali Mohan Gavara Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India

Seema Kumari Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India

Meher B. Gayatri Laboratory of Cell Signaling, Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India

Saikrishna L. Department of Zoology, Visvodaya Government Degree College, Venkatagiri, India

Sneha Govardhanagiri Department of Hematology and Medical Oncology, Winship

Bhaskar L.V.K.S. Sickle Chhattisgarh, Raipur, India

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Cell

Institute

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CONTRIBUTORS

Umair Majeed Internal Medicine Residency, Florida Hospital, Orlando, FL, United States Rama Rao Malla Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India Sathish Kumar Mungamuri Institute of Basic Sciences and Translational Research, Asian Healthcare Foundation, Asian Institute of Gastroenterology, Hyderabad, India Ganji Purnachandra Nagaraju Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States Maya Nair University of North Texas Health Science Center, Fort Worth, TX, United States Sushma P.S. Dr. NTR University of Health Sciences, Vijayawada, India Balney Rajitha Department of Bioscience and Biotechnology, Banasthali University, Banasthali, India Ganji Seeta Rama Raju Department of Energy and Materials Engineering, Dongguk University, Seoul, Republic of Korea

Mamoon Ur Rashid Department of Internal Medicine, Florida Hospital, Orlando, FL, United States Aramati B.M. Reddy Laboratory of Cell Signaling, Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India Prasuja Rokkam Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India Tabinda Saleem Islamic International Medical College, Riphah International University, Rawalpindi, Pakistan Sunil Shah University of North Texas Health Science Center, Fort Worth, TX, United States Waqas Ullah Abington Memorial Hospital, Abington, PA, United States Hammad Zafar Department of Internal Medicine, Florida Hospital, Orlando, FL, United States

About the Editor

Dr. Ganji Purnachandra Nagaraju, PhD is a faculty member in the Department of Hematology and Medical Oncology at Emory University School of Medicine. Dr. Nagaraju obtained his MSc and his PhD, both in biotechnology, from Sri Venkateswara University in Tirupati, Andhra Pradesh, India. He also

obtained a DSc degree in biotechnology from Berhampur University, India. Dr. Nagaraju’s research focuses on translational projects related to gastrointestinal malignancies. He has published over 65 research publications in highly reputed international journals and has presented more than 50 abstracts at various national and international conferences. He serves as an editorial board member of several internationally recognized academic journals. Dr. Nagaraju is an associate member of the Discovery and Developmental Therapeutics research program at the Winship Cancer Institute. Dr. Nagaraju has received several international awards. He also holds memberships with the Association of Scientists of Indian Origin in America (ASIOA), the Society for Integrative and Comparative Biology (SICB), the Science Advisory Board, and the American Association for Cancer Research (AACR).

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About the Series Editor factors in cancer cells, cell-signaling pathways mediated by therapeutic anticancer antibodies, and characterization of a dysregulated NF-κB/ Snail/ YY1/RKIP/PTEN loop in many cancers that regulate cell survival, proliferation, invasion, metastasis, and resistance. He has also investigated the role of nitric oxide in cancer and its potential antitumor activity. Many of the above studies are centered on the clinical challenging features of cancer patients’ failure to respond to both conventional and targeted therapies. The development and activity of various chemosensitizing agents and their modes of action to reverse chemo- and immunoresistance are highlighted in many refereed publications. Acknowledgments: The Series Editor, Benjamin Bonavida, wishes to acknowledge the significant effort and patience in the significant editing of this volume, both from the language and formatting changes. The assistance of Ms. Jazelle Bautista was pivotal in the final publication of this volume.

Dr. Benjamin Bonavida, PhD (Series Editor) is currently Distinguished Research Professor at the University of California, Los Angeles (UCLA). His research career, thus far, has focused on basic immunochemistry and cancer immunobiology. His research investigations have ranged from the mechanisms of cell-mediated killing and sensitization of resistant tumor cells to chemo-/immunotherapies, characterization of resistant

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Aims and Scope for Series “Cancer Sensitizing Agents for Chemotherapy” Current cancer management strategies fail to adequately treat malignancies with chemotherapy, with multivariable doserestrictive factors such as systemic toxicity and multidrug resistance, hence limiting therapeutic benefits, quality of life, and complete long-term remission rates. The resistance of cancer cells to anticancer drugs is one of the major reasons for the failure of traditional cancer treatments. Cellular components and dysregulation of signaling pathways contribute to drug resistance. If modulated, such perturbations may restore the drug response and its efficacy. The recent understanding of the molecular mechanisms and targets that are implicated for cancer chemoresistance have paved the way to develop a large battery of small molecules (sensitizing agents) that can target resistant factors and reduce the threshold of resistance, thus allowing their combination with chemotherapeutic drugs to be effective and to reverse the chemoresistance. A large variety of chemotherapy-sensitizing agents has been developed, and several have been shown to be effective in experimental models and in cancer patients.

The main objective of the proposed Series “Cancer Sensitizing Agents for Chemotherapy” is to publish individualized and focused volumes whereby each volume is edited by an invited expert editor(s). Each volume will dwell on specific chemosensitizing agents with similar targeting activities. The combination treatment of the sensitizing agent with chemotherapy may result in a synergistic/additive activity and the reversal of tumor cells resistance to drugs. The editor(s) will compile nonoverlapping review chapters on reported findings in various cancers, both experimentally and clinically, with particular emphases on underlying biochemical, genetic, and molecular mechanisms of the sensitizing agent and the combination treatment. The scope of the series is to provide scientists and clinicians with updated basic and clinical information that will be valuable in their quest to investigate, develop, and apply novel combination therapies to reverse drug resistance and, thereby, prolonging survival and even resulting in a cure in cancer patients.

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Dr. Benjamin Bonavida, PhD (Series Editor)

Preface Pancreatic cancer (PC) is the fourth most lethal cancer in the world and projected to become more adverse in the near future. Attempts at improving the outcome of PC by incorporating cytotoxic agents such as chemotherapeutic drugs have been disappointing. These results indicate that the main challenge remains in the primary resistance of PC cells to cytotoxic chemotherapy in the majority of patients. Therefore, improvement in the outcomes of these malignancies is dependent on the introduction of agents that can modulate the intrinsic and acquired mechanisms of resistance. In PC patients with innate chemoresistance, treatment is ineffective following the initial treatment. In comparison, acquired resistance advances only after PC cells have been exposed to chemotherapeutic drugs. In fact, PC cells primarily show chemosensitivity, but constant exposure eventually leads to PC cell resistance, relapse, and metastasis. The mode of resistance to chemotherapy in PC includes an extensive fibrotic stroma comprising the extracellular matrix (ECM), cancer stem cells (CSCs), cancer-associated fibroblasts (CAFs), and pancreatic stellate cells (PSCs). Furthermore, the activation of transcription factors (NF-κB, HIF-1α,

STAT-3, etc.) and growth factors also contributes to chemotherapy resistance pathways. All of these factors control the expression of several oncogenes involved in PC growth, metastasis, and inflammation. Therefore, sensitivity of PC cells to chemotherapy is an important phase toward effective therapy of PC patients. Gaining increased knowledge about these PC cell types and innate and acquired mechanisms could possibly help develop strategies and novel treatments for PC, which could finally accomplish better treatment results for PC patients. In this book, we will try to compile this information thoroughly and accurately. This book contains 11 chapters that cover different mechanisms of chemoresistance in PC and different approaches to sensitize refractory tumor cells to chemotherapy. The chapters will thus explore the clinical significance of chemosensitizing drugs, which are important for combating this malignant disease. It is my pleasure to present this book to the scientific community for a better understanding of chemoresistance and strategies to overcome it. I hope this will inspire new thoughts and novel research ideas for the benefit of PC patients. Dr. Ganji Purnachandra Nagaraju, PhD

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C H A P T E R

1 Overview of Pancreatic Cancer Biology Akriti Gupta Jain*, Tabinda Saleem†, Ranjeet Kumar*, Neelam Khetpal*, Hammad Zafar*, Mamoon Ur Rashid*, Saeed Ali*, Umair Majeed‡, Sarfraz Ahmad§,¶,k *

Department of Internal Medicine, Florida Hospital, Orlando, FL, United States †Islamic International Medical College, Riphah International University, Rawalpindi, Pakistan ‡Internal Medicine Residency, Florida Hospital, Orlando, FL, United States §Florida State University, Tallahassee, FL, United States ¶UCF Colleges of Medicine, Orlando, FL, United States k Gynecologic Oncology, Florida Hospital, Orlando, FL, United States

Abstract Pancreatic malignancy has been ranked as the fourth major cause of cancer-associated mortality in the United States, and the majority of its pathogenesis can be attributed to three stages of biological development. The inherited and acquired mutations in proto-oncogenes and tumor suppressor genes including KRAS, CDKN2A, Tp53, and SMAD4 lead to the initiation of pancreatic carcinoma. These mutations lead to the formation of a premalignant lesion in the ductal epithelium known as the pancreatic intraepithelial neoplasia (PanIN). The accumulation of incessant mutations leads to further clonal expansion and tumor spread into the local and distant microenvironment owing to its unique features of stromal proliferation and metabolic adaptation to obtain nutrients in a hypoxic environment. Moreover, the presence of a distinctive stem cell compartment is responsible for the rapid spread of the tumor and chemoresistance. An understanding of these molecular alterations along with successive changes in the signal transduction pathways can help in superior comprehension of the underlying molecular biology of pancreatic neoplasms.

Abbreviations BRCA CDKN EGFR FAMMM GTP KRAS PanIN

breast cancer susceptibility gene cyclin-dependent kinase inhibitor epidermal growth factor receptor familial atypical multiple mole-melanoma guanosine-50 -triphosphate Kirsten rat sarcoma virus oncogene pancreatic intraepithelial neoplasia

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00001-9

1

# 2019 Elsevier Inc. All rights reserved.

2 PRSS1 SPINK1 TGF-β TP TSG

1. OVERVIEW OF PANCREATIC CANCER BIOLOGY

protease serine 1 serine peptidase inhibitor kazal type 1 transforming growth factor beta tumor protein tumor suppressor gene

Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION Cancer is the foremost cause of mortality in the United States, and after lung, colon, and breast cancer, malignancies of the pancreas are ranked as the fourth cardinal cause of cancerassociated death (Fig. 1). Although it is relatively less common than the other three malignancies, the mortality rate for pancreatic cancer is highest among all. Around 53,670 new cases of malignancies of the pancreas and nearly 43,090 deaths were reported in the United States in the year 2017 [1–3]. Only 5% of patients diagnosed with pancreatic cancer are able to survive

Heart and circulatory disorders

Undetermined events Mental health disorders Transport accidents Suicide

Cancer Musculoskeletal disorders

Respiratory disorders

Diabetic disorders

Nontransport accidents

Infectious disorders Kidney disorders

Digestive system disorder

Nervous system disorders

FIG. 1 Leading causes of deaths in viewpoint.

PATHOGENESIS AND MOLECULAR BIOLOGY

3

for 5 years [4]. Much of this mortality is attributed to the inadequacy and the inefficiency of the available treatment options. Another reason for this morbid prognosis is delayed diagnosis with more than 90% cases being detected at later stages (III or IV) of the disease, with distant metastasis and/or local invasion [5]. Usually, pancreatic cancer presents with nonspecific symptoms like abdominal pain, diarrhea, jaundice, queasiness, and back pain but is often overlooked as they are not very distinctive to the ailment. Another way it can present is with the emergence of diabetes mellitus or exacerbation of prediagnosed diabetes [5]. Various studies have shown an association of obesity, smoking, and type II diabetes with the evolution of this cancer along with a complex interplay of genetic predisposition and environmental factors. Another well-known premorbid condition is the presence of another type of cancer (colon, biliary, and uterine malignancies) and subsequent development of a secondary cancer [6]. Understanding of the biology underlying this deadly cancer will help us identify markers for early detection and targets for better and directed treatment. Fig. 1 shows the causes of death in viewpoint, the chronic disease pandemic [7]. The statistics are taken from the 2017 NHS UK Database.

PATHOGENESIS AND MOLECULAR BIOLOGY Like all cancers, pancreatic malignancy is primarily a disease emanating from genes, and the majority of its pathogenesis can be attributed to the mutations in the cancer-associated genes; these mutations can be both acquired or inherited. Pancreatic cancer is a complex heterogeneous disease that is composed of multiple compartments and desmoplastic stroma. This multiplex stroma is built of collagen type I that is secreted by the activated fibroblasts and is arranged into fibrillar elements [8]. Among the mature and differentiated cell compartments, a small portion of the stem cell compartment is present. This relatively small compartment is believed to be involved in metastasis and is believed to be the key factor involved in treatment resistance to both chemotherapy and radiation therapy [9].

Three Biological Stages of Cancer Development In order to comprehend the biological development of pancreatic malignancy, it is best to characterize the stages into three main categories [10] (Fig. 2). The first is being the initiation of tumor by the acquisition of acquired or inherited mutations in the sentinel cell. The second step involves the clonal expansion and proliferation of that mutated cell into a multicellular FIG. 2 Three biological stages of cancer development.

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1. OVERVIEW OF PANCREATIC CANCER BIOLOGY

tumor, and then, the last step is being the establishment of these cancerous cells into the adjacent and remote microenvironment resulting in the clinical manifestation of pancreatic carcinoma [11].

Initiation In a pattern analogous to other neoplasms, as the genetic switches accrue in the pancreatic epithelium, it leads to an abnormal proliferation of the ductal epithelium and results in the formation of precursor lesions. These lesions that are initially nonneoplastic progress to noninvasive and then to invasive malignancy invading the small ducts and ductules of the pancreas. These premalignant lesions are known as “pancreatic intraepithelial neoplasias” (PanINs), and they are the best-characterized histological precursors of pancreatic adenocarcinoma, which is the most common type of pancreatic neoplasia [12]. These precursor lesions possess many genetic mutations that are similar to the infiltrating carcinoma, which along with telomere shortening predispose them to gather further chromosomal alterations and transform from a nonneoplastic to a neoplastic lesion. Other less common pancreatic antecedents include intrapancreatic mucinous and mucinous cystic neoplasias [13]. Around 50% of the pancreatic neoplasms are caused by intrinsic factors, but what causes these molecular changes is unknown. However, as mentioned above, some known extrinsic or environmental factors have been found to have a significant effect on the development of pancreatic neoplasms, and mainly, these include either the state of chronic inflammation or hyperinsulinism [14]. The strongest environmental influence is that of smoking. It increases the likelihood of developing pancreatic neoplasms by twofold through the production of mutagens that damage the DNA, which then act as an inciting event and lead to clonal expansion and accumulation of additional mutations in the pancreatic cells [15]. Likewise, obesity increases the risk by acting as a chronic proinflammatory state [16]. Type II diabetes, which is known as one of the major risk factors, is thought to substantiate the likelihood through hyperinsulinemia and hyperglycemia. Dysregulated blood glucose levels support the survival and proliferation of the mutant cells as these strains have divergent dependence on metabolism of glucose [17].

Clonal Expansion A tumor originates from the growth of clonal cells that have incurred mutations in four principal classes of regulator genes. These include genes that drive the process of cell maturation (like the proto-oncogenes and tumor suppressor genes (TSG)) and those that regulate the mechanism of apoptosis and DNA repair. The occurrence of the mutated genes does not assure the growth of the pancreatic neoplasm, as multiple mutations involving multiple genes must first become fixed and activate or suppress the downstream cell signaling pathways, which then lead to the phenotypic expression of the malignancy. Up to 33% of the pancreas at the time of autopsy contains PanINs indicating that not all precursor lesions result in a full-blown infiltrating disease [18]. The progression of the neoplasm as it evolves from minimal dysplastic changes (PanIN-1A and PanIN-1B) to severe epithelial dysplasia (PanIN-2 and PanIN-3) and, eventually, to invasive neoplastic disease corresponds to the sequential accretion of mutations in the key tumor

ONCOGENESIS

5

genes. These include the activation of the KRAS oncogene followed by the inactivation of the tumor suppressor genes CDKN2A and eventually TP53 and DPC4/SMAD4 [19]. Collectively, these genetic alterations aid the growth and survival of tumor cells over normal cells. Whether this gradual accumulation of somatic alterations occurs rapidly over a limited number of cycles or in a stepwise or linear advancement is unknown. Regardless of the way these alterations accumulate, these four molecular alterations KRAS, CDKN2A, TP53, and SMAD4 form the basis of the molecular biology of pancreatic neoplasm and form the cornerstone in understanding its pathogenesis.

ONCOGENESIS KRAS KRAS mutation is present in almost 95% of the pancreatic neoplasms, and it is considered as the most commonly mutated oncogene. Commonly, it is altered by a somatic activating mutation on chromosome 12p [20]. KRAS encodes for the RAS family of proteins that attaches to GTP and is involved in many key cellular operations, encompassing multiplication, cell survival, invasion, and cytoskeletal remodeling through the activation of several intracellular signal transduction pathways culminating in FOS and JUN transcription factors. KRAS also possesses an innate GTPase activity, which through GTP hydroxylation inactivates KRAS after a short transient activation. This vital process regulates the rate of cellular growth under normal circumstances. In 80%–90% of cases, point mutations chiefly transpire at codon 12 that codes for the protein’s intrinsic GTPase activity, thereby rendering the KRAS gene to become secured in its constitutively operational (GTP-latched) form. Less frequent mutations occur at codons 13 and 61 [21]. The downstream consequences of this mutation activate the signaling cascades that work as the principal effectors of KRAS and flood the nucleus with signals for clonal proliferation [22]. These are detailed below. Raf/Mitogen-Activated Protein Kinase (MAPK) Cascade Upon activation, RAS triggers the serine/threonine kinases, which are the member of the Raf family of kinases (Fig. 3). Upon activation, these kinases through a series of phosphorylation incidents activate MEK, which in turn turns on its downstream effector kinase known as the extracellular signal-regulated kinase (ERK). ERK then phosphorylates its substrate and results in cellular prosperity, multiplication, and differentiation. Both KRAS mutations and the growth factor receptor-ligand binding (like of EGFR) lead to the initiation of Raf/MEK signaling in pancreatic neoplasm and are responsible for its accelerated growth, survival, and invasion of the surrounding microenvironment. Phosphoinositide 3-Kinase/ATK Cascade Activated KRAS and other growth factor-related tyrosine kinases like the ATK pathways initiate the PI3K-directing cascade. Activation or overexpression of this pathway in pancreatic neoplasms results in increased cell proliferation, survival, and chemoresistance.

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1. OVERVIEW OF PANCREATIC CANCER BIOLOGY

Growth factor Receptor

Cell membrane

KRAS

RAF

ERK

MEK

Cell growth and division

Nucleus

FIG. 3 KRAS pathway.

Nuclear Factor Kappa B Signaling Cascade Activated nuclear factor-kappa B (NF-κB) acts as a transcription element that regulates genes that promote cell survival, infiltration, chemoresistance, and blood vessel formation and is essentially active in almost all pancreatic neoplasms as a result of the expressed RAS mutation.

TUMOR SUPPRESSOR GENES CDKN2A In 95% pancreatic neoplasms or more, the CDKN2A gene is nonoperational, and hence, it is the most commonly inactivated TSG in this type of neoplasm. Dissimilar to KRAS mutations, CDKN2A deactivation transpires because of a diverse apparatus. Forty percent of the neoplasms have both alleles deleted in a homozygous fashion, while 40% have an acquired

TUMOR SUPPRESSOR GENES

7

mutation of one of the alleles coupled with heterozygosity of the other allele. This reflects the Knudsonian mechanisms of gene inactivation. In the residual 10%–15% of neoplasms, CDKN2A gene is inactivated via promoter hypermethylation [23]. P16INK4A and p19ARF are encoded by CDKN2A by a shared locus on chromosome 9p, but these two proteins are not an isoform of each other as the CDKN2A gene encodes two different mRNAs for each protein [24]. The soaring incidence of the inactivation of this locus in pancreatic malignancy raises the question regarding which protein contributes more to the tumor progression [25]. Evidence-based studies in mice and human suggest that p16INK4A is the major culprit since mutations in exon 1 that encodes p16INK4A have been reported in melanoma and pancreatic neoplasms and these mutations do not affect the function of p19ARF. However, the giant homozygous deletions often make both proteins nonoperational suggesting that deprivation of either may play a role in the generation and progression of neoplasia through different ways. For instance, the function of p16INK4A is to inhibit the advancement of the cell cycle at the G1/S checkpoint. The G1/S checkpoint is regulated by CDKs such as CDK4 and CDK6, which mediate the activity of p16INK4A [26]. With deprivation of this crucial moderator, uncontrollable expression of CDK4 and CDK6 occurs, causing unregulated operation of the cell cycle through the G1/S checkpoint. This loss of an important checkpoint of the cell cycle in combination with telomere shortening facilitates an environment that leads to the lack of stability of chromosomes and the creation of structural derangements, also entailing fold-back inversions, which is a relatively specific mutation to neoplasms of the pancreas. In contradiction, p19ARF leads to halt of the cell cycle without the help of CDKs by attaching to the E3 ubiquitin ligase MDM2 to hinder p53 breakdown; forfeiture of p19ARF inhibits p53-promoted apoptosis and cell-cycle arrest [25]. Thus, the high frequency of CDKN2A abnormalities makes this gene as an appealing contender for biomarker evaluations.

TP53 One of the most frequently mutated gene in human neoplasms is a TSG called P53, which is found in 50%–70% of pancreatic neoplasms [27]. P53 is the principal tracker of stress in the cell and is triggered by tissue deprivation of oxygen, improper oncogene signaling, and DNA damage. After initiation, p53 is responsible for multiple tasks comprising of G1/S checkpoint supervision and G2/M arrest conservation to allow repair of DNA, and if restoration is not feasible, it leads to senescence or apoptosis of the cell. If both alleles of p53 are lost, DNA damage is not restored, and mutations become secured in the cells undergoing division and hence turn the cell into a one-way street of malignant metamorphosis [28]. The gene is situated on chromosome 17, and the most frequent mechanism of p53 inactivation nearly 66% is by homozygous missense mutations that result in a defective product that is not able to attach to DNA. Other mechanisms of inactivation in pancreatic cancers include nonsense mutations and frameshift homozygous deletions. In most cases, both of the p53 alleles are inactivated through acquired mutations in the somatic cells, but less commonly, some individuals inherit a mutant p53 allele, this disease is known as the Li-Fraumeni syndrome, and the loss of the second one in somatic tissues is associated with 25-fold increased risk of developing malignant tumors by the age of 50 years as compared with the general population [29].

8

1. OVERVIEW OF PANCREATIC CANCER BIOLOGY

SMAD4 The SMAD4 TSG is deleted in 55% of pancreatic neoplasms, either by homozygous inactivation (30%) or by an acquired mutation causing the loss of the other allele (25%) [30]. SMAD4 codes for a protein that plays a crucial part in the transduction of antiproliferative signals from the TGF-β receptors to the nucleus. TGF-β is a member of a lineage of dimeric growth factors that is connected with a composite range of operations, including inhibition of cellular proliferation and differentiation. It manages cellular processes by attaching to a composite made of TGF-β receptors I and II. Receptor dimerization after the ligand binding leads to a cascade of events that results in the activation of CDKIs with growth-suppressing activity and repression of growth-promoting genes like MYC, CDK2, CDK4, and cyclins A and E. The growth-restraining effect of TGF-β pathways is disabled by mutations in the TGF-β signaling pathways. These mutations affect either the type II TGF-β receptor or the SMAD4 molecules. Mutational deletion of SMAD4 is so frequent in pancreatic neoplasms that, in almost 100% of malignancies of the pancreas, one constituent of the TGF-β pathway is mutated. The resultant deletion of DPC4/SMAD4 in pancreatic neoplastic cells may permit them to break free the detrimental effects of TGF-β on growth and results in the promotion of invasion and angiogenesis [31, 32]. The TGF-β pathway is remarkable for its dual capacity in the course of pancreatic cancer such as suppressing the growth of neoplastic cells through premature phases of clonal expansion (PanIN-1 and PanIN-2) while it promotes growth through its signaling pathways in later phases of the clonal expansion (PanIN-3 and invasive neoplasms), mainly due to the loss of SMAD4 and the TGFβ pathways [33].

INTRODUCTION TO LOCAL AND DISTANT MICROENVIRONMENTS Metastasis is a multiplex affair necessitating a succession of steps. Human tissues are organized into a chain of compartments that are isolated from each other by the basement membrane and the interstitial connective tissue. Every one of these compartments is composed of collagens, glycoproteins, and proteoglycans. For its presence into the local and distant microenvironments, a neoplasm must first violate the integrity of the basement membrane traversing through the interstitial connective tissue and eventually gaining access to the nearby blood vessels by invading the vascular basement membrane. Similarly, the invasion of the neoplastic clonal population from the ductal system to the local stroma of the pancreatic tissue necessitates the launch of a species into a new environment [34]. Infiltration into the novel environment is not habitually promised, and the clonal population needs a threshold number of genetic and phenotypic changes to effectively infiltrate and colonize the surrounding local and distant microenvironment. The microenvironment of a primary pancreatic neoplasm is made of a dense stroma and restricted nutrients and oxygen gradient [35]. This stroma also named the desmoplastic reaction is formed by the pancreatic stellate cells or the myofibroblasts. Upon the stimulus of activating signals by the growth factors such as transforming growth factor beta type I (TGFβ-1), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), these cells produce collagen mainly the collagen type I and other constitutes of the extracellular

FAMILIAL PANCREATIC CANCERS

9

matrix. These stellate cells also function to supervise the resorption and replacement of the stroma through the activation of proteolytic enzymes known as the matrix metalloproteinases. They are also accountable for the inferior vascular supply of the pancreatic cancer and the distinctive metabolic feature to acquire nutrients under exacting hypoxic conditions. Moreover, the stroma is also responsible for the progression and the development of the overt tumor and the invasion of the surrounding structures and metastasis to distant organs. Another distinctive feature is the expression of multiple proteins that are responsible for the notorious resistance to treatment and ultimately the poor prognosis of the pancreatic carcinoma. These factors communally act as an inimitable selection force that shapes the growth and expansion of the parental clone and its unending adaptation [36]. Although metastasis is considered to be a distinct stage, it is believed that the process of dissemination begins from the most primitive phases of carcinogenesis [9]. It has been established that some exclusive genetic factors of the parental clone determine the magnitude to which the subclone survives or adapts into the foreign microenvironment. Moreover, following the development of the parental clone, a minimum of 5–10 years is required for the subclone to metastasize to distant areas. Nonetheless, what features of the pancreatic carcinoma dictate this distant spread to vital organs especially the liver, lungs, and peritoneum are still to be determined. The process that causes the epithelial cells to forfeit their characteristic features and leads to the acquisition of mesenchymal phenotypes that direct them to gain various attributes like motility and invasiveness is known as the epithelial to mesenchymal transition (EMT) [37]. EMT plays the fundamental role in pancreatic cancer metastasis. Snail1, Snail2, Zeb1 and Zeb2, and Twist1 and Twist2 are some of the core transcription factors controlling EMT [37]. Some major developmental signaling pathways include TGF-β as discussed with the SMAD4 tumor suppressor gene. Extracellular kinase-triggered pathways including Ras and Raf eventually culminate in TGF-β-mediated EMT. Another important mechanism is microRNAs (miRNAs), which can moderate metastasis through a number of different mechanisms including EMT, cancer stem cells, and matrix metalloproteinase activity [37].

FAMILIAL PANCREATIC CANCERS As mentioned earlier, pancreatic cancer arises through a progressive series of sporadic mutations especially in the somatic cells. Familial inherited disorders have been reported to have a strong association with the pathogenesis of pancreatic carcinoma, and a number of disorders are now recognized that increase the risk in susceptible patients [38, 39]. Like patients with chronic pancreatitis as a result of mutations in the protease, serine 1 (PRSS1), or the serine peptidase inhibitor, kazal type 1 (SPINK1) genes have a 50- to 80-fold escalated risk of developing the overt pancreatic malignancy [40]. This risk is attributed to the increased number of epithelial cell division that results from the process of repeated cell injury and repair or from DNA damage caused by the reactive oxygen species. Both of these processes result in successive mutations and, hence, the augmented risk of pancreatic malignancies. Similarly, inborn germ-line mutations of the BRACA1, the BRACA2, the Fanconi’s anemia, and the ataxia telangiectasia (ATM) genes can considerably increase the risk of pancreatic carcinoma by disrupting the repair mechanism of double-stranded DNA break and, hence,

10

1. OVERVIEW OF PANCREATIC CANCER BIOLOGY

increasing the genomic instability [41]. Although the p16 gene sporadic mutation is known for its characteristic role in the pathogenesis of pancreatic carcinoma as mentioned earlier, inherited germ-line mutations are also established and result in the disorder known as familial atypical multiple mole-melanoma (FAMMM). Patients with FAMMM have an increase tendency to develop multiple nevi and melanomas, and they have a 13- to 22-fold increase risk to develop pancreatic malignancies during the course of their lives. Other rare inherited disorders include the Peutz-Jeghers syndrome [42], and many more are still in research. An understanding of these familial conditions and the role of germ-line mutations can help to better comprehend the molecular biology of pancreatic carcinoma.

CONCLUSIONS Pancreatic carcinoma originates from the precursor lesions (PanINs) as a result of the sequential and uninterrupted accretion of the alterations in the key oncogenes and tumor suppressor genes. Several important advancements include the characteristic role of desmoplastic stroma in the growth and expansion of the disease and a barrier to effective chemotherapy. These, along with stem cell compartment, are involved in radiation and chemotherapy resistance. Another distinctive feature is the mechanism of metabolic adaptation and the attainment of nutrients under hypoxic environment. More information is required regarding these findings as these can be important therapeutic targets in formulating an effective treatment strategy for pancreatic carcinoma.

References [1] Siegel R, et al. Cancer statistics, 2014. CA Cancer J Clin 2014;64(1):9–29. [2] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68(1):7–30. [3] Zhang J, et al. Patterns and trends of pancreatic cancer mortality rates in Arkansas, 1969–2002: a comparison with the US population. Eur J Cancer Prev 2008;17(1):18–27. [4] Jemal A, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56(2):106–30. [5] Llop E, et al. Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma. World J Gastroenterol 2018;24(24):2537–54. [6] He X, et al. The impact of a history of cancer on pancreatic ductal adenocarcinoma survival. United European Gastroenterol J 2018;6(6):888–94. [7] Trewartha, J., 10 self-care strategies for any chronic health condition, in Lifestyle Prescriptions University Blog. 2018. https://lifestyleprescriptions.org/blog/10-self-care-strategies-for-any-chronic-health-condition/. [8] Mahadevan D, Von Hoff DD. Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther 2007;6(4):1186–97. [9] Hermann PC, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;1(3):313–23. [10] Vogelstein B, Kinzler KW. The path to cancer—three strikes and you’re out. N Engl J Med 2015;373(20):1895–8. [11] Makohon-Moore A, Iacobuzio-Donahue CA. Pancreatic cancer biology and genetics from an evolutionary perspective. Nat Rev Cancer 2016;16(9):553–65. [12] Takaori K. Current understanding of precursors to pancreatic cancer. J Hepatobiliary Pancreat Surg 2007; 14(3):217–23. [13] Maitra A, Kern SE, Hruban RH. Molecular pathogenesis of pancreatic cancer. Best Pract Res Clin Gastroenterol 2006;20(2):211–26. [14] Wu S, et al. Substantial contribution of extrinsic risk factors to cancer development. Nature 2016;529(7584):43–7. [15] Blackford A, et al. Genetic mutations associated with cigarette smoking in pancreatic cancer. Cancer Res 2009; 69(8):3681–8.

FURTHER READING

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[16] Gukovsky I, et al. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer. Gastroenterology 2013;144(6):1199–1209.e4. [17] Pannala R, et al. New-onset diabetes: a potential clue to the early diagnosis of pancreatic cancer. Lancet Oncol 2009;10(1):88–95. [18] Kimura W. How many millimeters do atypical epithelia of the pancreas spread intraductally before beginning to infiltrate? Hepatogastroenterology 2003;50(54):2218–24. [19] Feldmann G, et al. Molecular genetics of pancreatic intraepithelial neoplasia. J Hepatobiliary Pancreat Surg 2007;14(3):224–32. [20] Biankin AV, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 2012;491(7424):399–405. [21] Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011;11(11):761–74. [22] Abramson MA, et al. The molecular biology of pancreatic cancer. Gastrointest Cancer Res 2007;1(4 Suppl 2): S7–S12. [23] Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006;127(2):265–75. [24] Quelle DE, et al. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 1995;83(6):993–1000. [25] Sharpless NE, DePinho RA. The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev 1999; 9(1):22–30. [26] Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 2013;14(8):518–28. [27] Yachida S, et al. Clinical significance of the genetic landscape of pancreatic cancer and implications for identification of potential long-term survivors. Clin Cancer Res 2012;18(22):6339–47. [28] Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004;432(7015):316–23. [29] Sancar A, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004;73:39–85. [30] Hahn SA, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271 (5247):350–3. [31] Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003;113 (6):685–700. [32] Siegel PM, Massague J. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 2003;3(11):807–21. [33] Furukawa T, Sunamura M, Horii A. Molecular mechanisms of pancreatic carcinogenesis. Cancer Sci 2006; 97(1):1–7. [34] Rhim AD, et al. EMT and dissemination precede pancreatic tumor formation. Cell 2012;148(1–2):349–61. [35] Sousa CM, Kimmelman AC. The complex landscape of pancreatic cancer metabolism. Carcinogenesis 2014; 35(7):1441–50. [36] Stromnes IM, et al. Stromal reengineering to treat pancreas cancer. Carcinogenesis 2014;35(7):1451–60. [37] Hu J, et al. MiR-361-3p regulates ERK1/2-induced EMT via DUSP2 mRNA degradation in pancreatic ductal adenocarcinoma. Cell Death Dis 2018;9(8):807. [38] Rieder H, Bartsch DK. Familial pancreatic cancer. Fam Cancer 2004;3(1):69–74. [39] Cowgill SM, Muscarella P. The genetics of pancreatic cancer. Am J Surg 2003;186(3):279–86. [40] Weiss FU. Pancreatic cancer risk in hereditary pancreatitis. Front Physiol 2014;5:70. [41] van Asperen CJ, et al. Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. J Med Genet 2005;42(9):711–9. [42] Goldstein AM, et al. Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations. N Engl J Med 1995;333(15):970–4.

Further Reading Suarez AV, Tsutsui ND. The evolutionary consequences of biological invasions. Mol Ecol 2008;17(1):351–60.

C H A P T E R

2 Chemoresistance in Pancreatic Cancer: Emphasis on Age and Gender Myrna Hurtado, Sunil Shah, Maya Nair, Riyaz Basha University of North Texas Health Science Center, Fort Worth, TX, United States

Abstract Chemoresistance is a serious concern for treating cancer patients. It is especially important in the aggressively advancing malignancies such as pancreatic cancer. Patients can acquire chemoresistance to regimens that are specifically approved to treat a particular cancer or in a broad use. The resistance is often developed for the chemotherapeutic agents used in frontline therapy; however, it can occur even when treating for relapsed disease. Chemoresistance in relapsed patients is more serious and has grave consequences in prognosis. Several markers are known to be associated with chemoresistance in pancreatic cancer. Routine studies are mostly focused on understanding the role of biomarkers and molecular or genetic signatures in chemoresistance. A few studies observed to understand the influence of age or gender were inconclusive; consequently, such factors need further investigation. This chapter provides information on the role of age and gender in developing chemoresistance in pancreatic cancer. The presented information summarizes the details of the expression of major biomarkers, molecular and genetic signatures, and other factors associated with chemoresistance and potentially modulated in genders and among different age groups.

Abbreviations 5FU BRCA1 CA 19-9 CpG CTGF dCK FOLFIRINOX hENT1 IAP IGFBP2 MCP1 MMP7 MUC5AC NF-κB

fluorouracil breast cancer 1 carbohydrate antigen sialyl Lewis a 50 -cytosine-phosphate-guanine-30 connective tissue growth factor deoxycytidine kinase clinical trial with combination of 5FU, leucovorin, irinotecan, and oxaliplatin or gemcitabine human equilibrative nucleoside transporter 1 inhibitor of apoptosis protein insulin-like growth factor-binding protein 2 monocyte chemoattractant protein 1 matrix metalloproteinase 7 mucin 5AC, oligomeric mucus/gel-forming nuclear factor κB

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00002-0

13

# 2019 Elsevier Inc. All rights reserved.

14 PALB2 PDAC PLG RNA SEER sICAM1 sRAGE TIMP1 TNF TSP2 XIAP YAP

2. CHEMORESISTANCE IN PANCREATIC CANCER: EMPHASIS ON AGE AND GENDER

partner and localizer of BRCA2 pancreatic ductal adenocarcinoma plasminogen ribonucleic acid surveillance, epidemiology, and end results soluble intercellular adhesion molecule-1 soluble receptor for advanced glycation end products metallopeptidase inhibitor 1 tumor necrosis factor thrombospondin 2 X-linked inhibitor of apoptosis protein yes-associated protein

Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION Pancreatic cancer is deemed to be one of the most fatal malignancies worldwide. There are several factors that contribute to its poor prognosis, and a culmination of these challenges results in a high mortality rate. It is estimated over 331,000 people die from this disease worldwide every year [1]. Currently, it is the fourth leading cause of cancer-related deaths in the United States [2]. This is predicted to climb to be the second leading cause within the next couple of years. Predispositions for pancreatic cancer have been observed in an effort to identify high-risk groups for screening. Cigarette smoking is the leading preventable cause for this disease [3]. Smoking is associated with and increases the chances of development by more than two times [4]. Chronic pancreatitis has also been identified as a risk factor for pancreatic cancer [5]. The inflammation helps create an environment that becomes susceptible to cancer development. Since alcohol can cause chronic pancreatitis, alcoholism is also considered to be a risk factor [6]. While only a small percentage (10%) of pancreatic cancer cases has a family history of this disease, genetics is also a potential risk factor [7]. Additionally, obesity and diabetes are other important contributors [8, 9]. A major advancement in pancreatic cancer treatments has been surgical methods. Resectable surgery increases the patient’s survival time and is considered to be the most effective treatment [10, 11]. However, >85% of patients are deemed ineligible for surgery [2]. Resectable surgery can only be performed if the tumor has not metastasized, and unfortunately, most patients have advanced stage pancreatic cancer at the time of detection. The lack of established biomarkers leads to the late diagnosis of pancreatic cancer. The most promising biochemical marker, thus far, has been the carbohydrate antigen sialyl Lewis a (CA 19-9). Studies have shown an elevation of CA 19-9 in patient’s serum and a sensitivity of 70%–90% [12]. Unfortunately, CA 19-9 is not specific to just pancreatic cancer. Moreover, the sensitivity of the antigen drops significantly in the early stages of pancreatic cancer [13]. Thus, CA 19-9 cannot serve as a reliable biomarker for early detection. A late detection allows for the malignancy to grow, progress, and metastasize unopposed for years.

PANCREATIC CANCER INCIDENCES AND PROGNOSIS: ASSOCIATION OF AGE AND GENDER

15

PANCREATIC CANCER INCIDENCES AND PROGNOSIS: ASSOCIATION OF AGE AND GENDER Pancreatic ductal adenocarcinoma (PDAC) makes up 90% of all pancreatic cancer cases [14]. The incidence rates of pancreatic cancer vary among different countries [15]. While it is hard to determine the reason for these differences, the lifestyle risk factors previously mentioned (cigarette smoking, obesity, and alcoholism) may contribute to observed variations [16, 17]. Environmental and lifestyle factors can impact development of this disease. The highest rate of incidence was in Western European and North American countries, while the lowest rates were in Central African countries. Worldwide, the overall average of incidence in men was higher than in women, with 4.9 men per 100,000 compared with 3.9 women [1]. As shown in Table 1 and Fig. 1, 55,440 cases in the United States, 52.7% were men, and 47.3% were women [18]. Mortality in pancreatic cancer patients is also observed to be high in men than women [19, 20]. The cause for this difference is still unknown; however, it was believed to be related to the consumption of alcohol and tobacco. Additionally, there has been an observed correlation in the increase of incidence with age (Fig. 2). Ninety percent of pancreatic cancer patients are 55 years or older [22]. The average age of a patient at the time of diagnosis is 70, and the average age of a patient at the time of death is 72 [23]. Unsurprisingly, the increase in age has been found to have a decrease in overall survival time [24]. Patients 75 years old, do not receive the same high doses or as frequently as those younger than them [31]. Their older age raises concern when obtaining therapy as they may not be able to handle aggressive treatments as well. A major factor that may contribute to this is the comorbidities developed and associated with older age [32]. Another concern for ineffective treatment is a person’s pharmacokinetics altering with age. As a person ages, their drug distribution, absorption, and excretion capabilities are decreased and, thus, can cause resistance to chemotherapeutic therapies [33] A culmination of these issues creates a challenge when treating elderly patients. Nevertheless, perhaps, assessing elder patients and identifying novel positive prognostic factors could help combat this issue.

MECHANISMS ASSOCIATED WITH CHEMORESISTANCE Researchers have looked into various factors that can cause or add to this chemoresistance, like the tumor microenvironment caused by desmoplasia, altered genetics preventing delivery to the tumor, and insensitivity to apoptotic signals [34–36]. It is important to elucidate their mechanisms of action to successfully address issues associated with chemoresistance. However, the influence that gender and age can have on these mechanisms has not been greatly assessed.

18

2. CHEMORESISTANCE IN PANCREATIC CANCER: EMPHASIS ON AGE AND GENDER

As previously mentioned, there are no established predicators for pancreatic cancer or how a patient may respond to treatment. In 2014, a retrospective study performed by Hohla et al. attempted to identify a predictive biomarker for patients’ response to FOLFIRINOX treatment. They examined tumor protein p53, Ki67 antigen, carcinoembryonic antigen (CEA), and carbohydrate antigen (CA) 19-9 levels in males and females. Women were found to have a significantly higher response than men to this treatment, and this was also associated with elevated levels of CEA and CA 19-9. However, other researchers who also examined CEA and CA 19-9 serum levels found that there was no significant association with gender [37–39]. The observed differences in men and women are still unknown and worth investigating. Gemcitabine (20 ,20 -difluorodeoxycytidine) is a pyrimidine antimetabolite. Its structure is similar to DNA; thus, the drug works by being incorporated into DNA in cancer cells and halting synthesis during replication. The drug is first transported into cells by the human equilibrative nucleoside transporter 1 (hENT1) [40]. Once inside the cell, gemcitabine is then phosphorylated to its active form, gemcitabine di- and triphosphate, by deoxycytidine kinase (dCK) [41]. The phosphorylated gemcitabine is, then, what becomes incorporated into the DNA, and after inclusion of one more nucleotide, DNA synthesis is stalled [42]. Studies have demonstrated that decreased gene expression or activity of dCK resulted in resistance of gemcitabine treatment in cancer cells [43, 44]. One study in particular used immunohistochemistry to examine protein expression of dCK among different pancreatic cancer cell lines with varying ages [45]. They found a significant inverse relationship existed with age (70.3  8.1 vs 59.8  7.4 years) and expression of dCK; higher age correlated with decreased expression of dCK. The reduced expression of dCK in patients also resulted in shorter overall survival compared with higher expression. Furthermore, methylation of CpG islands, thus, gene silencing, has been observed in pancreatic cancer patients, and the amount of methylation increases with age [46, 47]. Because the low level of dCK in pancreatic cancer cells was not from a DCK gene mutation, the observed difference could perhaps be due to age-induced methylation and decreased expression of the gene.

FACTORS MODULATED IN VARIOUS AGE GROUPS Many studies have been dedicated to finding potential markers for pancreatic cancer and poor prognostic factors. Survivin (encoded by the baculoviral inhibitor of apoptosis protein [IAP] repeat containing 5 gene) and X-linked inhibitor of apoptosis protein (XIAP) are both part of the inhibitor of apoptosis protein family and prevent apoptosis by blocking caspase cleavage [48, 49]. Survivin and XIAP are found to be overexpressed in pancreatic cancer, and the inhibition of apoptosis renders tumor cells resistant to chemotherapy [50–52]. Their overexpression has been suggested to be used as a prognostic factor for patient survival; however, it has not been associated with age or gender [53, 54]. Efforts testing inhibitors of survivin and XIAP to induce chemosensitivity have been successful, and studies on this topic continue to grow [55–57]. There are many cytokines that have been implicated in the development and progression of cancer; among them is tumor necrosis factor (TNF). TNF is a pro-inflammatory cytokine, but it has also been found to play a role in cancer with cell proliferation, migration, and invasion [58]. In pancreatic cancer specifically, TNF is secreted by the tumor itself [59]. TNF

19

CONCLUSION

helps initiate a desmoplastic reaction and creates a protective microenvironment for the tumor [60]. This creates protection for the tumor against signal TNF-mediated apoptosis. TNF has been demonstrated to not only be involved with cancer progression but also add to the resistance of chemo agents [61]. One transcription factor activated by TNF that is also involved with pancreatic cancer is nuclear factor κB (NF-κB) [62]. NF-κB has activation that is transient and plays a role in the inflammatory system and tumorigenesis [63]. NF-κB expression serves as a positive feedback loop, and it promotes its own expression. This expression then causes pro-inflammatory and antiapoptotic responses in the tumor cell [64]. In pancreatic cancer, constitutive activation of NF-κB is common, and this contributes to chemoresistance [64]. Many studies have found that the expression of TNF and many other inflammatory cytokines increases with age [65–67]. Since TNF plays a role in NF-κB activity, during the aging process, an increase in TNF could also lead to an increase in NF-κB activity and ultimately promotes chemoresistance.

CONCLUSION After realizing the importance of molecular genetics in driving chemoresistance, a myriad of studies has been dedicated to elucidating the functions of associated markers, and some of them have the association with age or gender or stage (Table 2). While there are apparent differences among age and gender in pancreatic cancer patients, the causes for these are not as clear. Among males and females, it appears there are some differences in response to TABLE 2

Molecular markers associated with chemoresistance and influence on age, gender, and stage Influencing factor

Molecular marker dCK

Age

Gender

X

Progression/stage

Reference

X

[45–47]

X

[54]

X

[65–67]

XIAP

X

[53]

MUC5AC specific mucin species for PDAC

X

[68]

Carbohydrate antigen 19-9 (CA 19-9)

X

[69]

Multiple microRNAs performed in combination with CA 19-9

X

[70]

Survivin TNF

X

Chemokine MCP1

X

X

[71]

sRAGE, soluble advanced

X

X

[71]

BRCA2, BRCA1, and PALB2

X

[72]

MMP7, IGFBP2, TSP2, sICAM1, TIMP1, and PLG

X

[73]

X

[74]

YAP, CTGF

X

20

2. CHEMORESISTANCE IN PANCREATIC CANCER: EMPHASIS ON AGE AND GENDER

treatment. The literature on molecular differences between males and females in pancreatic cancer treatment is scarce and requires more research. However, among the different ages, there have been studies attempting to target chemoresistance in the elderly. One study in particular demonstrated that by inhibiting TNF, pancreatic cancer cells were sensitized to chemo treatment [75]. This was accomplished by diminishing the desmoplastic reaction and inflammation. This is significant as inflammation and desmoplasia (with its extensive stromal components) promote tumor progression and weaken chemotherapy treatment [76, 77]. Thus, targeting the increased expression of TNF in the elderly could perhaps help combat chemoresistance. Since chemotherapy-induced overexpression of NF-κB has been found to occur in pancreatic cancer, it could be useful to target their tumor-associated transcription factor. A study using pancreatic cancer cells demonstrated that treatment with doxycycline deactivated NF-κB [78]. Doxycycline prevented further activation of NF-κB and thus acted as an NF-κB “desensitizer.” This resulted in a higher response in tumor cells to chemo treatment. In another study, pancreatic cancer cells were treated with a variety of NF-κB inhibitors (gliotoxin, MG132, and sulfasalazine) to enhance their response to etoposide and doxorubicin treatments [79]. The inhibitors efficiently inactivated NF-κB, and this resulted in improved response to chemo treatments. Because an increase in TNF expression has been observed in the elderly and NF-κB is modulated by TNF, an increased expression of NF-κB could be occurring and contributes to chemoresistance (Fig. 3). Thus, targeting NF-κB could help sensitize older patients to chemotherapy. Finally, studies investigating the decreased expression of dCK and its association with chemoresistance in the elderly could be advantageous. Candelaria et al. used hydralazine FIG. 3 Schematic of the potential role in age-related effects of chemoresistance. There is an increased expression of tumor necrosis factor (TNF) seen in elderly pancreatic cancer patients. This increased expression may lead to increased NF-κB activity, thus eliciting an inflammatory response and contributing to the desmoplastic reaction. Inflammation and desmoplasia at the tumor site helps create a protective microenvironment, ultimately leading to increased chemoresistance.

TNF

NF-kB

Inflammatory response

Desmoplasia

Protective tumor microenvironment

Chemoresistance

REFERENCES

21

(a demethylating agent) in cervical cancer cells to enhance their response to gemcitabine [80]. Methylation in the tumor cells was found to be induced by gemcitabine and resulted in reduced expression of dCK. A combination treatment of hydralazine and gemcitabine sensitized cancer cells to the chemo treatment as hydralazine prevented gene silencing of DCK. However, it appears that epigenetic agents for dCK have yet to be investigated in pancreatic cancer and may be worth looking into. In conclusion, a more in-depth analysis is needed for the variations in chemoresistance among different ages and the two genders.

Acknowledgments RB is supported by National Institute for Minority Health and Health Disparities (grant#: 2U54 MD006882-06) and Shirley E. Noland Foundation (Melbourne, FL).

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C H A P T E R

3 EMT Contributes to Chemoresistance in Pancreatic Cancer Suresh Chava, Meher B. Gayatri, Aramati B.M. Reddy Laboratory of Cell Signaling, Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India

Abstract Pancreatic cancer tops fourth in cancer-related deaths in US adults because of its poor diagnosis and the lack of early markers thus resulted in increased metastasis cases. Treatment options for such cases are limited and exhibit minimum response to treatment. Research for the past five decades demonstrated that epithelial-tomesenchymal transition (EMT) is the major contributing factor for the cancer progression, invasion, and metastasis. Numerous xenograft animal- and cell line-based studies state that transcription factors (Snail, Slug, Twist, and ZEB1) that regulate EMT are involved in cancer progression. Recent insights show that various cell signaling pathways (TGF-β, Wnt/β-catenin, Notch, and TNF-α) that regulate EMT also contribute to invasion and metastasis in pancreatic ductal adenocarcinoma patients. The 50 years of study provide the fascinating inputs highlighting the role of EMT in pancreatic cancer progression, unraveling its role in chemoresistance. In particular, the impact of EMT in experimental therapies remains debatable, while the drug discovery strategies of combinational therapies by inhibition of EMT along with chemotherapy deserve consideration in pancreatic cancer. In this chapter, we encapsulate the critical research findings on EMT-associated chemoresistance and its mechanisms in pancreatic cancers.

Abbreviations 5-FU

5-fluorouracil chemoresistance

BNIP3 BxPC3 cc-RCC CDH1 Chk1/2 CK-1 COX2 CSCs CtBP CTCs

BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 human pancreas adenocarcinoma clear cell renal cell carcinoma cadherin-1 checkpoint kinase 1/2 casein kinase 1 cyclooxigenase-2 cancer stem cells C-terminal-binding protein circulating tumor cells

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00003-2

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# 2019 Elsevier Inc. All rights reserved.

26 CTLA4 ECM EGF EMT FdUMP FGF Gm-CSF GSK-3β HDAC HDAC1 HIF1-α LRP5/6 MDM2 MET NICD p300-p/CAF P53 PRRX S100A4 S100P SIN3A SMAD4 SNAIL SWI/SNF TCF/LEF TGF-β TNF-α TRADD TRAF2 Twist Wnt-1 ZEB1

3. EMT CONTRIBUTES TO CHEMORESISTANCE IN PANCREATIC CANCER

cytotoxic T lymphocyte-associated antigen 4 extracellular matrix epidermal growth factor epithelial-mesenchymal transition fluorodeoxyuridine monophosphate fibroblast growth factors granulocyte-macrophage colony-stimulating factor glycogen synthase kinase 3 beta histone deacetylase histone deacetylase 1 hypoxia-inducible factor 1-alpha low-density lipoprotein receptor-related protein 5/6 mouse double minute 2 homologue mesenchymal-epithelial transition Notch intracellular domain K (lysine) acetyltransferase 2B (KAT2B) tumor protein p53 paired-related homeobox 1 calcium-binding protein A4 calcium-binding protein P paired amphipathic helix protein Sin3a mothers against decapentaplegic homologue 4 zinc finger protein switch/sucrose nonfermentable T-cell factor/lymphoid enhancer factor transforming growth factor beta tumor necrosis factor tumor necrosis factor receptor type 1-associated DEATH domain protein TNF receptor-associated factor 2 class A basic helix–loop–helix protein 38 (bHLHa38) proto-oncogene protein Wnt-1 zinc finger E-box-binding homeobox

Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION The epithelial-mesenchymal transition (EMT) is a vastly conserved physiological phenomenon that is essential for embryonic development, wound healing, and pathophysiology of cancers in multicellular organisms [1]. Based on these functional differences, EMT has been classified into three types: (1) developmental EMT (2) wound healing/tissue regeneration EMT and (3) cancer metastasis EMT [2, 3]. EMT permits stationary epithelial cells to attain a mesenchymal cell phenotype as it undergoes multiple phenotypic and biochemical changes [3]. The EMT transition contains certain key events that are deliberated hallmarks of the process, which include increased cell migration capacity, improved resistance to apoptosis, invasiveness, and elevated expression of extracellular cellular matrix (ECM) components [4]. The loss of epithelial cell–cell junctions, degradation of basement membranes, and the loss of apical basal polarity simultaneously attain front-rear polarity that contribute to transition toward the mesenchymal stem cell that

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can invade and migrate away from where it is instigated [5]. EMT is characterized by harmonized repression of genetic profile of epithelial cells, predominantly downregulation of E-cadherin, and at the same time the activation of the mesenchymal genetic profile, which includes induction of N-cadherin, vimentin, and smooth muscle actin expressions [3, 6, 7]. These genetic alterations help in ECM degradation, which in turn facilitates migratory and invasive characteristics and reduced apoptosis [3, 7]. These substantive changes permit mesenchymal cells to relocate from their place of origin. Upon dissemination, the circulating tumor cells (CTCs) move to a suitable destination, wherein they resume to their epithelial phenotype, through a phenomenon known to be mesenchymal-epithelial transition (MET), allowing them to proliferate with subsequent formation of metastases at the new site [3, 6, 8–12]. Though the involvement of EMT in cancer metastasis is a well-known concept but still is a questionable one, mainly due to incompetence to screen the EMT phenotype in an in vivo system. Further, most of the CTCs show both epithelial and mesenchymal markers, which are sometimes being considered as partial EMT. In this chapter, we will describe an overview of the elementary mechanisms of EMT activation, which is associated with pancreatic cancer metastasis, EMT activators, and signaling pathways with key concepts behind the plasticity of epithelial-to-mesenchymal transition. Further, we will highlight the mechanisms behind chemoresistance of pancreatic cancer cells and review the ways to precisely target these chemoresistant cells.

PANCREATIC CANCER METASTASIS Pancreatic cancer is the fourth leading cause of cancer deaths in the world, mainly due to the lack of early diagnosis markers and specific drug targets. The patients are generally diagnosed at far late stages, which cause poor survival rates (13 mg/dL) found statistically significant improvement. In these patients, the 6- and 3month survival were 42% and 48% in the ruxolitinib arm and 11% and 29% in the placebo arm, respectively. These study subjects who received benefit were allowed to continue ruxolitinib, although no further studies were conducted.

Insulin-Like Growth Factor 1 Receptor Inhibitor Pancreatic cancer shows a high expression of insulin-like growth factor 1 (IGF-1) receptors, which are responsible for increased cancer cell growth and survival via downstream effectors involving KRAS-dependent or KRAS-independent mechanisms. A phase II trial evaluated ganitumab (anti-IGF-1R mAb) combination with gemcitabine in comparison with gemcitabine alone (57% vs 50%) [112]. The phase III GAMMA trial didn’t show any

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improvement in the OS or PFS in ganitumab plus gemcitabine arm over gemcitabine monotherapy. Moreover, selection based on the pretreatment biomarker levels was not associated with better outcomes [113].

Vascular Endothelial Growth Factor Receptor Inhibitors Vascular endothelial growth factor (VEGF) proteins are responsible for the regulation of angiogenesis, both normal and in cancerous conditions. VEGF receptor (VEGFR) has been shown to overexpress in pancreatic cancer, mainly responsible for angiogenesis in tumors [114]. Different VEGFR inhibitors such as bevacizumab (monoclonal antibody), axitinib (small-molecule inhibitor), and aflibercept (recombinant VEGFR fusion protein) have been evaluated in phase III trials showing no statistically increased improvement in the OS or PFS when combined with gemcitabine or gemcitabine and erlotinib [115–117].

TUMOR STROMA/DESMOPLASIA Transforming Growth Factor-β Transforming growth factor-β (TGF-β) is a peptide hormone released by essentially all cell lines and is found in a protein-bound inactive state in extracellular matrix. TGF-β is a pleiotropic hormone and can exert both pro- and antitumor effects. TGF-β is a good prognostic indicator in early-stage tumors and vice versa in the late tumors, a phenomenon known as “TGF-β paradox” [118]. TGF-β has a proneoplastic effect at microenvironment level in the tumor facilitating desmoplastic stromal reaction, which is responsible for the aggressive nature and high mortality of PAC patients. Therefore, TGF-β is a potential target for antitumor agents. In phase I/II studies, evaluating trabedersen (antisense oligonucleotide) that stops TGF-β2 expression and galunisertib, a small-molecule TGF-β receptor inhibitor, showed increased survival [118, 119]. Galunisertib is currently under investigation [120], while there are no studies evaluating trabedersen PAC.

Platelet-Derived Growth Factor Platelet-derived growth factors (PDGF) are four small glycoproteins, which undergo dimerization and subsequently activate one of the three isoforms of PDGF receptor (a transmembrane tyrosine kinase). This leads to activation of signaling cascade, which activates cellular processes such as angiogenesis and cellular migration. PDGF is involved in the carcinogenesis of many tumors, but its carcinogenic role in PAC is disputed [121]. PDGF overexpression was associated with lower growth, through enhanced pericyte recruitment in mice models. When PDGF was inhibited with imatinib in these mice models, enhanced cancer growth was observed [114]. On the other hand, worse prognosis and higher rate of metastasis have been found with the expression of stromal PDGFR beta expression in PAC patients [122]. No improved survival was found with the use of sunitinib (PDGFR inhibitor, also inhibits VEGFR, RET, KIT, and FLT3) in metastatic PAC patients who progressed on gemcitabine therapy [123]. Sunitinib was, however, shown to improve the PFS at 6 months

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(22% vs 3.6 months in the observation arm) when used as maintenance therapy after gemcitabine therapy. Furthermore, an increase in 2-year OS was also observed with sunitinib, but this outcome failed to achieve statistical significance [124].

Hyaluronic Acid Hyaluronic acid (HA) is glycosaminoglycan, found in the extracellular matrix of majority of tissues. HA is found in increased amount in the PAC, which imparts this tumor high metastatic potential and enhanced tumor growth [125]. Many mechanisms have been suggested for the procarcinogenic potential of HA including the creation of a physical barrier and lowering the density of tumor vascularity [126]. PEGPH20 is a recombinant human hyaluronidase, which enhances the drug delivery to tumor tissue and large amount of HA in the extracellular matrix. A phase II trial evaluated PEGPH20 in combination with nab-paclitaxel/gemcitabine versus nab-paclitaxel/gemcitabine in the fourth stage of metastasis PAC, showing an increased PFS in the PEGPH20 group (9.2 vs 6.3 months) [67]. PEGPH20 combined with gemcitabine, nab-paclitaxel, and rivaroxaban (NCT02921022) and gemcitabine plus nab-paclitaxel is under evaluation (NCT02715804).

Matrix Metalloproteinase Inhibitors Matrix metalloproteinases (MMP) remodel extracellular matrix through its endopeptidase activity [120]. Both tumor and stromal cells in the PAC are the producers of MMP, which are responsible for enhanced metastatic potential and aggressiveness of PAC [127, 128]. Phase III studies evaluated marimastat and tanomastat (MMP inhibitors), which failed to show superiority over gemcitabine [129, 130].

Hedgehog Inhibitors The hedgehog pathway has been known to involve in the regulation of embryogenesis, but its role in carcinogenesis has also been identified including in PAC. Hedgehog ligand acts by binding a transmembrane protein resulting in the release of smoothened homologue (SH). The SH facilitates the translocation of Gli transcription factors into the nucleus [127]. The SH exerts its pro-oncogenic effects by inducing a desmoplastic reaction in mice models and, thus, is a potential target for antineoplastic therapies [128]. IPI-926 is a smallsize molecule, the SH inhibitor causing enhanced intratumoral levels of gemcitabine and increased tumor vascularity in the initial studies [131]. IPI-926 was assessed in a phase I/II trial combined with gemcitabine therapy in comparison with gemcitabine monotherapy, but the study was halted after the median OS was found to be 1 year [78]. Gemcitabine plus nab-paclitaxel combination and gemcitabine monotherapy were investigated and compared in a phase III trial, the MPACT trial in patients with metastatic PAC. The combination arm was found to have an increase in the median overall survival (6.7 vs 8.5 months, HR 0.72, P < 0.001). Gemcitabine plus nab-paclitaxel was permitted by the FDA as the first-line treatment options in metastatic PAC patients.

PEP02 (MM-398) A nanoparticle lysosomal form of irinotecan is PEP02 (MM-398), which was investigated in a phase II clinical trial in metastatic PAC patients unresponsive to gemcitabine therapy showing 9 weeks of median PFS and overall survival of 21.6 weeks [92]. In a multinational phase III trial, 417 patients with gemcitabine-refractory metastatic PAC were randomly allocated to receive fluorouracil and folinic acid plus nanoliposomal irinotecan, fluorouracil, and folinic acid or nanoliposomal irinotecan monotherapy. The results showed an increase in median overall survival from 4.2 to 6.1 months with the addition of nanoliposomal irinotecan to fluorouracil and folinic acid. Nanoliposomal irinotecan plus fluorouracil/folinic acid combination was the first second-line treatment option permitted by the FDA [136].

Endo Tag-1 Although pancreatic cancer is generally not a highly vascularized tumor, it is found to have microangiogenic foci that show an overexpression of angiogenic factors [137]. EndoTag-1 is composed of positively charged liposomes incorporated in paclitaxel particles. Due to the absence of a glycocalyx coating in the tumor vascular endothelia rendering it negatively charged (normal endothelium is positively charged due to glycocalyx), this negatively charged endothelial surface allows the binding of positively charged liposomes resulting in higher selective concentration in tumor environment [138]. Evaluation in a phase II clinical trial showed increased median OS (9.3 vs 6.8 months) and PFS (4.4 vs 2.7 months) in comparison with gemcitabine monotherapy [139]. Phase III trials evaluating EndoTag-1 in combination with gemcitabine monotherapy are in progress (NCT03126435).

Trastuzumab Emtansine HER-2 overexpression in PAC is well documented, but attempts to target this receptor for PAC treatment have not been successful so far—a phase III trial didn’t show any survival

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benefit when trastuzumab was combined with gemcitabine. It has been suggested that this reduced response is due to the lower HER-2 expression in PAC as compared with other tumors such as breast cancer, which is highly responsive to trastuzumab therapy [105]. Therefore, to overcome this problem, attempts have been made. A newly developed conjugate of mAb trastuzumab has been developed, namely, trastuzumab emtansine (T-DM1), a derivative of maytansine (DM1), an inhibitor of microtubular assembly. T-DM1 complex upon interaction with the HER-2 receptor becomes internalized through endocytosis and breaks down in the lysosomes. The active product, DM1, then causes inhibition of microtubule assembly when released into the cell by the lysosomes [140]. It has been observed that this mechanism is more effective than trastuzumab as several trastuzumab-resistant breast and gastric cancers have been shown to be more responsive to trastuzumab emtansine [141]. Preclinical investigations have shown that gemcitabine induces an increased expression of HER-2 receptors, which is indicative of a higher potency due to the synergistic effect of gemcitabine and trastuzumab emtansine therapy [142]. Trastuzumab emtansine therapy in advanced PAC is under assessment in a phase II trial (NCT02999672).

TUMOR HYPOXIA Pancreatic cancer, like many other cancers, has the ability to create a hypoxic environment in the tumor tissue that contributes to bad prognosis and chemotherapy resistance [143]. This is mainly due to two features: (i) reduced uptake of drug by the tumor cells and (ii) the quiescence state of hypoxic cells that contributes to their resistance to the chemotherapeutic drugs, which are antiproliferative [144]. A prodrug of bromoisophosphoramide mustard, evofosfamide (TH-302), remains activated in the hypoxic conditions and is used to overcome the hypoxic resistance in tumor environment. Evofosfamide was assessed in combination with gemcitabine in a phase I/II clinical trial showing an increased PFS and tumor response in metastatic PAC patients in comparison with gemcitabine monotherapy [145]. Unfortunately, the phase III trial showed no increased over survival with evofosfamide plus gemcitabine combination as compared with monotherapy (median overall survival 8.7 vs 7.6 months, p0.059) [146].

CONCLUSIONS AND FUTURE PERSPECTIVES Pancreatic carcinoma continues to have poor prognosis despite the advancements in the development of chemotherapeutic drugs, radiological techniques, and targeted therapies. Its incidence continues to rise with relatively very low survival on the treatment strategies available. There are two major causes for this poor prognosis: firstly, late presentation with advanced-stage disease due to the delayed diagnosis and, secondly, nonavailability of specific/sensitive biomarkers to individualized treatment options. Therefore, not only new screening tests are required to detect PAC at an initial stage, but also focus on the development of individualized treatment strategies based on the predictive biomarker are also needed. Thus, we can say that early detection screening tests and “precision medicine” should be the focus of research in pancreatic cancer management and will hopefully improve the survival and prognosis in these patients.

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C H A P T E R

6 Curcumin and Genistein Enhance the Sensitivity of Pancreatic Cancer to Chemotherapy Begum Dariya*, Sneha Govardhanagiri†, Balney Rajitha*, Sheik Aliya‡, Afroz Alam*, Ganji Purnachandra Nagaraju† *

†

Department of Bioscience and Biotechnology, Banasthali University, Banasthali, India Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States ‡Department of Biotechnology, Jawaharlal Nehru Technical University, Hyderabad, India

Abstract Pancreatic cancer is one of the most lethal cancers in the United States because of its limited treatment options. PC has a high mortality rate because it is often diagnosed at the advanced stages due to its asymptotic nature. The most common form of treatment is adjuvant therapy with surgical resection combined with chemodrugs such as gemcitabine and 5-FU. However, PC cells develop a resistance to the chemodrugs; therefore, sensitizing them to the drugs is crucial. In addition to adjuvant therapy and sensitization, nutraceuticals such as curcumin, genistein, and resveratrol are used due to their natural anticancer properties. They provide protection to healthy pancreatic cells and control the signaling pathways of proteins and pancreatic stellate cells that are responsible for the growth and metastasis of PC cells. The nutraceuticals also potentiate the chemodrugs and sensitize the PC cells. Therefore, nutraceuticals are used in conventional therapies and in combination with chemodrugs. As explored in this chapter, curcumin and genistein downregulate the proteins (NF-κB, EGFR, VEGF, COX-2, miRNA-22, Bcl-2, Bcl-xL, etc.) responsible for growth, progression, invasion, and metastasis. In addition to these, they also upregulate proteins (Bax and caspase) responsible for apoptosis and inhibition of PC cells in vivo and in vitro. Although this provides much hope for more efficient treatment methods, the bioavailability of these natural products is a clinical hurdle; however, the use of metabolite forms could be promising to PC patients. Thus, they are to be tested in various clinical trials for toxicity and high plasma levels and may be considered as a novel therapy.

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00006-8

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# 2019 Elsevier Inc. All rights reserved.

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6. CURCUMIN AND GENISTEIN ENHANCE THE SENSITIVITY OF PANCREATIC CANCER

Abbreviations Akt AMPK AP AXP107-11 BAD Bax Bcl-2 BDMC BIRC cAMP CD CDF CDK Chk COX CSC CYP1B1 DMC DNMT EET EF31, UBS109 EGFR Egr-1 EMT ERK EZH Fbw FGF FLICE FoxM1 FOXO1 FU GO-Y030 GRP GSH HDAC Hes1 HIF-1α HS766-T ASPC-1 HSP-90 IAP IKK IL-1β iNOS ISG IкB JAK JNK LOX MAPK MDR

protein kinase B adenosine monophosphate-activated protein kinase activator protein combination of genistein and gemcitabine Bcl-2-associated death promoter apoptosis regulator B-cell lymphoma 2 bisdemethoxycurcumin baculoviral inhibitors of apoptosis repeat containing proteins cyclic adenosine monophosphate cluster of differentiation difluorinated curcumin cyclin-dependent kinase checkpoint kinase 1 cyclooxygenase cancer stem cells cytochrome P450 1B1 desmethoxycurcumin DNA methyltransferases epoxyeicosatrienoic acid synthetic curcumin analogues epidermal growth factor receptor early growth response protein epithelial-mesenchymal transition extracellular signal-regulated kinases enhancer of zeste homologue F-box/WD repeat domain fibroblast growth factor inhibitor of death receptor signaling forkhead box protein M1 forkhead box protein 01 fluorouracil a novel inhibitor of IKKβ glucose-regulated protein glutathione histone deacetylases hairy and enhancer of split 1 hypoxia-inducible factor-1α pancreatic cancer cell lines heat shock protein inhibitor of apoptosis protein inhibitor of nuclear factor kappa B interleukin-1 beta inducible nitric oxide synthase interferon-stimulated gene inhibitor of kappa B janus kinases C-Jun N-terminal kinases lipoxygenase mitogen-activated protein kinase multidrug resistance

INTRODUCTION

miRNA MMP NF-кB NICD PCDA PEG PGE2 PI3K PIK3 PKA PSC ROS SH siRNA SMA SP STAT TAM TGF-β1 TNF-α VEGF VPS34 XIAP

89

microRNA, small noncoding RNA matrix metalloproteinases nuclear factor кB notch intracellular domain poly(curcumin-dithiodipropionic acid) polyethylene glycol prostaglandin E2 phosphatidylinositol 3-kinase phosphoinositide 3-kinase protein kinase A pancreatic stellate cells reactive oxygen species small hydrophobic protein small interference RNA styrene-maleic acid specific protein signal transducer and activator of transcription proteins tumor-associated macrophages transcription growth factor-β1 tumor necrosis factor α vascular endothelial growth factor encoded by PIK3C3 gene X-linked inhibitor of apoptosis protein

Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION Pancreatic cancer (PC) is one of the most lethal cancers and may result in aggressive gastrointestinal tumors. In recent decades, PC diagnosis has become more prevalent worldwide, resulting in an increased death rate. The statistics from the global cancer data specify that PC is the seventh leading cause for death worldwide [1]. In developed countries, it is the fourth leading cause of death in women and fifth in men [1]. It is predicted that by 2030, it will be the second leading cause of death in the United States [1]. In currently developing countries, PC in women is the 10th leading cause of death and eighth in men with a less than 5% survival rate of patients 5 years after diagnosis [1]. PC is an invasive disease that rapidly metastasizes, caused due to hereditary factors, due to lifestyle habits, or due to the damaged DNA from oxidative stress. Diseases like obesity, diabetes, and chronic condition (chronic inflammation and chronic pancreatitis) cause 30% of PC. Patients receiving insulin therapy develop resistance against insulin and are prone to develop PC [2]. Multiple mutations in tumor suppressor and proto-oncogene genes are responsible for the initiation, proliferation, progression, and metastasis of PC. Due to the biological complexity of PC, efforts toward the development of better therapy solutions are being researched. Surgical procedures such as pancreaticoduodenectomy are suggested. This includes resection of the pancreas, which may increase the survival rate for 5 years; however, it is only eligible for patients whose PC was diagnosed at the early stages. The surgery can be followed by an adjuvant therapy including chemotherapy and radiotherapy. Though advancements in surgery have been made, as well as

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6. CURCUMIN AND GENISTEIN ENHANCE THE SENSITIVITY OF PANCREATIC CANCER

combination treatments with chemotherapy and radiotherapy, the mortality rate of PC has not decreased in patients in comparison with other cancers. The high mortality rate is due to the reappearance of the tumor postsurgery and the intrinsic resistance developed to chemo- and radiotherapy drugs. Therefore, sensitization of cancer cells against the chemoagent is necessary. Herbal products have been widely used for decades as chemopreventive agents and are effective at reducing and scavenging free radicals [3, 4]. The nutraceuticals are the herbal products, which considerably sensitize cancer-causing cells to chemotherapy and target their signaling pathways. They originate from nutrients and natural plant products (curcumin, resveratrol, isoflavone genistein, and metformin) and can be used as chemosensitizers. These nutraceuticals are shown to be advantageous in vivo and in vitro studies, whereby they regulate and modulate various signal transduction pathways. They upregulate cell signaling, epigenome, and tumor-suppressing miRNAs and are used in combination with chemoagents such as dimethylaminoparthenolide, hydroxycamptothecin, cisplatin, gemcitabine, erlotinib, and 5-FU.

CHEMOTHERAPY FOR PANCREATIC CANCER Chemotherapy targets the cancer cells that grow and divide rapidly. The chemodrugs used for the treatment for PC include 5-fluorouracil (5-FU), gemcitabine (Gemzar), cisplatin, paclitaxel (Taxol), irinotecan (Camptosar), capecitabine (Xeloda), oxaliplatin (Eloxatin), docetaxel (Taxotere), and irinotecan liposome (Onivyde). For three decades, 5-FU and gemcitabine have been used as standard drugs. Gemcitabine is a widely prescribed drug for patients suffering from advanced stages of PC. Advanced treatment for cancers now includes the combination therapy, a combination of multiple drugs. Unfortunately, this was unsuccessful due to the cancer cells developing resistance against the chemodrugs. Thus, the use of natural herb products is encouraged when used in combination with the conventional chemotherapy. This may augment the therapeutic efficacy against the cancer cells by sensitizing them and reducing the side effects of chemodrugs. This chapter focuses on the natural products (curcumin and genistein) and their effects on pancreatic cancer cells as sensitizers.

TUMOR MICROENVIRONMENT DEVELOPING CHEMORESISTANCE The pancreas can develop tumors in its exocrine or endocrine cells. Tumors in the exocrine cells are called adenocarcinomas and are very invasive, whereas tumors in the endocrine cells are neuroendocrine tumors or islet cell tumors and are less common. The mutation is the hurdle by which the PC cells develop resistance. The blocking therapy blocks the pathway; however, due to the mutation, the tumor evades the blockage and follows other pathways to grow. The microenvironment of the tumor includes the interstitial tissue with tumor cells, including pancreatic stellate cells, inflammatory cells, fibroblasts, nerve cells, and proteoglycans [5]. The fibrous stroma interacts with the cancer cells, allowing them to migrate, invade and develop resistance against chemodrugs [6]. The stroma produces proteins responsible for developing resistance against multiple drugs. The mechanism of multidrug resistance (MDR) can

TUMOR MICROENVIRONMENT DEVELOPING CHEMORESISTANCE

91

be separated into two classes: one in which it weakens the delivery system and suppresses their cytotoxicity due to the overexpression of drug efflux transporters and increased DNA damage repair and another in which it arises in the tumor cells due to changes in their genome and epigenome, which affect sensitivity to the drugs. Hypoxia is one of the proposed causes for drug resistance not only in PC but also in most tumors [7, 8]. PSCs or pancreatic stellate cells are one of the components from the fibrous stroma in the tumor microenvironment and play a major role in the development of tumor. The inactive PSCs in normal tissues are activated by TGF-β1 (an inflammatory signal), which develop into an intense stroma matrix-like myofibroblast phenotype [5] and include collagen, laminin, and fibronectin. Thus, these PSCs develop chemoresistance by affecting the tumor cell growth. Cao F et al. [9] observed from the coculture of PC cells and PSCs that PC cells develop chemoresistance against gemcitabine due to the overexpression of hairy and enhancer of split 1 (Hes1) through the Notch signaling pathway. The immune cells in the stroma that suppress or promote the tumor growth include tumorassociated macrophages (TAMs) responsible for the secretion of an enzyme called cytidine deaminase. This enzyme degrades gemcitabine in cancer cells, and the cancer cells develop chemoresistance [10]. TAMs also protract tumorigenicity by secreting interferon-stimulated gene 15 (ISG15) by the activity of IFN-β secreted by cancer stem cells [11]. The CSCs that possess abilities such as self-renewing, reoccurrence, and multilineage differentiation are hypothesized to initiate tumor development and also develop resistance against drugs. CSCs develop chemoresistance against gemcitabine and 5-FU due to the higher expression of c-Jun N-terminal kinases (JNK). CSCs are involved in the expression of CD47. This CD47 functions in phagocytosis that converses with protein-α, a signal regulatory protein, present on the macrophages of the stromal cells in the tumor environment [12]. The CD44 is a cell surface glycoprotein which plays a major role in cell-to-cell interactions, adhesion, and migration. In PC, CD44 has been modulated to multiple drug resistance (MDR). Targeting CD44 may be a strategy to suppress resistance toward drugs. The use of curcumin analogue CDF and HA conjugate of styrene-maleic acid (HA-SMA) nanomicelles against CD44 elicited a strong anticancer response. These nanomicelles also inhibited the expression of NF-κB, whose overexpression leads to proliferation and invasion responsible for the development of tumor [13]. Hypoxia is a condition that affects the amount of oxygen that reaches the tissues. This is a critical factor in tumor development and chemoresistance. These hypoxic cancer cells activate the NF-κB and stabilize the hypoxia-inducible factor-1α (HIF-1α), resulting in overexpression of cadherin, vimentin, and EMT [14]. The hypoxia condition also results in accretion of lactate dehydrogenase-A and promotes chemoresistance [15]. From the profile study of miR for sensitivity against gemcitabine and resistant PC cells, it was reported that there are 33 differentially regulated miRs [16] among which miR-497 is downregulated in PC cells and its upregulation will develop sensitization against the treatments with of gemcitabine and erlotinib [16]. Chemosensitizers are the compounds that make tumor cells susceptible to chemodrugs, and whereby repealing the activity of MDR makes chemotherapy more effective. Nutraceuticals are the plant products extracted and used as chemosensitizers such as curcumin, genistein, biochanin A, epigallocatechin-3-gallate (green tea), resveratrol, apigenin, and garcinol. This chapter focuses on curcumin and genistein and their mechanisms in chemosensitizing the PC cells against chemodrugs.

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CURCUMIN EFFECTING AS A CHEMO-SENSITIZER Curcumin (diferuloylmethane), derived from Curcuma longa (turmeric), is a natural polyphenolic pleiotropic compound extracted from perennial Asian herb C. rhizome and utilized as an additive in food. This naturally occurring compound is hydrophobic in nature and has various pharmacological benefits. It has been used as an antiinflammatory, antioxidant, and anticancer compound in vitro and in vivo. Curcumin alters various signaling pathways involved in controlling the proliferation and progression of cancer cells and is considered as a multifunctional drug because it targets various transcription factors like NF-κB, STAT3, AP-1, β-catenin, and HIF-1α [17–24]; protein kinases like Akt, MAPK, ERK, and JAK [25–28]; proteins involved in apoptosis like DNA topoisomerases, Bcl-2, p53, and survivin; growth factors like VEGF; proteins for cell cycle like cyclin D1, B, E, Chk1, P27, and P21; and various enzymes like COX-1, LOX, AMPK, and MMP [29–31] (Table 1). Li et al. demonstrated the antitumor nature of curcumin, which inhibits the growth and promotes apoptosis of tumor cells depending on the time and/or dose by downregulating the activity of NF-κB [32]. Curcumin shows its activity by damaging the DNA mediated by the arrest of the G0-G1 and/or G2/M cell cycle, upregulating cyclin-dependent kinase inhibitors p21 and p27, and downregulating the expression of cyclin B1/Cdc2 [33]. Curcumin targets various signaling pathways such as EGFR, ERK1/2, STAT3, Notch, NF-κB, COX-2, and miRNA-22 in vitro in PC cells. It potentiated the activity of the chemodrugs like gemcitabine and celecoxib when used

TABLE 1 Molecular Targets of Genistein and Curcumin Natural Compound

Cell Cycle

Transcription Factors

Apoptosis

Others

Genistein

" p27

#STAT3 and STAT5

"Caspases

#COX-2

"p21

#HIF-1α

"Bax

#MMP-9 and MMP-2

"p16

#Wnt

"PARP

#PTEN

#Cyclin D1 and Cyclin B1

#Notch-2

#Bcl-2

#ERK

#CDK

#NF-κB

#BCL-xL

#MAPK

#AP-1

#Survivin

#Cyclin B1

#NF-κB

"Caspases

#VEG

#CDK1

#Sp1, Sp3, and Sp4

"Bax

#E-cadherin

"p21

#Notch

#Bcl-2

#Vimentin

" p27

#STAT3

#Survivin

#MiR-21

Curcumin

"MiR-220 "GRP78 #Cullin3

GENISTEIN EFFECTING AS A CHEMO SENSITIZER

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in combination therapy. It enhanced the activity of gemcitabine in vitro by inducing apoptosis and inhibited the activity of NF-κB [34, 35]. In vivo, curcumin enhanced gemcitabine activity of anti-angiogenesis, antiproliferation, and proapoptosis by downregulating the functional pathway of NF-κB responsible for the expression for various genes like COX-2, VEGF, Bcl-2, Bcl-xL, cyclin D1, and inhibitors of apoptosis protein, thereby decreasing the size of tumors in mice [35], and in celecoxib, curcumin potentiated its antiproliferative and proapoptotic effects on PC cells [36]. Curcumin shows 31% inhibition against the cytochrome P450 2 J2(CYP2J20) enzyme, which is highly expressed in the tumor cells, promoting its growth [37]. This enzyme catalyzes the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs) and together regulates the progression and metastasis of the tumor [38].

GENISTEIN EFFECTING AS A CHEMO SENSITIZER Genistein (4,5,7-trihydroxyisoflavone) is a natural isoflavone derived from soy products and phytoestrogen with antineoplastic activity. The soy-derived isoflavone is a very good antioxidant, antiangiogenic, and immunosuppressive and is beneficial against some degenerative diseases in human. The naturally occurring compound shows various pleiotropic biological effects against cancer, in vitro and in vivo [39–43], and has low toxicity in normal cells. It functions by inhibiting tyrosine kinase proteins intracellularly, disrupting signal transduction, and inducing cell differentiation. It also shows its activity by inhibiting the topoisomerases I and II, causing fragmentation of DNA and apoptosis. It inhibits the protein histidine kinase and induces G2/M cell cycle arrest. From the study of population analysis, it was determined that consumption of genistein can decrease the mortality rate in cancer patients, predominantly in prostate and breast cancer [44–46]. Genistein brings changes morphologically in PC cells dependently. Yi et al. [47] showed that in the natural compound, flavonoid shows anticancer activity by arresting the cell cycle at the phase G0/G; changes in STAT3 pathway and apoptosis may be due to induction of ROS mediated by the mitochondria. Genistein shows its activity by regulating the pathways involved in cell proliferation, apoptosis, cell cycle, angiogenesis, and invasion. It plays a major role in suppressing the migration of PC cells and metastasis by downregulating the expression of miR-223. It targets various proteins and enzymes involved like caspases, Bcl-2, Bax, ERK1/2, NF-κB, Wnt, and AKT/PI3K (Table 1). They are advantageous because they can potentiate the functionality of chemodrugs such as gemcitabine, cisplatin, 5-FU, and oxaliplatin, when used in combinational therapy [48–50]. Genistein enhances the activity of 5-FU in inducing autophagic and apoptotic cell deaths, altering the expression of Bcl-2 and beclin-1. Bcl-2, an antiapoptotic protein, develops resistance against chemotherapeutic drugs [51]. It acts by altering the expression of proapoptotic protein caspase-9 in vitro, with an increase in rate when treated [52]. It functions by inhibiting autophagy, wherein it binds to the beclin-1 at its BH3 domain and negatively controls the beclin-1/VPS34 complex, which is an autophagy-promoting complex [53]. The matrix metalloproteinases (MMPs) function in remodeling tissues, homeostatic cell movement, cell growth, and proliferation. They develop an environment for the tumor to develop and release various growth factors. It was observed in vitro that treatment with genistein, the expression of MMP-9 in PC was decreased [54]. It controls the expression of

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MMP-9 by targeting the protein forkhead box protein M1 (FoxM1), which plays a role in progression of the cell cycle through the checkpoint G2/M phase by regulating the transcription of genes involved in cell cycle like cyclin B, cyclin D2, p21, p27, cdc25A, and cdc25B [54]. FoxM1 is also involved in cell invasion and is critically responsible for the development of the tumor and metastasis. Thus, genistein inhibited the activity of FoxM1 expression and its target genes. Zhiwei et al. [54] found that downregulation of FoxM1 activity is more effective if transfected with siRNA along with genistein in inhibiting growth and inducing apoptosis of PC cells. Small interference RNA (siRNA) is transfected into the target gene to silence its activity and also increases gemcitabine efficiency on the PC cells. In the adjuvant therapy, genistein controlled the activity of NF-κB by suppressing cisplatin and gemcitabine, which induced the activity of NF-κB, thereby enhancing the activity of chemotherapy [39, 40, 55]. Genistein shows an inhibitory effect on the CYP2J20 enzyme by 20% in order to suppress the progression of the tumor cells [37].

SIGNALING CASCADES Studies from various pancreatic cancer cases revealed that there are almost 63 genetic abnormalities including point mutations. These mutations led to the aberrant signaling pathways like EGFR, STAT3, and Notch and resulted in greater PC cell survival, proliferation, angiogenesis, and metastasis [56, 57].

EGFR The genetic variations in EGFR cause the overexpression and multigene mutations of various transcription factors and suppressor genes leading to PC and provoke resistance of PC cells against the chemodrugs and radiations. Thus, combining EGFR blockers like erlotinib and inhibitors of Akt/NF-κB would be more effective in inhibiting the EGFR signaling pathway and sensitizing PC cells to EGFR blockers/chemodrugs. The activated EGFR, the tyrosine kinase domain, phosphorylates PI3K/AKT and Ras to activate them. The activated AKT consecutively activates NF-κB, which in turn leads to the transcription of genes responsible for cell growth, progression, proliferation, angiogenesis, and apoptosis like COX-2, survivin, Bcl-2, and Bcl-xL. The Ras pathway phosphorylates MAPK and thus activates its translocation to the nucleus where it controls the expression of factors responsible for cell proliferation. Ninety to 95% of PC is caused in smokers due to mutations of the Ras pathway. Naveen et al. developed a novel form of curcumin, CMC2.24, to potentiate the chemodrug and observed the effect of CMC2.24 on Ras activation to induce apoptosis; in addition, it also showed its impact on decreasing the phosphorylation of ERK; it augmented the superoxide anion level in the mitochondria and reducing the ATP levels and inducing apoptosis by increasing the levels of caspase-9 [58]. In the intracellular cross talk between signaling of the EGFR and COX-2 pathways of COX2 [59, 60], the EGFR pathway is transactivated by the COX-2 product PGE2 through the four G-protein-coupled receptors leading to PC cell growth. Shahar et al. [61] showed from their study that curcumin inhibits cell survival of PC by downregulating the expression of EGFR

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and COX-2 and inhibiting the activity of Erk1/2. The cross talk of EGFR and COX-2 is in such a way that EGFR signaling activation leads to the transcription of AP-1-induced COX-2 and PGE2 production [62] whereas increase in COX-2 transcription eventually enhanced the production of PGE2, which in turn activated EGFR expression through various pathways [60, 63, 64]. When treated with curcumin, it inhibited EGFR in the COX-2-positive PC cells rather than the COX-2-negative cells [61]. Thus, curcumin inhibited COX-2, leading to downregulation of EGFR, modulating signaling molecules like ERK1/2, and inhibiting PC cell survival and enhanced apoptosis. The microarray experiment conducted by Jianfeng et al. on the PC cell lines showed that genistein alters the expression of almost 47 genes, inhibiting various pathways involved in carcinogenesis mainly targeting EGFR signaling affecting genes like EGFR, egr-1, CYP1B1, NELL2, and AKT2 involved in the pathway [65]. The chemopreventive activity of genistein is due to the inhibition of the tyrosine kinase of EGFR. It inhibits PI3K activity to suppress the growth of PC cells, induces apoptosis, and chemosensitizes PC cells consequently inhibiting AKT and NF-κB [39, 66–68]. The combination therapy of genistein and erlotinib is effective. Genistein potentiated erlotinib inhibition of NF-κB and Akt, which also enhanced the inhibition of the EGFR pathway by downregulating its expression. The PI3K signaling is activated by the EGFR leading to the phosphorylation of Akt. The phosphorylated Akt is involved in downregulating the proapoptosis of the molecules involved in apoptosis. Molecules like BAD, caspase-9, and FOXO1 are regulated. FOXO1 is categorized in the same group of transcription factors that are the critical regulators of cancer cells, control cell proliferation, survival, apoptosis, cell cycle, DNA repair, and stress resistance [69]. The overexpression of FOXO1 causes arrest of cells at the G1/S phase induced by the downregulation of cyclin D and upregulation of P21 and P27 [70, 71]. The active FOXO1 follows the mitochondrion-dependent caspase pathway for apoptosis, whereas the expression of Bcl-2 and Bax is downregulated and upregulated, respectively, thereby bringing an imbalance between them, eventually leading to the activation of caspase-9 and caspase-3 followed by apoptosis. However, the PI3K/AKT pathway is involved in inactivating it, whose activation is therapeutically advantageous. Curcumin causes the cell cycle arrest by promoting the expression of P21 and P27 and suppressing the expression of cyclin D1. Curcumin endorses apoptosis in PC by decreasing the ratio of Bax/Bcl-2 and increasing caspase-9/caspase-3. The natural compound inhibits siRNA-transfected FOXO1 and induces FOXO1 expression by increasing the activation of PTEN and inhibiting the PI3K and AKT pathways, which regulates the expression of FOXO1. This pathway promotes cell survival, suppressing the apoptosis [72], and mediates resistance against chemotherapeutic drugs. This pathway also regulates NF-κB expression through another mechanism. Curcumin and its analogues inhibit the activity of Akt signaling, resulting in inhibiting the pancreatic cancer cell survival through controlling the proapoptotic activity [73–76]. The AKT signaling pathway regulates the cell survival and is very essential for developing chemoresistance. Genistein enhances the chemotherapeutic effectiveness of gemcitabine by inhibiting the AKT pathway. Phenoxodiol (analogue of genistein) inhibits the AKT pathway, activates caspase, inhibits the expression of X-linked inhibitor of apoptosis protein (XIAP), an inhibitor of apoptosis, and dislocates the expression of FLICE/caspase-8 inhibitory protein to increase the sensitivity of PC cells to chemodrugs. Genistein is used in conventional therapy, in combination with erlotinib, and regulates EGFR activity by inhibiting PI3K, and with

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gemcitabine, it inhibits the activities of Akt and NF-κB. These result in the inhibition of PC cell growth by inhibiting cyclin, survivin, Bcl-2, Bax, enhancing apoptosis, controlling expression of COX-2, and MMP9 to regulate angiogenesis, invasion, and metastasis. This nuclear factor κB (NF-κB) is a transcription factor, ubiquitous in nature, and plays a central role in controlling the apoptotic pathway. It can be regulated by many conditions like hypoxia and chemodrugs and is responsible for gene transcription, which affects proliferation, inflammation, angiogenesis, and metastasis, and is also responsible for developing chemoresistance [77]; when compared with the normal cells, NF-κB is constantly activated in PC cells [78–80]. Thus, NF-κB has emerged as a novel target for cancer therapy to induce apoptosis of cancer cells against the chemodrugs, by activating gene transcripts. The dimeric NF-κB exists in five complex forms of Rel proteins like p65, c-Rel, RelB, p50, and p52 [80] among which the most common forms are p65 and p50. In general, NF-κB remains inactive in the cytoplasm due to the activity of its inhibitor IκB. This transcription factor (NF-κB) gets activated by TNF-α, IL-1β, ROS species, and chemotherapeutic agents by degrading IκB [81] translocating the active NF-κB to the nucleus, promoting the activation of various gene transcripts like Bcl-2, Bcl-xL, X-linked inhibitor of apoptosis protein (IAP), caspases, VEGF, and regulators of cell cycles like cyclin D1 mainly involved in developing resistance to apoptosis. Apoptosis of a cell is also dependent upon caspases, which are inhibited attached and inhibited by the family IAP (cIAP1, cIAP2, survivin, and XIAP) [82]. NF-κB regulates the production of these IAPs; their expression is also responsible for resistance against gemcitabine [35, 83–87]. Curcumin effectively reduces the expression of IAPs at the levels of mRNA and protein resulting in cell death in vitro, which is very critical in developing resistance. Thus, curcumin downregulates NF-κB and sensitizes the cancer cells to chemodrugs [88] since the signaling of NF-κB is significant to control cell growth, stress response, and apoptosis. Curcumin blocks the activation of IKK, thereby blocking the phosphorylation and degrading it. It downregulates the Notch signaling pathway in order to control the activity of NF-κB, which is a novel approach in the treatment of PC. NF-κB is also activated by the activation of Akt (protein kinase B) or the IKK (IκB kinase), which mediates in the degradation of IκBα (κB inhibitor). This lets NF-κB free in the cytoplasm and is translocated to the nucleus and binds to the DNA. The binding of this transcription factor to DNA at the target gene sites augment the expression of VEGF (angiogenesis), survivin (inhibitor of apoptosis), and matrix metallopeptidase 9 (MMP-9) responsible for cell growth, progression, invasion, metastasis, angiogenesis, and apoptosis of tumor cells. The PC cells upregulate the activity of NF-κB [78]. These transcription factors are overexpressed in PC and are sensitized by curcumin, used as a combinational treatment with gemcitabine in vitro and in vivo [35, 89]. Curcumin exhibits its activity by downregulating the activity of transcription factor specific proteins (Sp1, Sp3, and Sp4) and NF-κB p50 and p60. At the G0 phase of the cell cycle, curcumin blocks cyclin D1 and CDK complex formation essential for progression of the cell cycle from G1 to the S phase; thus, cyclin D is downregulated causing cell cycle arrest at G0-G1 phase along with transfected P65 siRNA [35]. Since the S phase is a major phase for DNA replication [90], curcumin along with the chemodrug may control the PC cell growth by controlling the pathways involved in DNA repair, increasing the apoptosis. The yellow curcumin shows its antiproliferative activity in prostaglandins (PGE); these are the autocrine and paracrine lipid mediators produced by the catalyzation of the cyclooxygenase (COX) enzyme. The COX enzyme regulates the cancer cell proliferation

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by regulating the function of PGE. The COX-2 enzyme production is deficient in healthy tissues but is produced by growth factors and promoters of cancer during carcinogenesis, which in general is upregulated; thus, this enzyme is to be inhibited in order to control the progression of cancer cells. Curcumin is a very strong inhibitor of COX-2 and various other growth and transcription factors like EGFR, NF-κB, lipoxygenase (LOX), ERK, and inducible nitric oxide synthase (iNOS) by downregulating their expressions [61, 91]. Curcumin has been used together with gemcitabine as a combinational therapy by inhibiting the proliferation and promoting the proapoptotic activity [34]. Its analogue difluorinated curcumin (CDF) also acts as an inhibitor in downregulating EGFR, NF-κB, COX-2, Bcl-xL, and VEGF with cancer stem cell (CSC) signatures [92, 93]. Curcumin also shows its effect on PC cells by damaging the DNA, mediated by the cell cycle arrest at the G2/M phase; inhibits the expression of cyclin and Cdk; and activates the checkpoint kinase 1 (Chk1) [94]. Genistein increases the apoptotic effect of gemcitabine and erlotinib by inhibiting the activity of NF-κB by downregulating the expression of EGFR, BCL-xL, Bcl-2, cyclin D1, MMP-9, and survivin and sensitizes PC cells [95]. Sanjeev et al. observed that genistein with gemcitabine potentiated the activity of inhibiting PC cell growth and chemosensitizing them by downregulating NF-κB and Akt and substantially downregulated antiapoptotic molecules Bcl-2 and Bcl-xL, where gemcitabine alone upregulated them and resulting in developing chemoresistance in PC cells. Their study also was confirmed in vivo by targeting NF-κB alone by genistein evading gemcitabine-induced activation in chemosensitizing cells of pancreatic tumor [39]. Yiwei et al. [66] found that the chemodrugs docetaxel or cisplatin promoted the binding activity of NF-κB to DNA, resulted in chemoresistance. However, pretreatment with genistein suppressed the activation of NF-κB, induced by docetaxel or cisplatin. Thus, genistein blocks the NF-κB activation to increase the rate of apoptosis and chemosensitizes PC cells. Genistein can also block the activity of NF-κB under an oxidative stress condition by oxidative stress inducers like H2O2 and TNF-α [96]. It was determined that the COX-2 inhibitor SC236 also suppresses the activity of NF-κB DNA binding and gene transcription mediated by NF-κB and is independent of IKK and IKBα and directly targets proteins responsible for translocation of NF-κB into the nucleus [97]. From the study of Y Li et al. [68], treatment of PC cells with docetaxel or cisplatin including NF-κB and cDNA transfection resulted in chemoresistance by increasing the expression of NF-κB p65 eventually inducing the binding activity of NF-κ B-DNA in PC cells; however, they found that PC cells treated with genistein and p65 siRNA inhibited p65 expression and NF-κB-DNA binding at a similar degree, which also suggested that they potentiated the chemotherapeutic agent activity. Thus, the inhibitors of NF-κB-curcumin and genistein inhibit the expression of VEGF, thereby suppressing the angiogenesis of PC cells [35, 85].

VEGF Angiogenesis is the growth propagation of tumor. In PC, hypoxia-inducible factors (HIF) and NF-κB are regulated by Hsp90 and act as angiogenic factors. VEGF is regulated by the hypoxia-inducible factor-1(HIF-1), which is activated by hypoxia. Genistein treated with PC cells inhibits the activity of tyrosine kinase and HIF-1 in a dose-dependent manner.

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Genistein blocked the binding of hypoxia to DNA that resulted in reduction of VEGF mRNA expression [98]. Curcumin analogues (EF31 and UBS109) control the expression of VEGF, COX-2, and HSP-90 by downregulating them [99]. However, factors like IL-α, EGF, FGF, and TGF-α also induce angiogenesis; therefore, a combination therapy that targets all the factors in multiple pathways is essential.

STAT Pathway Signal transducer and activator of transcription factors STAT1 and STAT3 act differently; STAT1 shows an antioncogenic activity and STAT3 an oncogenic activity by the activity of interferon-α and interferon-γ, respectively. STAT3 signal pathway regulates the signal transmission of various growth factors and proteins responsible for survival, development, differentiation, and angiogenesis. Interleukins and tumor necrosis factors activate the pathway. The pathway regulates cell proliferation by cyclin D1 upregulation and inhibits apoptosis by Bcl-2 and Bcl-xL upregulation. The PC cells’ own resistance against the chemotherapy and radiation therapies is because of the presence of active STAT3 in pancreatic CSCs. Genistein shows its activity on STAT3 pathway by preventing the phosphorylation of STAT3 and downregulating proteins like survivin and cyclin D1 [99]. Curcumin controls the STAT3 pathway and inhibits the expression of survivin or BIRC4, which are responsible for antiapoptotic activity in PC, through phosphorylating STAT3 [85]. The analogues of curcumin (FLLL31 and FLLL32) inhibit the phosphorylation of STAT3 by selectively binding to the JAK2 and Src homology 2 domain of STAT3 and also inhibit binding activity of DNA [100]. The strategy of the therapy should be in such a way that the curcumin analogue targets the EGFR and JAK upstream, inhibiting the SH2 domain of STAT3, restraining the translocation of STAT3 into the nucleus, and hindering its transcriptional activity since STAT3 acts in different signaling pathways.

Mitochondrial Pathway Apoptosis may be triggered through the mitochondrial pathway at various checkpoints in the cell cycle resulting in arrest at the particular phase [101]. In this pathway, Bcl-2 and Bax protein play a major role, wherein Bcl-2 acts as an antiapoptotic and proapoptotic protein, which regulates the permeability of the mitochondrial membrane [102]; on the other side, Bax is an apoptotic protein, which is in the mitochondrial outer membrane. Bax protein functions in discharging cytochrome c and stimulates caspase-9 and eventually activates caspase3 by the proteolytic cleavage. Genistein shows its activity as an apoptotic agent by reducing the potentiality of the mitochondrial membrane, Bcl-2 downregulation, and Bax upregulation, resulting in apoptosis enhancing the concentration of caspase-3 and caspase9. Genistein also targets matrix metalloproteinases, whose loss leads to changes in the potentiality of the mitochondria and also causes cytochrome c release into the cytoplasm [47]. Thus, genistein may target apoptosis by reducing MMP mediated by ROS, which are essential during the anticancer activity. MMP-2 and MMP-9 play their role in the migration of PC cells; therefore, genistein inhibits the expression of MMPs and VEGF of mesenchymal stem cells in order to inhibit metastasis [103].

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Curcumin, the natural chemopreventive agent, also shows its activity in the mitochondrial pathway. However, due to its limitation as the lower bioavailability of its analogue, bisdemethoxycurcumin (BDMC) promotes apoptosis against gemcitabine-resistant PC cells, detected to show dysfunction in the mitochondria and abnormality in the oxidative stress response. The oxidative stress is a disproportion between factors of antioxidant and prooxidant intracellularly, and the mitochondrial dysfunction causes an imbalance between the redox environment of cells leading to abnormality in the oxidative stress targeting damage in DNA of PC cells [104]. The work of H. yang et al. [105] illustrated that BDMC, when treated with or without gemcitabine, increases the levels of superoxide anions intracellularly and reduces the content of glutathione in PC cells. It also shows its activity in reducing the potentiality of the mitochondrial membrane and in Bax-to-Bcl-2 ratio, meant for cell death and survival. H. Yang et al. [105] also demonstrated about the relationship between cullin and glucose-regulated protein 78 (GRP78), cullin controlled by BDMC targeting GRP78 whose upregulation controls the expression of cullin3. Cullin3 is a scaffold protein substrate for E3 ligases and functions in stress responses, and GRP78 is a protein chaperon that mediates endoplasmic reticulum stress-induced apoptosis [105, 106]. In the case of the PC cell microenvironment, cullin3 is overexpressed and is absent in normal tissues [107]. Thus, cullin3 with GRP78 can be targeted by BDMC, wherein from the analysis of bioinformatics protein-protein interactions, it was observed that cullin3 and GRP78 are juxtaposed with one another and activation of GRP78 inhibits cullin3. Thus, BDMC assists apoptosis by upregulating GRP78 through the elF2α pathway. DJ-1 and prohibitin are the two proteins overexpressed in the microenvironment of tumor after gemcitabine treatment showed chemoresistance [108– 110]. These two are targeted by BDMC, wherein the activity of GRP78 can downregulate the expression of DJ-1 but prohibitin is independent of GRP78 activity. Thus, BDMC is highly effective in sensitizing PC cells resistance against gemcitabine.

MiRNAs MicroRNAs are the noncoding RNAs and do not code to any proteins. They hold nucleotides of about 18–22 and downregulate the expression of various target genes responsible for cell proliferation, progression, and apoptosis and eventually develop drug resistance. The development of drug resistance by miRNA is due to the EMT, cancer stem cells, and transporters related to developing resistance against multidrugs [111–113]. From the analysis of an miRNA array conducted using PC cells, healthy pancreatic epithelial cells, and serum, it was observed that miRNA expression was aberrant in the PC cells, their variation leading to the development of drug resistance. When treated with gemcitabine, the levels of miR-320 [111] is elevated, and the level of miR-200 reduced is in PC cells resistant to gemcitabine [114]. MiRNAs function in regulating various factors like EGFR, Akt, NF-κB, p53, TGF-β, and K-ras by controlling their cell cycle, repair DNA, apoptosis, metastasis, and invasion. MiR-21 and miR-221 are expressed at higher levels whereas miR-34 and miR-200 in reduced levels in the PC cells. Thus, targeting the miRNA for the therapy of PC is the better strategy; however, it is miR-21 that develops chemoresistance in the treatment of PC. Curcumin analogue CDF is used in combinational therapy with gemcitabine in order to sensitize the PC cells. CDF inhibits the expression of miR-21 and also upregulates the expression of various

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tumor-suppressive miRNAs (miR-146a, miR-200, miR-3656, and let-7 family) [93, 115]. During the process of sensitization, CDF inhibits the ability in the formation of sphere in PC cells and degrades the pancreatospheres. The curcumin analogue in the gemcitabine-resistant PC cells downregulates the CSC marker genes [93]. CDF can also sensitize the PC cells by controlling the CSC signature genes (cancer stem cells) mediated by miRNA, Notch-1, VEGF, AKT, and IL-6 signaling pathways [74, 93, 115]. The cancer stem cell develops resistance eventually against chemo- and radioexposure, since they have extensive DNA repair mechanisms. They slow the doubling rate and have a drug efflux pumping system. It was identified that the radiations have increased the count of CSC, due to high level of checkpoint kinases (Chk1/ Chk2 kinases). These CSCs have gained chemoresistance due to the activation of stimulation of IL-4 receptors, AKT pathway activation, and ATP-binding cassette transporters (ABC) like multidrug resistance transporter1 (MDR1). It was demonstrated from the reports that miRNAs play a key role in regulating EMT, wherein miR-223 inhibition could reverse the EMT and also can improve the sensitivity of PC cells against chemotherapeutic drugs. Jia Ma et al. [116] reported that genistein downregulates the expression of miR-223 in gemcitabine-resistant PC cells. They demonstrated that genistein inhibited the growth of PC cells depending on the dose by downregulating the expression of miR-223. Genistein with inhibitor miR-223 causes morphological changes (elongated to round) to reverse EMT of PC cells [117] and also upregulates the expression of E-cadherin (calcium-dependent adhesion). It was proved that F-box/WD repeat domain-containing protein 7 (Fbw7) is the target for miR-233 in order to regulate EMT since FBW7 suppresses EMT and also sensitizes the cells to chemodrugs. Thus, it was proven in vivo that the combinational treatment of miR-223 and genistein sensitizes the PC cells against gemcitabine, whereas genistein promotes the antitumor activity of miR-223 inhibitor by regulating the Notch-1 pathway and EMT. Genistein also inhibits the expression of miR-27a in PC cells by suppressing the growth of the PC cells, inhibiting invasion, and inducing apoptosis [118].

Notch Signaling Pathway The Notch signaling pathway plays a role in cell survival, progression, apoptosis, invasion, and migration of PC cells mediated by the ligand receptor [119]. The pathway gets activated when the ligand binds to the receptor; thereby, the Notch gets cleaved releasing the Notch intracellular domain (NICD) through a cascade by the multiple enzymes like γ-secretase [120], translocated to nucleus. In the nucleus, it activates its target genes like cyclin D1, Bcl-2, and C-myc [121]. The fate of CSCs is decided by the Notch signaling pathway; it is even reported that expression levels are high for Notch-1 and Notch-2 in pancreatic CSCs [122, 123]. Notch-1 is also involved in another apoptotic regulatory pathway, inducing NF-κB. MiR-34a regulated the expression of Notch-1 and Notch-2, which are the downstream genes of miR-34 and are responsible for self-renewal of CSCs. Thus, Notch signaling pathway is also involved in building up resistance against drugs and can be taken as a novel strategy for the therapy [124]. Using the natural compounds, curcumin and isoflavone inhibit Notch-1 signaling expression and its related genes like Hes1, NF-κB, Bcl-xL, and cyclin D1 responsible for developing resistance against chemodrugs [125, 126]. PC cell growth can be controlled by inhibiting Notch-1 expression by the upregulation of miR-34a [127].

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EPIGENOME CHANGES The chemical changes in DNA and histone proteins that are heritable are included in an epigenome. The changes are like methylation in DNA and histone, which causes changes in functionality of the genome. Thus, organ development and differentiation of tissue are controlled by the epigenome by regulating the expression of genes without changing the sequence of DNA. However, enzymes like histone deacetylases (HDAC), DNA methyltransferases (DNMT), and histone acetyltransferases control the regulation of the epigenome. These epigenetic regulations are observed in most of the cases of PC, and overexpression of HDAC in PC cells develops chemoresistance in the epigenome [128, 129]. In another way, DNMT is also highly expressed in PC cells, which causes methylation in DNA of the tumor-suppressing genes, thus inactivating them [129]. Fu S et al. found that curcumin acts as an epigenetic mediator and can interact with DNMT-1, HDAC, and histone acetyltransferases [130] and thus inhibits PC. Curcumin and its analogues (CDF) can downregulate the expression of EZH2 [enhancer of zeste homologue-2 (histone methyltransferase)]. EZH2 is an essential regulator of the epigenome and controls functions of proliferation, apoptosis of cells, and cancer stem cells (CSCs). Thus, CDF inhibits the cell survival, the formation of pancreatosphere capacity, and cell migration of PC cells [115]. The expression of DNMT is also regulated by the curcumin analogues EF31 and UBS109, which inhibit HSP-90 and NF-κB and, in turn, downregulating DNMT [131]. Genistein also modulates methylation of DNA by upregulating the expression of miRNA and tumor suppressor genes to inhibit the PC cells [132, 133]. Genistein shows its activity in protecting β cells that are associated with type 2 diabetes by regulating epigenetically the cAMP/PKA signaling [134].

BIOAVAILABILITY Curcumin is a safe, nontoxic agent and is the most effective agent to improve antitumor drugs. Its limitation in pharmacokinetic profile and low bioavailability are the major obstacles; however, the technique from nanotechnology has formulated curcumin as an encapsulated nanoparticle; the use of analogues of curcumin (CDF, FLLL11, and FLLL12) and liposomal formulated curcumin have been built up. The anticancer efficacy through the use of liposomes has been evaluated by Li et al. on various PC cell lines including HS766T, ASPC-1, and Capan-2. Due to its low availability, solubility, and poor physiological condition, curcumin as such has gain less importance in vivo. However, it has been used in combination bonded with a backbone. The nanoparticle poly(curcumin-dithiodipropionic acid) (PCDA)-polyethylene glycol (PEG)-biotin copolymer is used in order to protect the hydrophobic diarylheptanoid (curcumin) from hydrolysis at physiological pH [135, 136]. The PCDA backbone is then degraded due to the high concentration of GSH in the cytoplasm. The curcumin and the chemodrug get parted away; curcumin then shows its activity as a chemosensitizer and downregulates the expression of P-gp and inhibits the activity of ATP. The cytotoxicity of the chemodrug is increased by getting accumulated intracellularly by the asset of chemosensitization. Curcumin analogue GO-Y030 is used as in combinational

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therapy whose bioavailability is high when compared with curcumin and sensitizes the PC cells when used with gemcitabine. It shows its activity by inducing the activation of NF-κB by suppressing its transactivation. They target the JNK/AKT molecule upstream of IKK-NF-κB and TNF-α receptors. Other curcuminoid analogues are bisdemethoxycurcumin (BDMC) and desmethoxycurcumin (DMC) that are stable; however, investigation is still carried on to elucidate their efficacy in combination therapy against human cancers. The derivatives of genistein have been proven to augment the antitumor activity experimentally in vitro and in vivo and are used in the combinational therapies. The isoflavinoid analogues like phenoxodiol and triphendiol are more potent than genistein that induce apoptosis mediated by caspase-dependent and caspase-independent mechanisms in PC cell lines [137]. Wang X et al. from their work demonstrated the effectiveness of triphendiol monotherapy and as a sensitizer of gemcitabine. The synthetic isoflavone in monotherapy induces G2/M cell cycle arrest independent of P53 in order to inhibit the proliferation of PC cells and enhances the activity of the mitochondrial pathway for apoptosis of PC cells. Triphendiol is granted with investigational novel drug status by the US Food and Drug Administration to be used as a combo drug with an gemcitabine to treat patients with early and late stages of PC [138]. AXP107–11 is a multicomponent crystalline form of the soy protein genistein, said to possess better bioavailability and physiochemical properties when compared with the natural form of genistein. Combining AXP107-11 with gemcitabine may decrease the chemoresistance [139].

CONCLUSION PC remains one of the most complicated malignancies and a challenging therapy for the physicians and researchers. Malignancies and difficulties in treating pancreatic cancer may come from high metastasis, drug resistance, tumor relapse, and poor prognosis. The limitation for current therapies is because of their one-sided actions on one or two pathways, instead of multitarget of cancer pathways; thus, the traditional therapies have been encouraged. The studies of epidemiology showed that polyphenols and flavonoids sensitize the tumor cells to chemodrugs suppressing the signaling pathways involved in developing resistance. The use of natural bioactive products as novel preventive and chemotherapeutic drugs has been clinically proven due to their safety profile and PC cells developing resistance against conventional therapy. The development of combinational therapy is a very good approach for the abnormalities of multigenes in PC cells, but the toxicity is the concern. Natural flavonoids and polyphenols are considered the best chemopreventive agents in vitro and in vivo. They potentiate the conventional therapies and therapies like anti-EGF and antiVEGF in regulating various factors responsible for growth, proliferation, angiogenesis, and apoptosis. Both the natural compounds curcumin and genistein are highly involved in apoptosis and are complex in targeting multiple pathways. Curcumin can thus be considered as multifunctional drug performing multimolecular mechanisms, which exerts antitumor effects on PC cells. Curcumin synergistically reduces inhibition and migration of tumor cells, reversing EMT, suppression of COX-2, and miR221 and inhibiting CSCs [140, 141], which are responsible for developing resistance. They interrupt the pathways by blocking the phosphorylation of STAT1 and STAT3 (signal transducer and activator transcription protein), EGFR, and Notch-1 signaling pathway [115].

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Genistein has gained importance with its properties ranging from its antioxidative to antitumor nature and has been proven as a novel compound of interest in vitro and in vivo. The natural flavonoid is pleiotropic in nature and shows its activity in cell cycle arrest, angiogenesis, metastasis, and pathways involved in apoptosis by controlling the expression of Akt, NF-κB, Bax, Bcl-2, and MMP. The tumor cells are multifactorial in nature; thus, one-drug-one-target theory is no more a regime and not successful since the cancer cells developing chemoresistance and the healthy cells also developing toxicity due to the conventional therapies. The natural herbal products have been used in adjuvant therapy, but because of their bioavailability, they have given less importance though their usage is less toxic to normal cells. Thus, compounds with the similar molecular form and activity are to be identified, and better derivative and analogue forms of these natural compounds with better bioavailability are being developed to potentiate the chemodrugs and sensitize the PC cell to these drugs. Thus, from the reviews, the use of herbal products in the adjuvant therapy to sensitize the PC cells to chemodrug can be developed as a novel and promising strategy in the field of cancer therapy.

Acknowledgment We thank our institute for providing the resources required to complete this work.

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[103] Yazdani Y, Rad MRS, Taghipour M, Chenari N, Ghaderi A, Razmkhah M. Genistein suppression of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF) expression in mesenchymal stem cell like cells isolated from high and low grade gliomas. Asian Pac J Cancer Prev 2016;17:5303. [104] Mencalha A, Victorino VJ, Cecchini R, Panis C. Mapping oxidative changes in breast cancer: understanding the basic to reach the clinics. Anticancer Res 2014;34:1127–40. [105] Yang H, Fan S, An Y, Wang X, Pan Y, Xiaokaiti Y, Duan J, Li X, Tie L, Ye M. Bisdemethoxycurcumin exerts pro-apoptotic effects in human pancreatic adenocarcinoma cells through mitochondrial dysfunction and a GRP78-dependent pathway. Oncotarget 2016;7:83641. [106] Firczuk M, Gabrysiak M, Barankiewicz J, Domagala A, Nowis D, Kujawa M, Jankowska-Steifer E, Wachowska M, Glodkowska-Mrowka E, Korsak B. GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy. Cell Death Dis 2013;4:e741. [107] Haagenson KK, Tait L, Wang J, Shekhar MP, Polin L, Chen W, Wu GS. Cullin-3 protein expression levels correlate with breast cancer progression. Cancer Biol Ther 2012;13:1042–6. [108] Patel N, Chatterjee SK, Vrbanac V, Chung I, Mu CJ, Olsen RR, Waghorne C, Zetter BR. Rescue of paclitaxel sensitivity by repression of Prohibitin1 in drug-resistant cancer cells. Proc Natl Acad Sci U S A 2010;107 (6):2503–8. [109] Chen Y, Kang M, Lu W, Guo Q, Zhang B, Xie Q, Wu Y. DJ-1, a novel biomarker and a selected target gene for overcoming chemoresistance in pancreatic cancer. J Cancer Res Clin Oncol 2012;138:1463–74. [110] Thuaud F, Ribeiro N, Nebigil CG, Desaubry L. Prohibitin ligands in cell death and survival: mode of action and therapeutic potential. Chem Biol 2013;20:316–31. [111] Li Y, VandenBoom TG, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH. Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res 2009;69:6704–12. [112] Hage C, Rausch V, Giese N, Giese T, Sch€ onsiegel F, Labsch S, Nwaeburu C, Mattern J, Gladkich J, Herr I. The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer. Cell Death Dis 2013;4:e627. [113] Wang H, Zhan M, Xu S-W, Chen W, Long M-M, Shi Y-H, Liu Q, Mohan M, Wang J. miR-218-5p restores sensitivity to gemcitabine through PRKCE/MDR1 axis in gallbladder cancer. Cell Death Dis 2017;8. [114] Iwagami Y, Eguchi H, Nagano H, Akita H, Hama N, Wada H, Kawamoto K, Kobayashi S, Tomokuni A, Tomimaru Y. miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1. Br J Cancer 2013;109:502. [115] Bao B, Ali S, Banerjee S, Wang Z, Logna F, Azmi AS, Kong D, Ahmad A, Li Y, Padhye S. Curcumin analogue CDF inhibits pancreatic tumor growth by switching on suppressor microRNAs and attenuating EZH2 expression. Cancer Res 2011;. [116] Ma J, Zeng F, Ma C, Pang H, Fang B, Lian C, Yin B, Zhang X, Wang Z, Xia J. Synergistic reversal effect of epithelial-to-mesenchymal transition by miR-223 inhibitor and genistein in gemcitabine-resistant pancreatic cancer cells. Am J Cancer Res 2016;6:1384. [117] Ma J, Fang B, Zeng F, Ma C, Pang H, Cheng L, Shi Y, Wang H, Yin B, Xia J. Down-regulation of miR-223 reverses epithelial-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Oncotarget 2015;6:1740. [118] Xia J, Cheng L, Mei C, Ma J, Shi Y, Zeng F, Wang Z, Wang Z. Genistein inhibits cell growth and invasion through regulation of miR-27a in pancreatic cancer cells. Curr Pharm Des 2014;20:5348–53. [119] Ranganathan P, Weaver KL, Capobianco AJ. Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 2011;11:338. [120] Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene 2008;27:5124. [121] Miele L. Notch signaling. Clin Cancer Res 2006;12:1074–9. [122] Wang Z, Li Y, Banerjee S, Sarkar FH. Emerging role of Notch in stem cells and cancer. Cancer Lett 2009;279:8–12. [123] Ji Q, Hao X, Zhang M, Tang W, Yang M, Li L, Xiang D, DeSano JT, Bommer GT, Fan D. MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells. PLoS One 2009;4:e6816. [124] Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, Sarkar FH. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta Rev Cancer 2010;1806:258–67. [125] Wang Z, Zhang Y, Banerjee S, Li Y, Sarkar FH. Retracted: inhibition of nuclear factor κb activity by genistein is mediated via Notch-1 signaling pathway in pancreatic cancer cells. Int J Cancer 2006;118:1930–6.

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[126] Wang Z, Zhang Y, Banerjee S, Li Y, Sarkar FH. Retracted: Notch-1 down-regulation by curcumin is associated with the inhibition of cell growth and the induction of apoptosis in pancreatic cancer cells. Cancer 2006;106:2503–13. [127] Xia J, Duan Q, Ahmad A, Bao B, Banerjee S, Shi Y, Ma J, Geng J, Chen Z, Wahidur Rahman K. Genistein inhibits cell growth and induces apoptosis through up-regulation of miR-34a in pancreatic cancer cells. Curr Drug Targets 2012;13:1750–6. [128] Zhang J-J, Zhu Y, Zhu Y, Wu J-L, Liang W-B, Zhu R, Xu Z-K, Du Q, Miao Y. Association of increased DNA methyltransferase expression with carcinogenesis and poor prognosis in pancreatic ductal adenocarcinoma. Clin Transl Oncol 2012;14:116–24. [129] Gao J, Wang L, Xu J, Zheng J, Man X, Wu H, Jin J, Wang K, Xiao H, Li S. Aberrant DNA methyltransferase expression in pancreatic ductal adenocarcinoma development and progression. J Exp Clin Cancer Res 2013;32:86. [130] Fu S, Kurzrock R. Development of curcumin as an epigenetic agent. Cancer 2010;116:4670–6. [131] Nagaraju GP, Zhu S, Wen J, Farris AB, Adsay VN, Diaz R, Snyder JP, Mamoru S, El-Rayes BF. Novel synthetic curcumin analogues EF31 and UBS109 are potent DNA hypomethylating agents in pancreatic cancer. Cancer Lett 2013;341:195–203. [132] Wang Y, Zhou Y, Zhou H, Jia G, Liu J, Han B, Cheng Z, Jiang H, Pan S, Sun B. Pristimerin causes G1 arrest, induces apoptosis, and enhances the chemosensitivity to gemcitabine in pancreatic cancer cells. PLoS One 2012;7. [133] Li Y, Chen H, Hardy TM, Tollefsbol TO. Epigenetic regulation of multiple tumor-related genes leads to suppression of breast tumorigenesis by dietary genistein. PLoS One 2013;8:e54369. [134] Gilbert ER, Liu D. Anti-diabetic functions of soy isoflavone genistein: mechanisms underlying its effects on pancreatic β-cell function. Food Funct 2013;4:200–12. [135] Liu J, Chen S, Lv L, Song L, Guo S, Huang S. Recent progress in studying curcumin and its nano-preparations for cancer therapy. Curr Pharm Des 2013;19:1974–93. [136] Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed M, Alharbi SA, Tan BK, Kumar AP, Sethi G. The multifaceted role of curcumin in cancer prevention and treatment. Molecules 2015;20:2728–69. [137] Saif MW, Tytler E, Lansigan F, Brown DM, Husband AJ. Flavonoids, phenoxodiol, and a novel agent, triphendiol, for the treatment of pancreaticobiliary cancers. Expert Opin Investig Drugs 2009;18:469–79. [138] Wang X, McKernan R, Kim KH, Alvero AB, Whiting A, Thompson JA, Mor G, Saif MW, Husband AJ, Brown DM. Triphendiol (NV-196), development of a novel therapy for pancreatic cancer. Anti-Cancer Drugs 2011;22:719–31. [139] L€ ohr J-M, Karimi M, Omazic B, Kartalis N, Verbeke CS, Berkenstam A, Fr€ odin J-E. A phase I dose escalation trial of AXP107-11, a novel multi-component crystalline form of genistein, in combination with gemcitabine in chemotherapy-naive patients with unresectable pancreatic cancer. Pancreatology 2016;16:640–5. [140] Sarkar S, Dubaybo H, Ali S, Goncalves P, Kollepara SL, Sethi S, Philip PA, Li Y. Down-regulation of miR-221 inhibits proliferation of pancreatic cancer cells through up-regulation of PTEN, p27kip1, p57kip2 and PUMA. Am J Cancer Res 2013;3:465. [141] Sun X-D, Liu X-E, Huang D-S. Curcumin reverses the epithelial-mesenchymal transition of pancreatic cancer cells by inhibiting the Hedgehog signaling pathway. Oncol Rep 2013;29:2401–7.

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7 Terpenoids as Potential Targeted Therapeutics of Pancreatic Cancer: Current Advances and Future Directions Rama Rao Malla, Seema Kumari, Deepak K.G.K., Murali Mohan Gavara, Shailender Guganavath, Prasuja Rokkam Cancer Biology Lab, Department of Biochemistry, GIS, GITAM (Deemed to be University), Visakhapatnam, India

Abstract Pancreatic adenocarcinoma contributes to high mortality among solid malignancies, which is usually detected at an advanced stage of metastasis. Terpenoids are the major class of naturally occurring biologically active compounds and can be explored for the drug discovery with the anticancer property. The approach has to be made in identifying a variety of terpenoids against metastatic cancer. This chapter is aimed to discuss the current advances in pancreatic adenocarcinoma, including specific target of terpenoids that are associated with NF-κB signaling, caspases, DNA damage, and apoptotic proteins.

Abbreviations FAMM-PC FAP HBOC HNF1A HP KRT81 LFS PJS ROS

familial malignant melanoma and pancreatic cancer familial adenomatous polyposis hereditary breast and ovarian cancer hepatocyte nuclear factor 1A hereditary pancreatitis cytokeratin 81 Li-Fraumeni syndrome Peutz-Jeghers syndrome reactive oxygen species

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Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION Cancer is the principal cause of death worldwide, and pancreatic adenocarcinoma contributes to high mortality among solid malignancies. In spite of contributing only 3% of cancer diagnoses, the mortality for patients with pancreatic cancer has remained high [1]. Pancreatic cancers are typically diagnosed at the advanced metastatic stage. Terpenoids are the major class of natural products that are rich sources of biologically active compounds for drug discovery. Terpenoids have been traditionally used against different ailments in India and China and are being studied for treating cancer. Recent advancements in cancer drug discovery from natural sources have led to the exploration of the diverse type of terpenoids against metastasis of cancer.

PANCREATIC CANCER Pancreatic ductal adenocarcinoma presents a poor prognosis, and patients diagnosed at an advanced stage show a low rate of survival in contempt of treatment. A major reason for therapy to be unsuccessful in improving the survival rate might be due to the existence of pancreatic ductal adenocarcinoma subtypes with diverse treatments. In spite of aggressive nature, the clinical outcome has a slow progress. This is due to a shortage of novel prognostic markers that are required for the establishment of a personalized treatment strategy. Findings from different studies suggest that subtypes can be identified by immune histochemistry based on the expression of cytokeratin 81 (KRT81) and hepatocyte nuclear factor 1A (HNF1A) that have different survival outcomes and responses to chemotherapy. The other prognostic factors that are known to cause pancreatic cancer are clinicopathologic features of surgical resection, and the most prominent cancer biomarker, CA19-9, is restricted to prognostic prediction, which emphasizes the requirement of additional markers with improved prognosis [2–5].

Risk Factor of Pancreatic Cancer A risk associated with pancreatic cancer is an average of 1% chance to develop the disease. Generally, the most common causes of pancreatic cancers (90%) are considered as sporadic and somatic mutations with inherited pancreatic cancers (10%), which are transmitted through germ-line mutations. Other associated risk factors include age, gender, smoking, diabetes, obesity, and inherited conditions like hereditary pancreatitis (HP), a condition generally linked with recurrent pancreatitis; Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder; familial malignant melanoma, a condition where parents or relatives have melanoma; hereditary breast and ovarian cancer syndrome (HBOC), a condition where cases of breast and ovarian cancers are high in genetically related families; Lynch syndrome, an autosomal dominant genetic condition with high risk of colon and other cancers; LiFraumeni syndrome (LFS), an inherited condition where individuals are susceptible to rare

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cancers; and familial adenomatous polyposis (FAP), an autosomal dominant inherited condition diagnosed with polyps in large intestine. The exposure to pesticides or benzene or certain dyes or petrochemicals might increase the risk incidence of pancreatic cancer. Helicobacter pylori also causes symptoms associated with inflammation and ulcers that can increase the risk of pancreatic cancer [6–8].

TERPENOIDS Natural compounds exhibit enormous structural diversity with extensive biological activities toward various ailments including malaria and inflammation as well as cancer. Natural compounds such as “etoposide, vincristine, irinotecan, and paclitaxel” are widely being used as clinical therapeutics [9]. Terpenoids are a subclass of natural products that are used in treating skin, lung, pancreatic, colon, and prostate cancers [10]. On the basis of various number of isoprene subunits, terpenoids are categorized into hemiterpenoids with one isoprene unit (C5), monoterpenoids with two isoprene units (C10), sesquiterpenoids with three isoprene units (C15), diterpenoids with four isoprene units (C20), sesterterpenoids with five isoprene units (C25), triterpenoids with six isoprene units (C30), tetraterpenoids with eight isoprene units (C40), and polyterpenoids (>C40) [11]. Reports suggest that monoterpenoids and triterpenoids are extensively studied as anticancer agents [12], as they suppress cancer cell growth by affecting tumor cell differentiation and by inducing apoptosis or by inhibiting tumor angiogenesis, invasion, and metastasis [13].

Molecular Targets of Terpenoids in Pancreatic Cancer Terpenoids as NF-κB Signaling Inhibitors Terpenoids can inhibit the signaling of NF-κB, a key regulator in inflammation and cancer. Various pathways were discovered as targets involved in the anticancer activity of terpenoids, including induction of apoptosis. Some therapeutic indications on various terpenoids are described in the subsequent section. Monoterpenoids are composed of two isoprene units with a general molecular formula of C10H16. They exist in acyclic, monocyclic, or bicyclic forms. Monoterpenoids act as NF-κB signaling inhibitors through IκB degradation and DNA binding or by translocation of p65 [14]. Some of the monoterpenoids are described below. Aucubin, a glycoside iridoid, is a class of monoterpenoids, and the most common iridoid glycoside is aucubin (Fig. 1). According to some studies, degradation of IκBα is prevented by aucubin. Aucubin also obstructs the translocation of p65 and NF-κB complex into nucleusactivated mast cells. It has been revealed through different studies that aucubin could be a useful agent in the prevention of pancreatic cancer [15]. Moreover, α-pinene, catalposide, genipin, and limonenes are other monoterpenes with an ability to induce NF-κB-dependent apoptosis in pancreatic cancer and inhibit proliferation and metastasis [16]. Terpenoids in the Regulation of Caspase Activity Diterpenoids and triterpenoids like andrographolide, geranylgeraniol, bacoside A, and waltonitone were reported to enhance the expression pattern of proapoptotic proteins like caspase-3 and caspase-9, which suggest the involvement of the mitochondrial apoptotic pathway in the chemopreventive effect [17]. Oridonin, a diterpenoid isolated from Rabdosia

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Terpenoids

NF-kB signaling inhibitor

Caspase activator

Aucubin α-pinene

Andrographolide

Catalposide

Bacoside A Waltonitone

Genipin

Geranylgeraniol

DNA damage

β-ionone Lutein

Apoptosis activator

Diterpenoid Paclitaxel Docetaxel

Limonenes

FIG. 1 Molecular targets of terpenoids in pancreatic cancer.

rubescens, was reported to promote cytotoxic activity against pancreatic cancer, by enriching apoptotic cell death through the generation of reactive oxygen species (ROS). Oridonin appears to show the apoptotic activity by elevating the expression of p53, caspase-3, caspase9, cytochrome c (cyt c), and p38 protein accompanied by a reduced mitochondrial membrane potential (△ Ψm). Terpenoids With Targeting DNA Damage β-Ionone is one of the dietary cyclic isoprenoid derivatives rich in grapes and also wine; it represents a novel class of therapeutics against various cancers. It is mainly associated with the inhibition of pancreatic and hepatic preneoplastic lesions with reduced cell proliferation, plasma cholesterol levels, and enhanced DNA damage at initial phases of carcinogenesis triggered by DENA and 2-AAF in a rat model [18]. The study showed that β-ionone is mainly involved in the reduction of cell proliferation and regulation of HMG-CoA reductase activity [19], thus defining β-ionone as the biologically active chemopreventive agent. Also, lutein a carotenoid is a chemopreventive agent against DENA-mediated and 2-AAF-induced carcinogenesis in Wistar rats with decreased DNA damage [20]. Terpenoids Targeting Apoptotic Proteins Paclitaxel, a diterpenoid, and docetaxel are in clinical usage against well-documented pancreatic cancers. These terpenoids are mainly involved in the inhibition of proliferation and induction of cell death. The effectiveness of these terpenoids toward breast or pancreatic cancers in preclinical studies suggests the use of terpenoids for the treatment of human cancers related to hormones [21].

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Terpenoids With Differential Targets in Pancreatic Cancer Recent studies show that terpenoids arrest cell cycle at G0/G1 followed by massive apoptotic cell death [22]. Curcuminoids, in association with mono-, sesqui-, di-, and triterpenoids, exhibited significant anticancer properties that are related to the increase in drug absorption, which imparts high physiologically active than single-drug treatment [23]. MicroRNAs (miRNAs), the epigenetic regulators of cancer, are evolving as an important target in cancer therapy. Studies show that curcumin and curcuminoids are important terpenoids that are used against miRNA in pancreatic cancer [24, 25]. Moreover, ponicidin and oridonin have been associated with pancreatic stem cell differentiation. Nimbolide diminishes CD44-positive cells and enhances mitochondrion-mediated apoptosis in pancreatic cancer cells [26].

CONCLUSION Currently, terpenoids are being investigated as anticancer agents in clinical studies. Recent studies on natural products have led to the exploration of a variety of terpenoids against metastasis in cancer. A large number of terpenoids such as monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, and tetraterpenoids are being used as anticancer agents as they are target-specific. The potential activity of terpenoids against different cancers appears promising and will open up opportunities for pancreatic cancer therapy. Further, systemic exploration of epigenetic modulation leads to the discovery of novel therapeutics with high efficacy and minimal side effects against pancreatic cancer.

Acknowledgment We would like to thank DST-FIST, New Delhi (SR/FST/LSI-568/2013) and GITAM University for providing lab facilities.

References [1] Teague A, Lim K-H, Wang-Gillam A. Advanced pancreatic adenocarcinoma: a review of current treatment strategies and developing therapies. Ther Adv Med Oncol 2015;7(2):68–84. [2] Noll EM, Eisen C, Stenzinger A, Espinet E, Muckenhuber A, Klein C, Vogel V, Klaus B, Nadler W, R€ osli C, Lutz C, et al. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. Nat Med 2016;22(3):278–87. [3] Waddell N, Pajic M, Patch A, Chang DK, Kassahn KS, Bailey P, Johns AL, Miller D, Nones K, Quek K, Quinn MC, et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 2015;518:495–501. [4] Humphris JL, Patch AM, Nones K, Bailey PJ, Johns AL, McKay S, Chang DK, Miller DK, Pajic M, Kassahn KS, Quinn MC, et al. Hypermutation in pancreatic cancer. Gastroenterology 2017;152(1):68–74. [5] Schlitter AM, Segler A, Steiger K, Michalski CW, J€ager C, Konukiewitz B, Pfarr N, Endris V, Bettstetter M, Kong B, Regel I, et al. Molecular, morphological and survival analysis of 177 resected pancreatic ductal adenocarcinomas (PDACs): Identification of prognostic subtypes. Sci Rep 2017;7. [6] Raphael BJ, Hruban RH, Aguirre AJ, Moffitt RA, Yeh JJ, Stewart C, Robertson AG, Cherniack AD, Gupta M, Getz G, Gabriel SB. Integrated genomic characterization of pancreatic ductal adenocarcinoma. Cancer Cell 2017;32(2):185–203.e13. [7] Humphrey ES, Su SP, Nagrial AM, Hochgr€afe F, Pajic M, Lehrbach GM, Parton RG, Yap AS, Horvath LG, Chang DK, Biankin AV, Wu J. Resolution of novel pancreatic ductal adenocarcinoma subtypes by global phosphotyrosine profiling. Mol Cell Proteomics 2016;15:2671–85.

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[8] Sturgeon CM, Duffy MJ, et al. National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in liver, bladder, cervical, and gastric cancers. Clin Chem 2010;56(6):e1–e48. [9] Morland B, Platt K, Whelan JS. A phase II window study of irinotecan (CPT-11) in high risk Ewing sarcoma: a Euro-E.W.I.N.G. study. Pediatr Blood Cancer 2014;61(3):442–5. [10] Degenhardt J, Kollner TG, Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 2009;70(15–16):1621–37. [11] Wang L, Yang B, Lin XP, Zhou XF, Liu Y. Sesterterpenoids. Nat Prod Rep 2013;2013(30):455–73. [12] M H, Lu JJ, Huang M-Q, Bao J-L, Chen X-P, Wang Y-T, et al. Terpenoids: natural products for cancer therapy. Expert Opin Investig Drugs 2012;21(12):1801–18. [13] Kuttan G, Pratheeshkumar P, Manu KA, Kuttan R. Inhibition of tumor progression by naturally occurring terpenoids. Pharm Biol 2011;49(10):995–1007. [14] Dinda B, Debnath S, Harigaya Y. Naturally occurring iridoids. A review, Part 1. Chem Pharm Bull 2007;55(2):159–222. [15] Crowell PL. Prevention and therapy of cancer by dietary monoterpenes. J Nutr 1999;129:775S–778S. [16] Zhou JY, Tang FD, Mao GG, Bian RL. Effect of alpha-pinene on nuclear translocation of NF-kappa B in THP-1 cells. Acta Pharmacol Sin 2004;25:480–4. [17] Yoshida M, Matsui Y, Iizuka A, Ikarashi Y. G2-phase arrest through p21(WAF1/Cip1) induction and cdc2 repression by gnidimacrin in human hepatoma HLE cells. Anticancer Res 2009;29(4):1349–54. [18] de Moura Espı´ndola R, Mazzantini RP, Ong TP, de Conti A, Heidor R, Moreno FS. Geranylgeraniol and betaionone inhibit hepatic preneoplastic lesions, cell proliferation, total plasma cholesterol and DNA damage during the initial phases of hepatocarcinogenesis, but only the former inhibits NF-kappaB activation. Carcinogenesis 2005;26(6):1091–9. [19] Janani P, Sivakumari K, et al. Chemopreventive effect of bacoside A on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats. J Cancer Res Clin Oncol 2010;136(5):759–70. [20] Tharappel JC, Lehmler HJ, et al. Effect of antioxidant phytochemicals on the hepatic tumor promoting activity of 3,30 ,4,40 -tetrachlorobiphenyl (PCB-77). Food Chem Toxicol 2008;46(11):3467–74. [21] H Y, Dou QP. Targeting apoptosis pathway with natural terpenoids: implications for treatment of breast and prostate cancer. Curr Drug Targets 2010;11(6):733–44. [22] Chen HL, Lin KW, et al. Terpenoids induce cell cycle arrest and apoptosis from the stems of Celastrus kusanoi associated with reactive oxygen species. J Agric Food Chem 2010;58(6):3808–12. [23] Afzal A, Oriqat G, Akram Khan M, Jose J, Afzal M. Chemistry and biochemistry of terpenoids from Curcuma and related species. J Biol Active Prod Nat 2013;3(1):1–55. [24] Biersack B. Current state of phenolic and terpenoidal dietary factors and natural products as non-coding RNA/ microRNA modulators for improved cancer therapy and prevention. Noncoding RNA Res 2016;1(1):12–27. [25] Chitkara D, Mittal A, Mahato R. miRNAs in pancreatic cancer: therapeutic potential, delivery challenges and strategies. Adv Drug Deliv Rev 2015;81:34–52. [26] Kumar S, Inigo JR, Kumar R, Chaudhary AK, O’Malley J, Balachandar S, Wang J, Attwood K, Yadav N, Hochwald S, Wang X, Chandra D. Nimbolide reduces CD44 positive cell population and induces mitochondrial apoptosis in pancreatic cancer cells. Cancer Lett 2018;413:82–93.

Further Reading Huang J, Wu L, et al. Reactive oxygen species mediate oridonin-induced HepG2 apoptosis through p53, MAPK, and mitochondrial signaling pathways. J Pharmacol Sci 2008;107(4):370–9.

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8 Small Molecules and Pancreatic Cancer Trials and Troubles Sneha Govardhanagiri, Shipra Bethi, Ganji Purnachandra Nagaraju Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States

Abstract The American Cancer Society estimated that around 44,000 people will die of pancreatic cancer (PC). Resistance to chemotherapy drugs may be caused by various reasons such as inactivation of the drug, modification in the target molecule, drug efflux, reversal of induced DNA damage, inhibition of cell death, metastasis through an epithelial-mesenchymal mechanism, and epigenetics. New studies have examined multitargeted cancer treatments, suppressing tumor hypoxia, and inhibiting stroma-derived insulin-like growth factors, just to name a few. In novel cancer therapeutics, small-molecule inhibitors interrupt certain protein pathways, and this can lead to decreased cancer cell development and proliferation. Small molecules are used to inhibit heat shock proteins such as HSP90 in pancreatic cancer. This leads to downregulation of oncogenes, followed by PC cell apoptosis and decreased PC cell proliferation. Currently, clinical trials using HSP90 inhibitors include luminespib, tanespimycin, XL888 + pembrolizumab, and more combination treatments.

Abbreviations 5-FU

5-Fluorouracil

ABS ATP BCL-2 CAFs CDH1 DDR EMT FAK FDA GEM HA

transporters ATP-binding cassette transporter superfamily Adenosine triphosphate B-cell lymphoma 2 Carcinoma-associated fibroblasts Cadherin 1 DNA damage response Epithelial to mesenchymal transition Focal adhesion kinase Food and Drug Administration Gemcitabine Hyaluronan ()

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118 HIF HOP HSP90 IGF LAPC MDR MDSCs MMP2 MUC4 NETs NF-κB NSAIDs P13K PC PD1 PDACs PD-L1 PI3K R121 RR RTKs SFN siRNA SMI TGF-β UV VEGF

8. SMALL MOLECULES AND PANCREATIC CANCER TRIALS AND TROUBLES

Hypoxia-inducible factor Homeodomain-only protein Heat shock protein 90 Insulin-like growth factors locally advanced pancreatic cancer Multidrug resistance Myeloid-derived suppressor cells Metalloproteinase-2 Mucin 4 Pancreatic neuroendocrine tumors Nuclear factor κB Nonsteroidal Anti-inflammatory Drugs Phosphatidylinositol-4,5-bisphosphate 3-kinase Pancreatic cancer Programmed cell death-1 Pancreatic ductal adenocarcinomas Programmed death-ligand 1 Phosphatidylinositol-3-kinase Reversin 121 Respiratory rate Receptor tyrosine kinases Sulforaphane Small (or short) interfering RNA Small molecule inhibitor Transforming growth factor beta Ultraviolet Vascular endothelial growth factor

Conflict of Interest No potential conflicts of interest were disclosed.

PANCREATIC CANCER: CAUSES AND TREATMENTS There are >100 types of cancer affecting people’s daily lives including pancreatic cancer, lung cancer, prostate cancer, leukemia, breast cancer, and colorectal cancer [1]. Pancreatic cancer is one of the most challenging cancers to treat. Some therapeutic methods recommend single treatments; however, combination treatments are more common [2]. The treatments include surgery, chemotherapy, and radiation therapy; less common treatments include immunotherapy, targeted therapy, and hormone therapy [2]. The American Cancer Society estimated that around 44,000 people will die of pancreatic cancer and 10,000 more will be diagnosed with it in 2018 alone [3]. The pancreas is made up of two types of cells—exocrine and endocrine. The pancreas consists of a much smaller percentage of endocrine cells than exocrine cells; therefore, many pancreatic cancers originate from mutations in exocrine cells [3]. Endocrine cells maintain blood/glucose homeostasis by secreting insulin and glucagon to control blood sugar levels [3]. This is important because glucose is the main source of energy for the body, and its levels must be maintained to prevent damage of body organs. Exocrine cells function as digestive cells that break down proteins, carbohydrates, lipids, and nucleic acids in food [3].

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Endocrine pancreatic cancers, though rare, are called pancreatic neuroendocrine tumors (NETs) or islet cell tumors [3]. NETs can be classified into three types—functioning, nonfunctioning, or carcinoid tumors. Gastrinomas and insulinomas are the most common functioning NETs [3]. Nonfunctioning NETs are more difficult to detect because they don’t make enough excess hormones to show significant symptoms [3]. It is much more common for carcinoid tumors to originate in parts of the digestive system when compared with the pancreas [3]. In general, exocrine pancreatic cancer is more common than the endocrine pancreatic cancer. Because of this, these tumors can grow large before they are detected [3]. Exocrine pancreatic cancer types include adenocarcinomas, adenosquamous carcinomas, squamous cell carcinomas, signet ring cell carcinomas, undifferentiated carcinomas, undifferentiated carcinomas with giant cells, and ampullary cancer [3]. Within exocrine pancreatic cancers, 95% of the cases diagnosed are adenocarcinomas [3].

Causes and Prevention Although pancreatic cancer (PC) has low incidence rates, it has extremely high mortality rates; it is the fourth most common type of cancer that causes death in developed countries [4]. Some factors that influence the onset of PC include age, gender, and race [4]. Environmental lifestyle risk factors are smoking, alcohol consumption, diet, obesity, and drug use [4]. PC can also be affected by certain diseases including diabetes mellitus, chronic pancreatitis, allergies, and infection [4]. Finally, there are genetic predispositions for PC such as certain blood groups, germ-line diseases, and genetic polymorphisms [4]. PC is strongly age-dependent; patient numbers will increase as Western populations age [5]. In the United States, diagnosis of PC occurs at an average age of 72 years [4]. Genetic predispositions may cause earlier onset of PC; 5%–10% of cases are diagnosed before the age of 50 [4]. The Black population is most affected by pancreatic diseases and disorders when compared with any other races, about two- to threefold higher than the White population; rates are lowest in Asian populations [4, 6]. Some causes for this may be nongenetic risk factors including smoking, diabetes mellitus, and vitamin D insufficiency [4]. Cigarette smoking accounts for about 25% of all pancreatic cancer cases [5]. Studies have found that heavy smoking (25 or more cigarettes per day) causes a fourfold increase in PC risk compared with nonsmokers (RR ¼ 3.9, CI ¼ 1.5–10.3) [7]. Both smoking and alcohol use combined were associated with an even elevated risk (RR of 6.6, CI ¼ 2.2–20.5) [7]. Drugs can affect the onset of pancreatic cancer as well. One study explored the relationship between nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, and the frequency of pancreatic cancer [8]. It was found that among nonusers of aspirin, 43% of PC cases could be prevented [8]. Because of this, NSAIDs were thought to be chemopreventive drugs [8]. However, high consumption of animal foods (except eggs and dairy), especially salted/ smoked meat and fish, has shown increased risks of PC, although not significant [7]. PC is also connected to certain chronic diseases. One study found that in the 3 years before PC diagnosis, 40% of patients had a diagnosis of diabetes (compared with only 3%–5% in other forms of cancer) [9]. Patients with well-established diabetes have a twofold increased risk of PC, especially in adult-onset and gestational diabetes [10]. Additionally, chronic pancreatitis has been found to have a minor link to PC, as about 5% of patients with chronic pancreatitis will develop into PC [11]. Genetic predispositions that affect PC onset include certain

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blood groups; non-O blood groups are at higher risk for PC [6]. Hereditary pancreatitis has a penetrance of 80% and is a autosomal dominant disease [12]. Patients with early-onset pancreatitis, especially hereditary and tropical pancreatitis, have a 50-fold greater risk of PC than the general population [11]. One study found that the frequency of PC in patients with hereditary pancreatitis was significant, leading to the conclusion that hereditary pancreatitis is in fact a risk factor for PC [12]. Because there are many pathways and risk factors that cause pancreatic cancer to develop, there are some indicators that may prevent PC. The incidence of PC is not high enough for universal screening methods in the general population according to the US Preventative Services Task Force [13]. The only group that would clearly benefit from screening methods is those with hereditary or tropical forms of pancreatitis [13]. Still, studies have been done to look into key screening predictors for PC malignancies. The mutation of KRAS oncogenes is present in 75%–95% of pancreatic ductal adenocarcinoma (PDAC); one study found that analysis of the KRAS mutation combined with endoscopic ultrasound-guided fine-needle aspiration biopsy was able to differentiate between PDAC and chronic pancreatitis [14]. Some tumor-infiltrating immune cells can reinforce tumor growth and can advance angiogenesis [15]. Some tumor-infiltrating immune cells are regulatory T cells, myeloid-derived suppressor cells (MDSCs), and alternatively activated macrophages [15]. Alternatively, activated macrophages can be classified as M1 or M2 [15]. In patients with pancreatic cancer, it was found that high levels of M2 were associated with a shorter survival rate and high levels of M1 were associated with a longer survival rate [15]. Tumor-infiltrating immune cells can provide information about the immune microenvironment of pancreatic cancer and can be used to develop more efficient immunotherapy methods [15].

Therapeutic Treatments There are various types of treatment for pancreatic cancer that are available including surgery, chemotherapy, radiation therapy, chemoradiotherapy, and immunotherapy. If the PC is localized and is benign, surgery can be done to remove tumors. The pancreas consists of the head, body, and tail, and specific surgeries have to do with removing specific parts of the pancreas. If the tumor is located in the pancreatic head, a Whipple procedure (pancreaticoduodenectomy) is done [16]. If the tumor is located in the pancreatic body or tail, then a distal pancreatectomy is done [16]. Sometimes, removing the entire pancreas is necessary, which is called a total pancreatectomy [16]. In advanced stages of PC, these procedures are not ideal, and parts of blood vessels are removed and reconstructed [16]. Chemotherapy is a drug that kills cancer cells, and it works by stopping or mitigating the growth of cancer cells, which can divide quickly. Chemotherapy can also be used in conjunction with surgery or radiation therapy for many reasons, including to make tumors smaller or destroy cancer cells that have returned or spread around the body [16]. The US FDA approved 15 drugs for pancreatic cancer treatment [17]. Some common chemotherapy drug combinations include FOLFIRINOX, gemcitabine-cisplatin, gemcitabine-oxaliplatin, and OFF [17]. Ongoing studies are also examining new combinations of chemotherapy drugs such as sequential 5-fluorouracil/leucovorin combined with etoposide (FELv); 36% of patients in this chemotherapy group had an improved quality of life [18]. Other studies investigated singleagent gemcitabine (GEM) as a treatment for advanced and metastatic PC; these studies found

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that combining gemcitabine with capecitabine or platinum analogs (cisplatin or oxaliplatin) led to significantly longer survival rates [19]. In contrast to chemotherapy, radiation therapy kills cancer cells using high-energy beams from x-rays and protons. Radiation therapy can be done before or after surgery and is most often recommended in combination with chemotherapy. Specific types of radiation therapy such as C-ion RT for locally advanced pancreatic cancer (LAPC) have been found to be especially favorable for tumors not in close proximity to the gastrointestinal tract [20]. Studies have found that combining radiation therapy with chemotherapy leads to increased survival rate in advanced PC. One study investigated the drug 5-FU combined with 2000 rad courses of supervoltage radiation as adjuvant therapy and found that the 2-year actuarial survival rate was 46% in comparison with control treatment at 18% [21]. Another study showed that the 5-year survival rate was 10% in patients who received adjuvant chemoradiotherapy and 21% in patients who received adjuvant chemotherapy [22]. The results also showed that adjuvant chemoradiotherapy does not benefit patients when given prior to chemotherapy [22]. Immunotherapy (biotherapy) utilizes the body’s own immune system to combat PC in a more efficient way [23]. Synthesized immune system proteins can also be given to strengthen and aid the immune system in fighting PC [23]. There are four types of immunotherapy that can be used to combat cancer: monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines, and other nonspecific immunotherapies [23]. It is possible to combine immunotherapy methods as well. One study investigated how immunotherapies such as anticytotoxic T-lymphocyte-associated protein 4 and α-programmed cell death 1 ligand 1 were traditionally ineffective against pancreatic ductal adenocarcinomas (PDACs) but found that depleting FAP + stromal cells by depleting carcinoma-associated fibroblasts (CAFs) could allow the T-cell checkpoint antagonists to function [24]. AMD3100, a CXCL12 receptor chemokine receptor 4 inhibitor, helped induce rapid T-cell accumulation in cancer cells and led to reduction in cancer cells [24]. Another study found that Mucin 4 (MUC4) nanovaccine could be useful in immunotherapy because it shows suppressive effects in pancreatic tumors [25]. PD1 and PD-L1 interaction prevents T cells from attacking [25]. By using MUC4 and PD-L1 together, it creates an immunosuppressive environment for the tumor cells so that MUC4 nanovaccine could be used in combination with immune checkpoint blockade agents [25].

PANCREATIC CANCER RESISTANCE Causes of Treatment Resistance Pancreatic cancer (PC) diagnoses have continually had to battle with low survival rates that have changed little in recent years. Because of a lack of screening or biomarkers available, patients are often found with advanced local disease, with only about 4% of patients being alive 5 years after this initial diagnosis [26]. PC cells show initial sensitivity to drug therapies, but this is followed by the active development of resistance by PC cells to these treatments [27]. Resistance to chemotherapy drugs may be caused by various reasons such as inactivation of the drug, modification in the target molecule, drug efflux, reversal of induced DNA damage, inhibition of cell death, metastasis through epithelial-mesenchymal mechanism, and epigenetics [27].

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In order for a chemotherapy drug to work, it must first be metabolically activated [27]. If the rate of activation is decreased, then the cancer cells will continue to grow, ultimately developing resistance [27]. In pancreatic adenocarcinomas, MUC1 is a transmembrane protein that plays a role in metabolism for cancer cell growth [28]. By regulating the metabolism in hypoxic environments, tumor cells can thrive [28]. Some drugs are made to target a specific molecule to prevent cancer cell proliferation [27]. The cancer cells can become resistant to the drug if the target molecule is modified [27]. One target in particular is signaling kinases, which promote rapid cell growth [27]. Mutations and gene overexpression can cause the overactivation of the kinases [27]. Another way that drug response can alter is through variations in the level of modified enzyme expression [27]. Alterations of the activity of enzymes can affect the response of gemcitabine, which is a chemotherapy drug used to treat pancreatic, breast, ovarian, and other cancers [27]. Ribonucleotide reductase (RR) is an enzyme that is associated with the M1 and M2 subunits that are involved in gemcitabine metabolism [29]. The reaction that RR catalyzes limits the synthesis of dNTPs, which is essential for DNA synthesis [29]. Because the activity of RR is controlled by M2 subunit expression, increased levels of M2 subunit expression lead to greater activity of RR that accelerates DNA synthesis [29]. The greater M2 subunit expression and RR activity have been correlated with gemcitabine resistance in pancreatic adenocarcinoma [29]. Efflux is a mechanism that moves compounds and molecules out of cells [27]. The genetic code for efflux pumps may be incorporated into DNA that contributes to natural and acquired resistance [27]. The multidrug resistance (MDR) phenotype can be caused by drug efflux pump overexpression that leads to a decrease in drug accumulation [30]. An important mediator of drug efflux and MDR is the ATP-binding cassette transporter superfamily (ABS transporters) [30]. If ABS transporter expression is increased, then drug resistance can develop [30]. Some chemotherapy drugs specifically have a role to cause DNA damage [27]. However, the DNA damage can be reversed by a DNA damage response (DDR) [27]. For example, chemo drugs with cisplatin, a platinum-based drug, cause apoptosis [27]. Cancer cells can develop a resistance to the platinum-based drugs through DNA repair mechanisms such as homologous chromosome recombination and nucleotide excision repair [27]. An important phase in cells for cancer is cell death [27]. Apoptosis, one form of cell death, can occur through two pathways: intrinsic or extrinsic [27]. BCL-2 family proteins, Akt, and caspase-9 are involved in the intrinsic pathway, and receptors that induce cell death are involved in the extrinsic pathway [31]. In many cancers, antiapoptotic proteins such as BCL-2 family proteins and Akt are greatly expressed; therefore, these are good targets for drugs [31]. Occasionally, BCL-2 protein production is increased and results in the gene being altered [31]. One of these alterations could be the loss of the p53, a tumor suppressor [31]. Normally, when BCL-2 protein is increased, the p53 tumor suppressor regulates it by decreasing BCL-2 expression [31]. However, if the gene is altered, then the p53 tumor suppressor does not function, therefore allowing the cells to proliferate [31]. Solid tumors can become metastatic in a process called epithelial-mesenchymal transition (EMT) [27]. During metastasis, cancer cells and stromal cells can grow new blood vessels (angiogenesis), and cell to cell attachment occurs through proteins called integrins and cadherins [27]. In EMT, this cell to cell attachment is decreased by reduction of cell adhesion receptors [27]. Metastasis occurs to a greater degree because there is also increased expression in cell adhesion receptors that increase cell motility [27]. In pancreatic cancer, E-cadherin, which aids in cell-cell adhesion, expression is decreased, and vimentin is increased [32]. Decreased

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E-cadherin expression can result from a mutation or methylation of CDH1 in the promoter region [32]. Vimentin is a structural protein that improves flexibility of cells and allows movement; therefore, it is often used as a mesenchymal marker to detect EMT in cancers [32]. Similar to the methylation of CDH1 in the above passage, other epigenetic modifications can affect carcinogenesis and cause resistance to cancer treatments [27]. The main changes are DNA methylation and histone modification [27]. In DNA methylation, methyl groups can be added throughout a genome in various loci [27]. Histone modifications change chromatin conformation through acetylation and regulate gene expression [27]. These epigenetic modifications result in cancer growth and resistance to treatments.

Combatting the Various Chemotherapy Resistance Pathways PC treatment resistance pathways are being studied to investigate the mechanisms of action responsible and subsequently combat these pathways. Important areas of research to overcome drug resistance include targeting abnormal expressed genes, signaling pathways, and microenvironments that cause drug resistance [30]. New studies have examined everything from multitargeted cancer treatments, suppressing tumor hypoxia, and inhibiting stroma-derived insulin-like growth factors, just to name a few. Important targets include the MDR proteins (MRP1, MRP3, MRP4, MRP5, and Pglycoprotein), which are chemotherapy resistance proteins that occur in high expression in PC cells [30]. High-affinity peptide reversin 121 (R121) can bind to these MDR proteins and allows cells to be more sensitized to treatment [33]. R121 has been found to reduce tumor cells with MDR proteins both in vivo and in vitro and decrease tumor size and prevalence of metastases [33]. Studies have also examined how using multiple drugs (5-FU, gemcitabine, and cisplatin) in addition to R121 was better at decreasing drug resistance when compared with R121 with a single drug alone [30]. When activated, nuclear factor κB (NF-κB) induces cell proliferation, angiogenesis, metastasis, and drug resistance in PC cells [28]. Therefore, inhibitors of NF-κB are important targets for combating drug resistance. Chemopreventive compounds, including green tea polyphenols, curcumin, and caffeic acid phenethyl ester, and siRNA-mediators have helped suppress NF-κB activation and NF-κB-regulated gene expression in various studies [34, 35]. Phosphatidylinositol-3-kinase (PI3K)/Akt is another signaling pathway for which several small molecules have been developed. This pathway is deregulated/altered in pancreatic cancers, causing amplification of gene encoding and mutations, leading to increased cell proliferation [36]. For example, studies have looked sulforaphane (SFN) to inhibit phosphorylation of Akt, induced cell cycle inhibitors p21 and p27, inhibited cyclin D1 expression, and induced apoptosis in PANC-1 cells [37]. Notch signaling facilitates anchorage-independent growth in PC cells, so sustained Notch activation is necessary to maintain PC through cell proliferation and differentiation [38]. Studies have investigated how downregulating Notch-1 in BxPC-3, HPAC, and PANC-1 cell lines using genistein inhibited cell growth and induced apoptosis increased cell count in the G0-G1 phase [39]. Cells transfected with siRNA showed reduced expression of cyclin A, cyclin D1, and cyclin-dependent kinase 2 and upregulation of p21 and p27. Finally, this study examined how NF-κB was a downstream target of Notch (downregulation of Notch decreased NF-κB activity) [39].

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Epithelial-mesenchymal transition (EMT) has connections to abnormal gene expression (including P13K, Snail, Xeb1, and TGF-β), signaling pathways previously mentioned, cancer stem-like cells, and hypoxia [30]. EMT causes a dense microenvironment and reduces vasculature, making it much more challenging for drug treatments to reach PC cells [40]. The transition also increases migratory and invasive functions of PC cells, causing metastasis [40]. Additionally, antihypoxia and antistroma cell strategies have been developed to help increase sensitivity of drug treatments to PC cells [41]. Studies have examined intra-arterial chemotherapy to deliver higher drug concentrations to the pancreas, using PEGPH20 to target and deplete extracellular matrix component megadalton glycosaminoglycan hyaluronan (HA) and inhibiting insulin-like growth factors (IGFs) 1 and 2 [41–43].

PANCREATIC CANCER AND SMALL MOLECULES General Overview of Small Molecules Small molecules are molecules with a molecular weight of 80% in mouse models

Does not interfere with ATP binding to HSP90

[52, 59]

Geldanamycin + herbimycin A

Inhibits formation of src-HSP90 heteroprotein complex; destabilizes v-src (tyrosine kinase that transfers phosphate groups), raf-1 (serine/threonine protein kinase that transfers phosphate groups), and ErbB2 (epidermal growth factor receptor)

In vivo toxicity; the lack of stability

[60, 61]

XL888

Competes for HSP90 ATP-binding site; subsequent client protein degradation; causes cell cycle arrest, apoptosis, tumor regression in carcinomas; orally bioavailable

[62, 63]

HSP90 Inhibitors in Clinical Trials A few HSP90 inhibitors that are currently not in clinical trials are geldanamycin (radicicol), alvespimycin (17-DMAG), gamitrinib, BIIB021/CNF2024, celastrol, and geldanamycin + herbimycin A. Clinical trials for some HSP90 inhibitors have been conducted and later terminated such as ganetespib and luminespib (NVP-AUY922) [64]. Ganetespib did not complete phase II clinical trials [64]. It was tested on stage IV adenocarcinoma PC patients [64]. Out of 13 participants analyzed, eight showed grade 3 toxicity [64]. Because the participants were at high risk for blood and lymphatic system disorders, gastrointestinal disorders, and metabolism disorders, the study was terminated in April 2013 [64]. Luminespib (NVP-AUY922) also did not complete phase II clinical trial; the study was with metastatic pancreatic adenocarcinoma patients who were resistant to first-line chemotherapy [65]. The disease control rate

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H

127

O O

H

O H

O O

H N

O

H

H

O O H N H O

FIG. 1 Two-dimensional and 3-D structure of HSP90 inhibitor geldanamycin [68].

H

O O

H

N H

H

H

O

N O

H O

H O O H

N H O

FIG. 2 Two-dimensional and 3-D structure of HSP90 inhibitor tanespimycin (17-AAG) [63].

in the 15 participants was 18–20 months. The study was terminated in August 2013 due to adverse events [65]. Tanespimycin (17-AAG) completed phase II clinical trial on stage IV/recurrent/adenocarcinoma pancreatic cancer patients in 6-month survival rate from 2008 to 2010 [66]. Twenty-one total patients were tested with combination chemotherapy of gemcitabine hydrochloride IV and tanespimycin IV [66]. There were 25, 67, and 33% 6-month survival rates in arms I, II, and III of the study, respectively [66]. However, there were also 75, 33, and 33% patients affected by a serious, adverse event in arms I, II, and

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H

N O

H N

O

O N O

O H

FIG. 3 Two-dimensional and 3-D structure of HSP90 inhibitor luminespib (NVP-AUY922) [68].

III, respectively [66]. Other HSP90 inhibitors, XL888 + pembrolizumab, are currently being tested in patients with advanced gastrointestinal malignancies that spread to other areas of the body; this phase I clinical trial is currently taking patients with metastatic pancreatic adenocarcinoma and recurrent/stage III/stage IV pancreatic cancer [63, 67]. The combination HSP90 inhibitor, XL888 PO and pembrolizumab IV, was administered over 30 days and repeated treatment cycles every 21 days [63, 67]. The clinical trial began in 2017 and its completion predicted to be in 2021 [67].

Acknowledgment We thank our institute for providing the resources required to complete this work.

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[62] Zhong H, et al. Modulation of hypoxia-inducible factor 1α expression by the epidermal growth factor/ phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000;60(6):1541–5. [63] Zundel W, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev 2000;14(4):391–6. [64] Haarberg HE, et al. Inhibition of Wee1, AKT and CDK4 underlies the efficacy of the HSP90 inhibitor XL888 in an in vivo model of NRAS mutant melanoma. Mol Cancer Ther 2013;12(6):901–12. [65] El-Rayes B. Pembrolizumab and XL888 in patients with advanced gastrointestinal Cancer. Available from: https://clinicaltrials.gov/ct2/show/study/NCT03095781?term¼HSP90&cond¼Pancreas&rank¼3; 2017. [66] Cardin D. PhII study STA-9090 as second or third-line therapy for metastatic pancreas Cancer. Available from: https://clinicaltrials.gov/ct2/show/results/NCT01227018?term¼Ganetespib&cond¼pancreatic+cancer& rank¼1§¼X0123456&view¼results#all; 2010. [67] Bussenius J, et al. Discovery of XL888: a novel tropane-derived small molecule inhibitor of HSP90. Bioorg Med Chem Lett 2012;22(17):5396–404. [68] Huang LE, Bunn HF. Hypoxia-inducible factor and its biomedical relevance. J Biol Chem 2003;278(22):19575–8.

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9 Targeting the Epigenome as a Therapeutic Strategy for Pancreatic Tumors: DNA and Histone Modifying Enzymes Sathish Kumar Mungamuri Institute of Basic Sciences and Translational Research, Asian Healthcare Foundation, Asian Institute of Gastroenterology, Hyderabad, India

Abstract Pancreatic cancers, because of its highly aggressive nature, display one of the highest mortality rates. Further, this devastating disease is difficult to treat because of its late-stage diagnosis. Most research efforts have primarily focused on how genetic alterations lead to altered progression, contribute to diagnosis, and influence pancreatic cancer progression. However, none of these findings have turned into effective treatment for this dismal malignancy, and most patients cannot be treated by surgical resection. Both genetic and/or epigenetic perturbations are known to contribute toward the pathogenesis of pancreatic cancer. Indeed, the PDAC phenotype is defined by perturbations in epigenetic events, which drive the aberrant gene expression patterns and altered signaling from mutated oncogenes and tumor suppressors. Unlike mutations, epigenetic alterations are reversible. Given this feature of epigenetic mechanisms, it is conceivable to target epigenetic-based events that initiate, promote, and/or maintain pancreatic cancer. The conceptual understanding of the epigenetic perturbations could offer novel therapeutic avenues for treating patients affected with pancreatic cancer. In fact, extensive investigations are currently in progress for developing small-molecule inhibitors that could modify the epigenome in a reversible manner. In this new era of “epigenetic therapeutics,” the topics discussed herein could provide novel therapeutic and diagnostic approaches for this dismal disease to the medical community. The epigenetic processes can be divided mechanistically into the regulation of methylation of DNA, posttranslational modifications (PTMs) of histones, remodeling of nucleosomes, and transcriptional regulation or translation by noncoding RNAs. This chapter focuses on enzymes responsible for DNA and histone posttranslational modifications and the current knowledge we have on how these events are altered and their possible use as targets for epigenetic therapies during the pancreatic cancer progression.

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00009-3

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# 2019 Elsevier Inc. All rights reserved.

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Abbreviations 5mC ChIP DNMT EMT GEF HAT HDAC HMT KMT MBD Nupr1 PDAC Rb TET TSS

5-methylcytosine chromatin Immunoprecipitation DNA methyltransferases epithelial-mesenchymal transition guanine nucleotide exchange factor histone acetyl transferase histone deacetylase histone methyltransferase lysine methyltransferase methyl-CpG-binding domain proteins nuclear protein 1 pancreatic ductal adenocarcinoma retinoblastoma ten-eleven translocation proteins transcription start site

Conflict of Interest No potential conflicts of interest were disclosed.

INTRODUCTION In the present biomedical research backdrop, the “epigenetic” field offers as one of the most promising and expanding fields, and understanding global DNA methylation changes and histone posttranslational modification alterations etc., characterizes the cancer epigenome [1, 2]. Among all, one of the most widely studied and stable epigenetic modifications is the methylation of DNA. DNA methylation generally hinders the transcription process, and cancers in general show a global hypomethylation and a promoter-specific hypermethylation patterns [3–6]. Eukaryotic DNA is condensed into chromatin with the core histone proteins, H2A, H2B, H3, and H4. Each histone octamer, consisting of one H3-H4 tetramer (two copies each of H3-H4) and two H2A-H2B dimers, is blanketed with
146 base pairs of DNA to form a nucleosome [7, 8]. These histones are the principle constituent of chromatin and play an essential role in instructing the DNA scaffold to respond to the external cues. Multiple posttranslational histone modifications have been reported, mainly at their N-terminal tails [9], whose functions we are just beginning to understand [1, 9–11]. The enzymes that carry out these modifications include histone deacetylases (HDACs and sirtuins), histone acetyl transferases (HATs), histone demethylases (HDMs), histone methyltransferases (HMTs), phosphatases, kinases, small ubiquitin-related modifier (SUMO)-conjugating enzymes, E3 ubiquitin ligases, and ADP-ribosyl transferases (ADPRT) [5, 12]. Genome-wide studies and promoter-specific chromatin immunoprecipitation (ChIP) analysis revealed that a combination of these modifications can lead to either activation or repression of the expression of genes through a more “open” or “closed” chromatin conformation. Markedly, trimethylation of H3K27 (H3K27me3) and di- or trimethylation of H3K9 (H3K9me2 and H3K9me3) often causes repression of gene expression, while acetylation of H3K9 and H3K14 (H3K9ac and H3K14ac), monomethylation of H4K20 and H2BK5 (H4K20me and H2BK5me), and most importantly trimethylation of lysines (K) 4, 36, or 79 on H3

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(H3K4me3, H3K36me3, and H3K79me3, respectively) usually result in the activation of gene expression [13–16]. Collectively, these abnormal epigenetic events found in a localized region of the chromatin function through multiple mechanisms as part of a “chromatinbased signaling” system, resulting in unusual gene expression and altered metabolism, leading to cancer [8, 10, 17–19]. Even though, in most of the cancers, substantial progress has been made in increasing the survival rates, pancreatic cancer still remains highly fatal, killing
43,090 people in the 2017 alone [20]. In pancreatic cancer, the current 5-year survival rates are estimated to be of only 4% [21] and are likely to be the 2nd leading cause of cancer deaths in the western world by 2020 [22]. Pancreatic ductal adenocarcinoma (PDAC), which represents
90% of pancreatic cancers [21], is one of the most malignant types of pancreatic cancer with a dismal prognosis and is commonly referred as pancreatic cancer. Pancreatic neuroendocrine tumors, cystadenocarcinomas, and papillary mucinous tumors represent other histopathologic spectrum of pancreatic cancers. Pancreatic cancers are known to have activating point mutations in the K-ras oncogene [23–25] and to overexpress the HER2/neu gene product [26]. These alterations are believed to be “early” genetic events in the pancreatic neoplasia development because they occur in pancreatic duct lesions with minimal atypia [27]. In addition to K-ras and HER2 alterations, pancreatic cancers also display a loss of DPC4 gene expression [28, 29], abnormalities of tp53 gene expression [30–32], and biallelic inactivation of the BRCA2 tumor suppressor gene [33, 34]. However, these gene abnormalities happen almost exclusively in duct lesions with significant cytological and architectural atypia [27]. Cross talk among genetic alterations with epigenetic perturbations manifests the cancer phenotype. Indeed, the PDAC phenotype is defined by perturbations in epigenetic events, which drive the aberrant gene expression patters and altered signaling from mutated oncogenes and tumor suppressors. The classical initial transforming genetic proceedings include mutations in proto-oncogenes and/or tumor suppressor genes and genomic instability. In cancer progression, it is worth to appreciate the notion that epigenetic deregulation precedes the genetic changes, since the typical epigenetic features like promoter-specific hypermethylation and global DNA hypomethylation were witnessed in benign neoplasia’s and in early-stage tumors [35, 36]. Although we are increasingly understanding the means of PDAC progression, till date, there are no effective conservative therapeutic strategies, raising the need to define novel epigenetic targets.

DNA METHYLATION AND DEMETHYLATION The carcinogenesis process includes the cooperation between alterations in genetic events and DNA methylation patterns. Understanding the latter requires consideration of the methylation distribution across the human genome [10, 17, 37]. In the vertebrate genome, >50% of the genes comprise CpG islands (CGIs) that are short (approximately 1 kb) CpG-rich regions [38]. Initially, two studies independently suggested that in the context of CpG dinucleotide repeats, the methylation status of the cytosine residues in the DNA serves as an epigenetic mark in vertebrates [39, 40]. Methylation of other sequences is also established in plants [41], in fungi [42], and in mammals [43]. However, in mammals, as of today, the function of non-CpG methylation is not yet established. In animals and in the CpG sequence context,

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maximum research has been focused on 5-methylcytosine (5mC). Methylome-based genomewide studies have highlighted that the methylation position in the transcriptional unit will have influence on gene expression. For example, methylation at the transcription start site (TSS) vicinity, but not in the gene body, is generally known to block transcriptional initiation. However, methylation in the gene body aids in stimulating transcriptional elongation and impacts the splicing phenomenon [37, 43]. It is worth to note that such CpG methylation at the centromere repeat regions also contributes to chromosomal stability [44]. Among all, the most well-established function of CpG islands and associated methyl groups are present in the promoter regions. Such CpG-island methylation(s) interferes with the transcription factor biding to the promoters, thus essentially blocking transcription, and also invite methylCpG-binding domain proteins (MBDs), essentially leading to gene silencing and chromatin compaction [3]. In the genome, the addition and maintenance of the methylation patterns are conducted by DNA methyltransferases (DNMTs). The mammalian genome codes for three DNMTs, and each of which is required for embryonic or neonatal development [45, 46]. Initial studies have interpreted that DNMT1 is the maintenance methyltransferase, that is, responsible for preserving the patterns of DNA methylation from the parent to daughter strand [4, 6], while DNMT3a and DNMT3b employ de novo methyl groups to DNA [46]. However, recent studies disclosed that DNMT3a and DNMT3b participation is required as well for methylation maintenance [38] (Fig. 1). DNA demethylation is not as direct as addition of methyl groups to the DNA. Loss of DNA methylation occurs in response to abrogation in the activity of DNMT or during the mechanisms of DNA repair [47–49]. In mammalian cells, ten-eleven translocation (TET) proteins

FIG. 1 Genes under expressed in PDAC due to aberrant hypermethylation. Schematic diagram of conversion of normal epithelium to PDAC is shown. Bona fide list of tumor suppressor genes that are downregulated in pancreatic cancers due to promoter hypermethylation.

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facilitate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine [50], which serves as an intermediate in the CpG demethylation phenomenon. 5-hydroxymethylcytosines are not acceptable for 5-methylcytosine-binding proteins, and the hydroxy group will not be recognized by DNMT proteins, leading to the loss of methylation maintenance patterns [51–53]. DNA methylation is one of the well-established tumor suppressor gene inactivation mechanisms. Combined with the genetic alterations, tumor suppressor gene inactivation through CpG methylation of DNA aids in PDAC progression. Several investigators have reported that DNMT1, DNMT3a, and/or DNMT3b are elevated in PDAC [54–56]. One mechanism for DNMT overexpression in PDAC is that DNMTs may be elevated by oncogene signaling [57]. In addition, nuclear protein 1 (Nupr1), a chromatin protein that is overexpressed in pancreatitis and PDAC, binds to the DNMT1 promoter, enhancing the expression of this gene [58]. The DNMT3b promoter is reported to be hypomethylated with coordinate overexpression of DNMT3b in some PDAC samples [59]. In addition, DNMT3b is amplified in some PDAC samples, leading to enhanced activity [60]. Thus, it is possible that DNMT overexpression may drive hypermethylation at gene loci, silencing the essential gene expression in pancreatic cancers. In PDAC, the p16 tumor suppressor gene promoter inactivation via methylation is well established. Greater than 95% of PDACs have a loss of p16Arf expression, due to promoter hypermethylation [61]. Such methylation interferes with the SP1 transcription factor binding and results in MBD recruitment, which along with HDACs leads to chromatin compaction [62]. Loss of p16Arf leads to release of D-family of cyclins binding to their cyclin-dependent kinase (CDK) partners, thus increasing the retinoblastoma (Rb) phosphorylation, essentially releasing E2F1–3 transcription factors and subsequent G1 to S phase of the cell cycle progression [61, 63]. Furthermore, significant MBD1 upregulation was shown in pancreatic cancer tissues and in vitro loss- and gain-of-function studies authenticated that MBD1 is a potent oncogene and promotes epithelial-mesenchymal transition (EMT) and invasion of pancreatic cancer cells [64]. Other than p16Arf, PDAC also shows increased promoter methylation of secreted apoptosis-related protein 2 (SARP2), neuronal pentraxin II (NPTX2), and claudin 5 (CLDN5) leading to their transcriptional repression [65, 66]. Inactivation of secreted apoptosis-related protein (SARP) family of proteins aids cancer development and progression by counteracting the oncogenic Wnt signaling pathway [67, 68]. Methylation analysis of NPTX2 and SARP2 promoters, either from bile juice or from serum, has been successfully established as a prognostic marker for pancreatic neoplasms or diagnostic marker for pancreatic cancers, respectively [69–72]. NPTX2, a tumor suppressor gene, is a second member of the neuronal pentraxin protein family and executes a role in cellular uptake of materials [73]. Lower expression of NPTX2 was observed in numerous pancreatic cancer tissue lines, and treatment of such cells with 5-aza-20 -deoxycytidine, a DNA methyltransferase inhibitor, was shown to restore its expression [74]. Further, NPTX2 ectopic expression inhibits cell cycle progression and induces apoptosis, further substantiating its role as a tumor suppressor in the pancreatic cancer tumorigenesis. The claudin (CLDN) genes encode a protein family that are essential formation and function of tight junctions and are found to be overexpressed in several cancers [75]. PDACs also express high levels of CLDN4 [76, 77] and CLDN18 [78, 79]. However, CLDN5 was silenced in PDACs due to promoter hypermethylation [65, 66], for which the exact role needs to be characterized. Initially, some of these tumor suppressors are thought to be methylated solely in pancreatic lesions that become malignant. However, growing evidence suggests that even the preneoplastic stage itself, such methylations do occur.

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Genome-wide approaches were used by number of laboratories to detect genes, which are differentially methylated in pancreatic cancer. For example, a study by Vincent and colleagues detected several genes silenced by DNA methylation, including genes involved in stem cell pluripotency (BMI1, BMP3, FOXD3), Wnt signaling (WNT5A, APC2, and SOX1), and cell adhesion (CDH2, CDH4, PCDH1, and PCDH10) [80]. A recent study compared the methylation profiles of 167 resected PDAC samples from previously untreated patients with 29 samples from untransformed regions of the pancreas and identified
3500 aberrantly methylated genes in PDAC. Pathway analysis of affected genes indicated the axonal guidance signaling pathway as one of the most affected processes with hypermethylation of the ROBO1, ROBO3, SLIT3, and SLIT2 genes. Other abrogated pathways include TGFβ, integrin, and Wnt signaling in their study [81]. Exome sequencing of human PDAC tumors have recently revealed frequent mutations in SLIT and ROBO genes [82]. Although the functions of SLIT and ROBO proteins in PDAC are still under investigation [82, 83], some studies suggest that these proteins can play a tumor suppressive role [84, 85]. Dutreal et al. compared gene promoter methylation patterns between PDAC; chronic pancreatitis (CP), a condition predisposing to PDAC; and normal pancreas and identified some genes that were hypermethylated in PDAC only and some that were methylated in both PDAC and CP [86]. One of the genes hypermethylated in both PDAC and CP was WNK2, a cytoplasmic serine/ threonine kinase. Further study showed that WNK2 mRNA and WNK2 protein expression decreased progressively from normal pancreas, through PanIN to PDAC. In pancreatic neuroendocrine tumors (PanNETs), with regard to promoter methylation, Ras association domain family 1 (RASSF1) is by far the largely investigated. RASSF1 tumor suppressor family of genes consists of multiple isoforms produced as a result of transcription from diverse promoters containing CpG islands and of alternative splicing [87]. RASSF1 regulatory region includes two CpG islands: CpG island “A” in the regulatory region common to RASSF1A, RASSF1D, RASSF1E, RASSF1F, and RASSF1G and CpG island “C” in the regulatory region of RASSF1C [88]. In the case of RASSF1B, not much is known about how its expression and function are influenced by the CpG islands [88–90]. Out of all, RASSF1A controls cellular apoptosis, proliferation, and microtubule stabilization. Further, RASSF1A gene promoter is frequently methylated in PanNETs. Isoform RASSF1C is overexpressed in PanNETs, where it is thought to inhibit β-catenin degradation and modulate Wnt signaling [90]. House et al. detected an aberrant RASSF1A promoter hypermethylation in 75% of 48 nonfunctioning and well-differentiated PanNETs. These authors found that tumors larger than 5 cm and those with either liver or lymph node metastasis exhibited a greater methylation frequency, compared with PanNETs without malignant features, histologically [91]. Furthermore, in PanNETs, Malpeli et al. exemplified that there is inverse correlation between gene methylation and RASSF1A expression [89]. In pancreatic cancer, cells also show abnormal expression of otherwise normally silenced genes, due to the loss of promoter methylation. Sato and colleagues analyzed the importance of hypomethylation in PDAC progression and found seven genes in PDAC samples and cell lines that were overexpressed in comparison with normal pancreatic duct samples [92]. For example, Vav1, a guanine nucleotide exchange factor (GEF), was shown to be overexpressed in pancreatic cancers due to promoter demethylation. Further, Vav1 induces cellular proliferation by promoting oncogenic KRAS activity and by Rac1, PAK1, and NF-kB activation [93]. Genome-wide analysis had identified several overexpressed genes in PDAC in comparison

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with normal cells due to promoter hypomethylation including genes for chromatin enzymes (e.g., histone methyltransferase SETD8, histone deacetylase KDM6A, and the histone acetyl transferase EP400) and oncogenes (JUNB, MYB, and FOS) [80]. These findings establish that the importance of not only hypermethylation but also hypomethylation is associated with the PDAC disease process. Furthermore, DNA methylation also acts in concert with other epigenetic and histone modification mechanisms for arbitrating pancreatic pathogenesis [94, 95].

HISTONE DEACETYLATION AND ACETYLATION Eukaryotic DNA is enwrapped around an octamer, made up of histones, into nucleosomes. Chromatin is nothing but the regular and repeating organization of the nucleosomes. Histone acetylations alter the chromatin structure and modify gene expression profile [96–98]. Histone acetylations are coordinated by two classes of enzymes, namely, histone deacetylases (HDACs) and histone acetyltransferases (HATs) [97–101]. HDACs are known to carry out normal cellular functions by associating with transcription factors, tumor suppressors, and oncogenes. To date, in humans, 18 HDACs are recognized, and depending on how much homologous they are to yeast, HDACs are categorized into four distinct classes [5]. Classes I, II, and IV are similar in their structure and have zinc-dependent active sites. Class III, consisting of the sirtuins (SIRTs), which are zinc-independent, requires nicotinamide adenine dinucleotide (NAD) as a cofactor for their function. Class I human HDACs include HDAC1, HDAC2, HDAC3, and HDAC8 and have homology to Rpd3, a yeast HDAC [101]. Class II HDACs are homologous to HDA1, another yeast HDAC [101], and include HDAC4, HDAC5, HDAC6, HDAC7, and HDAC9 [102]. Class III of human HDACs consists of Sirt1, Sirt2, and Sirt3, which are homologues of Sir2 of yeast and mouse and are insensitive to a HDAC inhibitor, trichostatin A (TSA) inhibition [103, 104]. The fourth class of HDACs, consisting solely of HDAC11, has just recently been proposed to have functions similar to those of class I [5]. Class II HDACs, whose functions are regulated by class I HDACs, are also required for genomic organization and developmental stage transcriptional silencing, while class III enzymes have a specific role in gene silencing and serve in acetylation level maintenance [105]. Histones are the first identified HDAC substrates. ε-amino group of lysines present at the N-terminal tail of histones gets deacetylated by the HDACs, which leads to charge neutralization and establishment of repressive chromatin conformation (heterochromatin) resulting in the loss of gene expression [106–108]. Interestingly, activating HDAC mutations is rare, but HDAC overexpression is frequently observed in cancer patients [109]. Histones are not the only substrates of HDACs, but the increasing list includes α-tubulin, HSP90 and cortactin (HDAC6), p53 (HDAC5), and ERRα (HDAC8). Further, proteins involved in tumor migration, metastasis, and growth are also HDAC substrates [110, 111]. Finally, HDACs are known to function in multiprotein complexes, which contain transcriptional corepressors and coactivators. HDAC1, HDAC2, HDAC3, and HDAC7 are shown to be overexpressed in PDACs [112–115]. Nearly, 56% of PDACs have shown HDAC1 positive immune histochemical staining, and the coexpression of HIF1α and HDAC1 was strangely correlated with poor patient prognosis [116]. HDAC1 also forms a protein complex with E2F1, Rb, and DNMT1, leading to E2F-dependent transcription repression, thus regulating G1/S cell cycle progression [117]. Further, HDAC1 was also shown to control G2/M checkpoint through its interaction

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with Hus1 and Rad9 proteins [118]. HDAC2 gets upregulated by cMyc in pancreatic cancer cells, which in turn cooperates with cMyc to downregulate cyclinG2 transcription, enabling pancreatic cancer cell proliferation [119]. Transcriptional repressor ZEB1 downregulates expression of E-cadherin through HDAC1 and HDAC2 recruitment, thus promoting epithelial and mesenchymal progression (EMT) in pancreatic cancer [120]. Fritsche et al. noted that pancreatic cancer cells that acquire etoposide resistance show HDAC2 upregulation and HDAC2 inhibition with valproic acid restores etoposide sensitivity in these cells [113]. HDAC3, through histone modification of p53, Bax, and p27 promoters, enhances PC cell proliferation and PC cell invasion and escalates PC drug resistance [121]. High expression of sirtuin 1 (SIRT1), NAD-dependent histone deacetylase, is associated with poorly differentiated PDAC and is negatively correlated with PDAC patient survival [122]. SIRT1 expression in PDAC cells increased their viability and lymph node metastasis, while small-molecule inhibition of SIRT1 repressed growth in these cells [123]. Kugel et al. showed that SIRT6 suppresses pancreatic cancer progression and metastasis through controlling Lin28b expression, a let7 microRNA negative regulator. In their study, they showed that loss of SIRT6 leads to pronounced upregulation of Lin28b and downstream let-7 targets, HMGA2, IGF2BP1, and IGF2BP3, due to histone hyperacetylation at the Lin28b promoter favoring Myc promoter occupancy [124]. Since HDACs control cellular apoptosis, cell proliferation, migration, angiogenesis, and differentiation in cancer [108, 125], these enzymes represent attractive therapeutic targets [115, 126–128]. For efficient and successful PDAC treatment, currently HDAC inhibitor-dependent combination therapeutic strategies are being verified both in preclinical and clinical scenarios [129]. A recent study showed that AR-42, a histone deacetylase inhibitor, by affecting multiple biochemical pathways exhibits antitumor activity in pancreatic cancer cells [130] (Fig. 2).

FIG. 2

Histone-modifying enzymes and histone modifications that are altered during PDAC progression. Schematic picture of nucleosome with histone octamer and DNA wrapped around it along with the important histone tail posttranslational modifications is shown. Bona fide list of histone-modifying enzymes and histone modifications during pancreatic cancer progression is shown.

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HATs counteract histone deacetylation and relaxes of the chromatin fibers, generating an open chromatin structure (euchromatin). Histone H3 lysine 27 acetylation (H3K27Ac) and histone H3 lysine 9 acetylation (H3K9Ac) are the well-studied histone marks that are usually correlated with gene activation [131]. Lysine acetylation neutralizes the positive charge on histone tails, which is proposed to release chromatin compaction, thus opening up chromatin and facilitating transcription factors binding and activating their target genes [132]. HATs are classified into two different groups, type A and type B. Type “A” HATs are mainly localized in the nucleus; type “B” HATs are essentially cytoplasmic. Type A HATs acetylate nuclear histones and chromatin-associated proteins, while type B HATs function to modify newly synthesized histones before they assemble into the genome [133, 134]. Based on the homology in the HAT domain, type A HATs fall into four main families: (1) the GNAT family with the first identified HAT, which consists of general control of amino acid synthesis protein 5 (GCN5) and p300/CBP-associated factor (PCAF); (2) the p300/CREB-binding protein (CBP) family; (3) the MYST family, which was so-called according to the founding members monocytic leukemia zinc-finger protein (MOZ), yeast-binding factor 2 (YBF2), something about silencing 3 (SAS3), SAS2, and Tat interactive protein-60 (TIP60); and finally (4) the regulator of Ty1 transposition gene product 109 (Rtt109) family [133–135]. Apart from the above mentioned, several other nuclear proteins, like general transcription factor TAF250, steroid receptor coactivator family (e.g., AIB1), and the ATF-2 transcription factor, also hold HAT activity. Several transcription factors, in addition to histones like E2F1, p53, p73, and c-Jun, are controlled and directly regulated by HATs [135]. In spite of the HDAC and HAT functions in various cancers that are relatively known [12], the relevance of HATs in PDAC progression is less well established. One report evaluated H4K12 and H3K18 acetylation in PDAC samples by immunohistochemistry and found that these acetylation marks were indicators of lower overall survival in patients [136]. This study suggests that significant alterations in the acetylation machinery occur in PDAC that impact upon tumor biology. However, the overall assessment with respect to the role of HATs in PDAC initiation and progression is conflicting and confusing and can be best illustrated by understanding the role of p300 in pancreatic progression. p300 has been shown as a metastatic suppressor of pancreatic cancer growth [137] and as a PDAC growth promoter [138]. To twig HAT functions in the PDAC carcinogenesis, we need future findings, especially at the genetic and epigenetic levels.

INACTIVATING HISTONE METHYLATIONS As mentioned earlier, H3K9me3 and H3K27me3 histone marks represent the most important inactivating histone marks. While the histone acetylation mediated by HATs and HDACs are known to facilitate short-term responses [139, 140], the histone methylations generally mediate long-term effects. Further, it is evident that for certain lysines present on the histone Nterminal tails, the acetylation machinery shows increased specificity than the methylation machinery. In organisms varying from yeast to humans, histone 3 lysine 9 dimethylation and trimethylation (H3K9me2 and H3K9me3) marks the constitutive heterochromatin [141, 142]. In mammals, the SET-domain-containing family of lysine methyltransferase (KMT) members catalyzes the H3K9 methylations. These enzymes are broadly expressed and

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include SET domain bifurcated 1 (SETDB1; KMT1E) [143] and SET domain bifurcated 2 (SETDB2; KMT1F) [144], and the related enzyme suppressor of variegation 3-9 homologue 1 (SUV39H1; KMT1A) [141] and suppressor of variegation 3-9 homologue 2 (SUV39H2; KMT1B) [145] contribute to both H3K9me2 and H3K9me3 [141, 143], while GLP (euchromatic histone-lysine N-methyltransferase 1, EHMT1; KMT1D) [146] and G9a (euchromatic histonelysine N-methyltransferase 2, EHMT2; KMT1C) [147] catalyze H3K9me1 and H3K9me2 [146– 148]. H3K9 methyltransferases do not make any specific DNA base contacts. In agreement, it is well established that site selectivity of H3K9me3 and deposition and developmental dynamics of its domains are known to be regulated by additional factors [149, 150]. Heterochromatin protein 1 (HP1; exists in three isoforms in mammals, α, β, and γ) through their chromo-domains binds to H3K9me2/me3. These proteins contribute to heterochromatin spread and compaction through self-oligomerization and recruite repressive histone modifiers [151, 152]. Further, these histone methyltransferases also contribute to the establishment of high DNA methylation intensities at CpG islands and low histone acetylation levels, two other events required to achieve heterochromatinization [153, 154]. Loss of H3K9me3 was observed to increase the metastatic progression in pancreatic cancer [155, 156]. Mucin1 is overexpressed in >90% of PDAC samples [157, 158], and the mechanism involves the loss of H3KK9me3 on its promoter [159]. In contrast, many PDAC cells show hyperactivation of G9a. Further, it has been shown that decreasing the G9a function by various approaches is an appropriate target for PDAC therapy. H3K9 methyltransferase inhibition as a monotherapy has been successful, yielding in vitro promising antitumor results. Inhibitors of G9a induce ER stress and apoptosis and decrease the proliferation and/or overall cellular viability of pancreatic cancers overexpressing G9a [160, 161]. Furthermore, siRNA-mediated and pharmacological inhibition of G9a activity in pancreatic cells has shown to trigger autophagy, ultimately lowering cell viability [162]. Treatment of PC cells with BRD-4770, which decrease H3K9me marks, results in reduced cell proliferation via G2/M arrest and boosted cellular senescence. This small-molecule inhibitor shows greater specificity for G9a compared with other methyltransferases [161, 163]. Finally, gemcitabine resistance, commonly observed in pancreatic cancer, has been shown to be overridden by inhibiting G9a activity [164]. Similarly, SUV39H1/2 inhibition was shown to negatively regulate the PDAC cell growth in cell culture, in monolayer, in spheroids, and in in vivo organoids and grafts [165]. Finally, in various aforementioned PDAC models, it has been shown that a combination treatment of MLN8237, an Aurora kinase A (AURKA) inhibitor with chaetocin, a pan-histone methyltransferase inhibitor [166], induces mitotic catastrophe [165]. H3K27me3 is yet another histone tail modification responsible for repressive chromatin conformation that is also altered in many cancer types. Poor pancreatic cancer patient outcome is correlated with the loss of H3K27me3 [155]. In fact, in PDAC, H3K27me3 level together with lymph node status and tumor size prognostically have an independent and strong influence [155, 167]. H3K27me3 methyl mark on the chromatin is deposited by the enhancer of zeste homologue 2 (EZH2), which is a part of the polycomb repressive complex 2 (PRC2) and a catalytic subunit. EZH2 is fundamental and necessarily contributes to PC cell stemness [168]. Although the H3K27me3 mark is downregulated in PDACs, EZH2 is generally elevated in PDAC. The reasons for these seemingly anomalous findings are not known,

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but might involve imbalances in other components of the PRC2 complex (e.g., EED and SUZ12) that could affect the EZH2 activity. PDAC poor prognosis has been correlated with EZH2 overexpression [83]. In PDAC, poorly differentiated PDAC exhibits more frequent nuclear accumulation of EZH2, in which context, p27Kip1 is implicated to be the target gene [87]. Pretreatment with EZH2 inhibitors was shown to resensitize the resistant pancreatic cancer cells. Ougolkov et al. observed that chemoresistant pancreatic tumor cells have increased EZH2 nuclear accumulation. The authors showed that the pancreatic cell lines, which were resistant to chemotherapeutics, for example, doxorubicin- and gemcitabine-resistant PC cells, can be resensitized by artificial EZH2 depletion [167]. Much of the preclinical work using EZH2 inhibitors being done as a monotherapy has shown promise. In numerous in vitro (monolayer and spheroid cultures) and in vivo (PDX models) PDAC prototypes, EZH2 inhibitors as a single agent showed great promise. PDAC cells in response to either UNC1999 or GSK126, both EZH2 specific inhibitors, showed a decreased proliferation accompanied by reduced anomalous H3K27 methyl marks [169–171]. In addition, GSK126 also inhibits angiogenesis and increases apoptosis [170, 171]. Finally, short pretreatment of 3-deazaneplanocin A, another EZH2 inhibitor as a chemotherapeutics delivered through nanoparticles, induced apoptosis in both poorly and well-differentiated cancers and in both gemcitabine-resistant and sensitive PC cells [172]. It is worth to note that small-molecule inhibitors targeting various PRC complex binding domains (apart from EZH2) are also recently showing similar effects in the EZH2-inhibitor desensitized cell lines [173]. Overall, the PCR complex connection with poor survival of PDAC patients not only inspires to learn much more about this pathway but also makes this research area one of utmost importance. Lysine (K)-specific demethylase 6B (KDM6B), also known as jmjC domain-containing protein 3 (JMJD3), is one of the histone demethylases known to remove the H3K27me3 methyl mark and is generally annotated to transcriptionally activate its target genes [174]. In human fibroblasts, oncogenic RAS induces KDM6B, which contributes to oncogene-induced senescence (OIS) by enhancing p16/INK4a gene transcription [175, 176]. KDM6B expression was found to be downregulated in various human cancers [175, 176]. Yamamoto et al. observed that in low-grade PanINs, KDM6B was upregulated. However, its expression was inversely correlated with malignant disease progression, that is, poorly differentiated PDAC showed lowest KDM6B expression [177]. The same authors showed that the expression profiles of KDM6B and CCAAT-enhancer binding protein alpha (CEBPA) and KDM6B’s downstream target were correlated well in human PDAC tumors [177], suggesting that mitigation of KDM6B-C/EBPα pathway axis will aggravate pancreatic neoplastic progression.

ACTIVATING HISTONE METHYLATIONS Trimethylation of H3K4 (H3K4me3) is typically an activating histone mark, often found abnormal and correlated with poor outcome in various cancers. Genome-wide studies have shown that H3K4me3 is concentrated largely at the transcription start site (TSS) of the promoters [8, 15]. In higher eukaryotes, the repressive chromatin atmosphere levied by the H3K9 and H3K27 methylations is mainly opposed by the H3K4 methylation. In mammalian cells, six families of enzymes deposit H3K4me3 mark in the chromatin. They include

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SET domain-containing 1A (SET1a) and SET domain-containing 1B (SET1b), which are mammalian orthologs of yeast Set1 [178] and four MLL-family HMTs, MLL1 (KMT2A; lysine (K)-specific methyltransferase 2A), MLL2 (KMT2A; lysine (K)-specific methyltransferase 2B), MLL3 (KMT2C; lysine (K)-specific methyltransferase 2C), and MLL4 (formerly known as MLL2; KMT2D; lysine (K)-specific methyltransferase 2D), which share limited similarity with yeast Set1 beyond the SET domain [179–183]. RbBP5, Ash2L, and WDR5 are the three structural proteins that are common in all mammalian MLL and SET1 complexes and contain the required structural information for substrate specificities and enzymatic activities of SET1-family HMTs [184]. Other than the three core conserved components, Set1 and MLL1 complexes contain HCF1; MLL1 and MLL2 complexes contain HCF2 and menin proteins. Further, MOF (member of MYST family histone acetyl transferases, homologue of drosophila MOF) is exclusively present in MLL1 complex; ASC2 is exclusively present in MLL3 complex, while MLL4 complex is known to contain DPY-30 and RPB2 exclusively [184]. Even though individual functions of each of the individual conserved proteins are reported [185–187], exhaustive analysis of this fundamental arrangement in both yeast and higher eukaryotes needs further studies. H3K4 methylation is also tied to the activities of other chromatin-modifying enzymes, such as HATs [188], chromatin remodelers [189, 190], and HDACs [191, 192]. Furthermore, in certain settings, it has been reported that H3K4 HMT complexes also function as coactivators for transcription. For example, in both in vitro and in vivo settings, MLL1 acts cooperatively with MOF and CBP [180, 193]. MLL1 and MLL4 also interact with the basic transcription machinery [181, 194]. In deciphering this “epigenetic code” of cellular memory and transcription regulation, understanding the H3K4 HMT functions and their interacting partners is going to be a key factor. Genome-wide sequencing studies have demonstrated that PDAC tumors contain mutations in MLL proteins [82, 195–197]. One study reported that out of
100 PDAC samples tested, mutations in MLL3, MLL4, and MLL1 occurred in 7%, 5%, and 2% of patients, respectively [198]. In addition to point mutations, truncations, frameshifts, and amplification of MLL genes have been reported in PDAC [198–200]. Sausen et al. reported that mutations in MLL1, MLL3, and MLL4 were correlated with improved overall survival in PDAC [198]. In addition, Dawkins et al. found that decreased MLL3 and MLL4 expression in tumors was correlated with increased survival time in PDAC patients [201]. Studies by this same group showed that knockdown of MLL4 in PDAC cells in vitro led to reduced cell growth with an inhibition of the cell cycle and increased apoptosis. Thus, it appears that loss of expression or mutation of MLL proteins may have a negative impact on tumorigenesis or prognosis and that the presence of the wild-type protein in some way supports oncogenesis. However, further studies are needed to refine these observations. Alterations in H3K4me3 mediate PDAC formation, resistance to therapy, and immune evasion [202, 203]. PDAC cells utilize H3K4me3 for the activation of antiapoptotic promoters Bcl-xl, FLIP, and Mcl-1 and utilize H3K9me3 to silence proapoptotic promoters Bak, Bax, and Bim, thus acquiring an apoptosis-resistant phenotype [204]. Targeting MLL1 in the pancreatic cells to alter the epigenetic landscape might be used successfully to decrease the pancreatic islet tumor formation [203]. Carugo et al., across multiple PDAC samples and using an unbiased target discovery approach in vivo, identified WDR5 as a top hit required for tumor maintenance. Mechanistically, these authors observed that in PDAC cells, WDR5 functions to sustain proper DNA replication execution by interacting with cMyc [205, 206]. Further, the same authors observed

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that the effects of WDR5 suppression are mirrored by ATR inhibition [206]. These data provide a rationale to examine the hypothesis of utilizing ATR and WDR5 inhibitors for PDAC therapeutics. SMYD2 (SET and MYND domain-containing 2) catalyzes the histone H3 at lysine 4 monomethylation (H3K4me1) and histone H3 at lysine 36 dimethylation (H3K36me2) [207, 208]. This enzyme is also known to decrease p53 DNA binding and associated transactivation of its target genes by monomethylation of p53 at lysine 370 [209]. SMYD2 also methylates Rb at lysine 860, which provides a direct methyl-binding domain binding site for transcriptional repressor L3MBTL1 [210]. Reynoird et al. observed that SMYD2 levels were elevated in PDACs and monomethylates MAPKAPK3 at Lys355. In the same study, the authors showed that PDAC growth can be limited by MAPKAPK3 inhibition and either genetic or pharmacological SMYD2 blocking [211]. Further, inhibition of SMYD2 aids typical chemotherapy for PDAC cells and tumor treatment [211]. Whole genome/exome sequencing efforts have led to the identification of mutations in the gene MEN1, in nonfunctioning and functioning PanNETs [212]. It has been reported that over 1300 mutations, most of which are truncating, have been identified in MEN1 [213–215]. Men1 loss in pancreatic cells leads to alterations in gene expression and is partially reversed by the loss of JARID1A (Jumonji, AT-rich interactive domain 1A; KDM5A; Rbp2) in these cells [216]. These data support the notion that in Men1 deletion-mediated PanNET tumorigenesis, histone methylation plays a key/essential role. Although loss of function of Men1 protein plays an important role in PanNET initiation and tumor progression [217], the mechanisms of linking menin functions in HMT complexes are limited. KDM2B (also known as Ndy1, FBXL10, and JHDM1B), an H3K36 histone demethylase, has been associated in somatic cell reprogramming [218, 219] and in cellular senescence bypass [220, 221]. KDM2B is overexpressed markedly in PDAC patients, and its expression level correlates with tumor grade with the highest level of expression in metastases [222]. KDM2B drives tumorigenicity through two different transcriptional mechanisms. In the first mechanism, KDM2B co-occupies the TSS along with polycomb group (PcG) proteins and represses developmental genes. In the second mechanism, KDM2B along with histone demethylase KDM5A and Myc oncogene activates a module of metabolic genes [222]. Thus, KDM2B weakens the cellular differentiation program and drives PDAC pathogenesis.

CONCLUSIONS AND FUTURE PERSPECTIVES In contrast to genetic modifications, epigenetic alterations are reversible. Recent studies demonstrated that DNA methylation and chromatin-based mechanisms are highly dysregulated in pancreatic cancers. Further, rather than operating independently, these epigenetic processes interact with each other, establishing a multilevel regulatory network altering the expression of genes and proteins beyond the changes imposed by genetic alterations alone. Thus, small-molecule inhibitors that target epigenetic mechanisms could be of potential use to reset the epigenetic state of PDAC cells [223, 224]. Indeed, several clinical trials utilizing compounds to target DNMTs and HDACs in PDAC are ongoing or completed [20, 223, 225]. Given the reversibility of histone H3K4me3, there is a high demand to develop small-molecule inhibitors targeting the MLL proteins [203]. The EZH2 inhibitor such as CPI1205 is shown to decrease pancreatic cell proliferation and increase apoptosis, by decreasing

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the H3K27 trimethylation marks. Some of the EZH2 inhibitors were successful preclinically and are in clinical trials for medulloblastomas [226] and B-cell lymphomas [227]. Further work is needed in case of PDACs, with these inhibitors, before proceeding to clinical trials. New inhibitors of EZH2, such as GSK2816126 and tazemetostat, are developed and are progressing toward clinical trials for various cancers, although large-scale trials have yet to be performed in PDACs [223]. Currently, there are no “targeted therapies” available for PanNET patients harboring MEN1 mutations. Mice deficient in Men1 take up to a year for tumor formation. It is assumed that such a time laps is required to accumulate sufficient genetic and epigenetic alterations, because of which pancreatic islet cells transform into hyperplastic and finally into a malignant from their normal state [203]. It is worth to mention that this time lap period displays us a chance to probe the early events in pancreatic neuroendocrine tumorigenesis. Specifically, this window of time should be used for identifying how menin-HMT complexes modulate cell proliferation and behavior at this precancerous stage and early cancer progression. In addition to the tumor cells, tumor-associated fibroblasts, endothelial cells, and immune cells have been demonstrated to undergo extensive epigenetic alterations in PDAC [228]. Thus, caution needs to be taken whether these epigenetic drugs have unanticipated negative effects on stromal fibroblasts or immune cells. Note that in pancreatic cancers, immunotherapy with anti-PD-L1/PD-1 antibodies was not successful. Lu et al. showed that MLL1 inhibition in combination with immunotherapy (anti-PD-L1 or anti-PD-1 antibody), in a FasL- and CTL-dependent manner, effectively suppresses pancreatic tumor growth [202]. Finally, since PDAC is an extremely heterogeneous disease, we need to understand if there are any subsets of PDAC patients who would most benefit from certain epigenetic drug treatments. In conclusion, our increased understanding of the genetic and epigenetic aberrations that drive pancreatic cancer suggests novel avenues for treatment, but extensive basic and clinical studies are needed to translate these findings into improved patient outcome.

Acknowledgments I would like to acknowledge the contributions of the authors for their excellent research studies that I have cited in this chapter. I apologize that, due to space constraints, I had to omit some of the studies. I thank Vijayasarathi and Sheethal Galande for critical reading of this manuscript and V. Namratha for the secretarial assistance.

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C H A P T E R

10 Are Nanocarriers Effective for the Diagnosis and Treatment of Pancreatic Cancer? Prameswari Kasa*, Batoul Farran†, Ganji Seeta Rama Raju‡ *

Dr. LV Prasad Diagnostics and Research Laboratory, Hyderabad, India †Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States ‡Department of Energy and Materials Engineering, Dongguk University, Seoul, Republic of Korea

Abstract Pancreatic cancer ranks as the fourth cause of cancer-associated deaths in the West. It is an aggressive cancer with poor prognosis and a huge mortality rate. Current treatment modalities used for treating PC (surgery and chemoradiotherapy) have limited outcomes due to stroma-induced chemoresistance. These shortcomings stress the need for new therapeutic solutions with increased efficacy and cytotoxicity for PC. In recent years, nanotechnology has emerged as a promising solution for improving diagnosis, drug delivery, and treatment in many cancers including PC. In this chapter, we explore the various nanotechnologies developed to date, their chemical composition, pharmacological properties, and activity. We also discuss their potential as drug delivery solutions for PC.

Abbreviations 5-FU

5-Fluorouricil

ACS CNT EBRT FDA GEM GITSG EORTC MWNT OS

American Cancer Society carbon nanotubes external-beam radiation therapy Food and Drug Administration Gemcitabine Gastrointestinal Tumor Study Group European Organization for Research and Treatment of Cancer multiwalled carbon nanotubes overall survival rate

Breaking Tolerance to Pancreatic Cancer Unresponsiveness to Chemotherapy https://doi.org/10.1016/B978-0-12-817661-0.00010-X

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# 2019 Elsevier Inc. All rights reserved.

160 PC PDAC PEG PM PNP RAFT SBRT SEER SWNT

10. NANOCARRIERS: DIAGNOSIS AND TREATMENT OF PANCREATIC CANCER

pancreatic cancer pancreatic ductal adenocarcinoma polyethylene glycol polymeric micelle polymeric nanoparticle reversible additional fragmentation polymerization stereotactic ablative radiotherapy surveillance, epidemiology, and end results single walled carbon nanotubes

Conflict of Interest No potential conflicts of interest were disclosed.

CURRENT STATUS ABOUT PANCREATIC CANCER Cancer is caused by uncontrolled abnormal cell growth and is categorized as the most hazardous class of diseases [1, 2]. It is a major cause of cancer-related deaths, with around 8.2 million deaths observed worldwide [3]. Pancreatic cancer (PC), also known as pancreatic ductal adenocarcinoma (PDAC), is the fourth most frequent cancer in Western countries, thus exceeding breast, prostate, and colorectal cancers. Furthermore, it is expected to become the second cause of cancer-related deaths after lung cancer by 2030 [4]. In 2018, the American Cancer Society estimated that out of 55,440 people, 29,200 men and 26,240 women were diagnosed with PC. Moreover, out of 44,330 people, 23,020 men and 21,310 women are projected to die from the same malignancy in the United States. PC accounts for around 3% of all cancers in the United States and around 7% of cancer deaths (https://ACS). The most recent Surveillance, Epidemiology, and End Results (SEER) database has estimated an overall 5-year survival rate (OS) of only 8.5% (years 2008–14) for the various stages of PC. This dismal prognosis ranks among the lowest in all solid cancers (https://SEER). Over the last decade, advanced treatments have been developed for breast cancer. However, no similar clinical advances in the treatment of PC have been achieved, and no satisfactory results have been obtained. PC is an aggressive malignancy with a high mortality burden. PC is typically diagnosed at advanced stages because its symptoms only manifest at late stages. These symptoms include jaundice, increased body temperature, abdominal pain, and weight loss, which are usually correlated with a significant tumor size that has a high probability of metastasizing. Around 10%–15% of pancreatic cancers are diagnosed following surgery, and the prognosis is very poor in patients who undergo the surgery. At best, only 25%–30% of pancreatic cancer patients will remain alive after 5 years of surgery. One of the most commonly used clinical treatments for pancreatic cancer is gemcitabine. This chemotherapeutic drug hinders tumor growth and reduces the metastasis of tumor cells in the body. Unfortunately, it is therapeutically effective in a small fraction of treated patients (about 23.8%) and is the only alternative method for surgical treatment [5–7]. These limitations highlight the urgent requirement to increase the efficacy of chemotherapeutic treatments and to explore alternative and more effective therapies in pancreatic cancer.

COMMONLY USED THERAPEUTIC AGENTS

161

COMMONLY USED THERAPEUTIC AGENTS Several methods are currently used for treating cancer including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Other procedures or techniques include stem cell transplantation, hyperthermia, photodynamic therapy, blood transfusion, blood donation, and lasers in cancer treatment. However, the three most commonly used methods for cancer treatment remain (1) conventional cancer therapy (limited to surgery), (2) radiation therapy, and (3) chemotherapy. Unfortunately, all these strategies damage healthy tissues and result in an incomplete eradication of cancer, thus stressing the urgent need for novel therapies and drug delivery methods that overcome resistance to traditional treatments and exhibit improved antitumor activity.

Chemotherapy Chemotherapy is used to slow down or stop the growth of cancerous tissues in the body but lacks drug solubility and selectivity for cancer cells. Furthermore, repeated administration of the same chemical/drug can lead to the development of multidrug resistance [8]. Due to their high toxicity and low specificity, these chemotherapeutic drugs are also harmful to healthy cells. In fact, they can reach cancerous regions and cancer-free normal tissues, thus causing multiple drug-related side effects [9].

Radiation Therapy Radiation therapy can be defined as a high-energy X-ray therapy that eradicates the cancer from the body. The most common type of treatment is external beam radiation therapy (EBRT), which is mostly used to treat pancreatic cancer. This therapy consists of a different number of treatments given in a set of time periods using different modes of delivery. Seeing as most PC patients are not completely cured by surgical methods, chemoradiation has emerged as a standard therapy for patients suffering from local advanced disease, although its rates of failure are high and its role in disease management remains unclear and controversial. Stereotactic ablative radiotherapy (SART) or radiation therapy offers an alternative method of treatment because it allows the administration of elevated doses of conformal radiation. The high doses coupled to the reduced treatment time achieved with SART enhance local disease control and outcomes in life quality and cost-effectiveness. When pancreatic cancer surgery is performed, surgeons remove around 95% of the pancreas along with the tumor, leaving only a small portion of the pancreas to ensure the production of insulin. Due to the poor survival rate of pancreatic cancer patients following pancreatectomy, researchers have developed an adjuvant and neoadjuvant therapy program to treat pancreatic cancer. Although their success is limited, these regimens have delineated important principles in the therapeutic application of new-generation treatments such as gemcitabine and docetaxel. In the northern region of the United States, adjuvant EBRT and chemotherapy were adopted as the standard treatment for PC based on the positive results of the Gastrointestinal Tumor Study Group (GITSG, 1987). Another study further reinforced the implementation of

162

10. NANOCARRIERS: DIAGNOSIS AND TREATMENT OF PANCREATIC CANCER

adjuvant EBRT and chemotherapy. In contrast, the European Organization for Research and Treatment of Cancer (EORTC) did not detect significant improvements in survival rates following the randomized study of chemoradiotherapy [10], thus highlighting the limitations of these therapeutic methods. Furthermore, PC treatment is hindered by the emergence of chemoresistance. PDACs characteristically produce a dense stroma that obstructs the penetration of drugs into the cells and exhibits a poor response to most chemotherapeutic agents. The administration of increasing doses of chemotherapy may lead to increased therapeutic holdings. However, these toxic agents do not selectively target specific tumor cells and result in a wide spectrum of side effects, stressing the necessity of developing more targeted therapies and methods of drug delivery.

NANOSCIENCE AND NANOTECHNOLOGY As the previous sections illustrate, extensive surgery, intensive chemotherapy, and radiation therapy have only achieved limited improvements in PC treatment (Fig. 1). Additionally, targeted therapies for pancreatic cancer might not become available in the short term. In light of these current limitations and the shortcomings of traditional chemotherapy, nanotechnology has emerged as an attractive strategy for developing innovative materials characterized by enhanced therapeutic properties and promising applications for disease treatment. These nanotechnologies have paved the way for the design of new treatment methods with improved pharmacokinetic properties. For instance, an attractive strategy to improve the antitumor effects of chemotherapeutic agents and reduce their side effects is to package them into nanoparticles. Since these nanocarriers

FIG. 1 Schematic representation of pancreatic cancer with different treatment methods.

NANOSCIENCE AND NANOTECHNOLOGY

163

are 50–800 nm in size, they cannot cross the vessel wall of healthy cells (15–30nm). In tumor regions, on the other hand, the endothelial cells are loosely packed compared with healthy cell regions, which allow the accumulation of nanoparticles in tumor cells near blood vessels. The bioavailability of nanoparticle-based drug carriers can be ameliorated using two targeting methods, that is, (1) passive and (2) active targeting. The first mechanism, passive targeting, allows nanoparticles to escape through the wall of a blood vessel into cancerous tissues due to the enhanced permeability and retention phenomenon (EPR) described by Maeda and Matsumura [11]. This phenomenon depends on the size of nanoparticles and two key features of cancerous tissues, that is, (1) the leaky vasculature and (2) abnormal lymphatic drainage, which facilitates the penetration and accumulation of nanoparticles in cancerous cells [12]. This feature enables the protection of healthy tissue and can significantly decrease injurious side effects. Passive targeting, however, fails to enhance nanoparticle uptake by tumor cells. This key feature can be accomplished by the second mechanism, namely, active targeting of nanoparticles to receptors or signaling molecules overexpressed at the cellular membrane of cancerous cells. In fact, the addition of targeting ligands to a nanoparticle drug delivery system allows it to uniquely adhere to specific cells or subcellular foci in a given tissue [13, 14] [14]. This in turn can activate the cells by triggering receptor-mediated endocytosis of the nanoparticles [15]. Receptor-based active targeting of nanovehicles can thus limit the nonspecific systemic exposure of cytotoxic agents and can bypass multidrug resistance by overcoming P-glycoprotein-mediated drug efflux [16]. Given their mechanistic advantages, both active and passive targeting can be combined to reduce drug interactions with healthy tissues. Furthermore, nanocarrier-based technologies can increase the effectiveness of chemotherapeutic drugs by improving delivery mechanisms, thus achieving higher tumor reduction with smaller drug doses [17].

Nanocarriers Nanoparticles can be described as colloidal drug transporters or carriers containing submicron sizes of particles nearly A

180

11. MOLECULAR MARKERS FOR TREATMENT AND TOXICITY OF GEMCITABINE

TABLE 1 Polymorphism genes involved in the gemcitabine transport Location on gene and rsID and characteristics

Affinity for GEM

Reference

+419 (rs17215836) insertion/ deletion

Not associated with response rate, mTTP, mST, and hematologic toxicity in NSCLC patients

[37]

+565G>A (rs2290272) V189I

Not associated with response rate, mTTP, mST, and hematologic toxicity in NSCLC patients

[37]

+709C>A (rs8187758) Q237K

Not associated with response rate, mTTP, mST, and hematologic toxicity in NSCLC patients

[37]

+1368G>A (rs2242048) Q456Q

Not associated with response rate, mTTP, mST, and hematologic toxicity in NSCLC patients

[37]

+1528C>T (rs2242047) R510C

Not associated with response rate, mTTP, mST, and hematologic toxicity in NSCLC patients

[37]

Associated with mTTP in PC patients

[41]

The response rate, mTTP, and mST were not significantly different among patients with different genotypes. However, GG genotype is associated with decreased risk of hematologic toxicity

[37]

+65C>T (rs11854484) P22L

Not associated with response rate and mTTP in NSCLC patients. However, CC genotype is associated with lower mST and decreased risk of hematologic toxicity

[37]

+225C>A (rs1060896) R75S

Not associated with response rate and mTTP in NSCLC patients. However, CC genotype is associated with lower mST and decreased risk of hematologic toxicity

[37]

+338C>T (rs10868138) Y113C

Not associated with response rate, mTTP, mST and hematologic toxicity in NSCLC patients

[37].

rs7867504

Associated with increase in the formation clearance of dFdCTP

[43]

Increased OS in metastatic BC patients

[44]

+600G>Ad (rs1128870) E200E

Monomorphic for G allele in Asian BC patients

[39]

706G>C (rs61758845)

Compared with the GC or CC genotype, GG genotype showed higher response rate and increased OS

[46]

IVS12 201A>G (rs760370)

Associated with tumor response

[27]

As a part of a 2-SNP haplotype (rs747199 and rs760370), rs760370 “A” allele is associated shorter OS compared with G allele in metastatic BC patients receiving GemTaxol

[44]

SLC28A1

+1561G>A (rs2242046) N521D SLC28A2

SLC28A3

SLC29A1

181

NEUCLEOSIDE TRANSPORTER GENES

TABLE 1

Polymorphism genes involved in the gemcitabine transport—cont’d

Location on gene and rsID and characteristics

Affinity for GEM

Reference

IVS2 549T>C (rs324148)

Carriers of CC genotype associated with decreased postinduction OS and DFS

[49]

IVS2 +913C>T (rs9394992)

Associated with grade 3 and 4 neutropenia

[27]

706G>C (rs747199)

As part of a 3-SNP haplotype (rs731780 and rs70914 and rs747199), both alleles of rs747199 showed 37% higher RNA expression

[45]

As part of a 2-SNP haplotype (rs747199 and rs760370), rs747199G allele is associated with significantly shorter OS in metastatic BC patients receiving GemTaxol

[44]

(rs2242047) was shown to be associated with median time to progression (mTTP) or tumor progression [41]. SLC28A1 1543C > T, 1576G > A, and SLC28A2 283A > C variants were not associated with pharmacokinetic or toxicity profile of drug in advanced NSCLC patients receiving GemCis [32]. The SLC28A3 haplotypes were associated with OS of NSCLC patients receiving GEM [42]. A pharmacokinetic study in patients with solid tumors receiving GEM treatment revealed the association between the formation clearance of dFdCTP and the C allele carriers of SCL28A3 rs7867504 [43]. A twofold increase in the formation clearance of dFdCTP was found in individuals with the CC genotype. Further, SCL28A3 rs7867504 CC or CT genotypes are associated with the increased OS in metastatic BC patients receiving combination chemotherapy with GemTaxol [44].

SLC29 Family The hENT1 (SLC29A1) mRNA is detected mainly in the liver, kidney, and small intestine. SLC29A1 mediates the cellular uptake of GEM and its expression correlated with cell chemosensitivity. Screening 1.6 kb upstream of the transcription initiation site of SLC29A1 revealed the presence of three functional promoter polymorphisms [1345C > G (rs731780), 1050G > A (rs70914), and 706G > C (rs747199)] [45]. Of the four naturally occurring haplotypes from these polymorphisms, only two are major haplotypes, CGG/CGG and CGG/CGC. Compared with the haplotype CGG/CGG, the CGG/CGC haplotype showed 37% more RNA expression [45]. Polymorphisms in SLC29A1 gene are indecently or in combination with other genes associated with the outcome measures of GEM therapy in patients with locally advanced PC [27]. SLC29A1 rs747199 and rs9394992 polymorphisms and haplotypes were associated with OS of GEM therapy in patients with NSCLC [42]. Analysis of SLC29A1 rs61758845 in GEM chemotherapy responders and nonresponders revealed that the response rate and OS are higher in patients with the GG genotype compared with those with the GC or CC genotype [46]. In metastatic BC patients receiving combination chemotherapy with GemTaxol, the GA haplotype of rs747199 and rs760370 polymorphisms was associated with a significantly shorter OS [44]. Reduced median survival that is noted in PC patients with low SLC29A1 expression from the ESPAC-3 trial indicated that the GEM is not the appropriate choice for

182

11. MOLECULAR MARKERS FOR TREATMENT AND TOXICITY OF GEMCITABINE

patients with low tumor SLC29A1 expression [47]. Higher SLC29A1 expression is correlated with increased PFS and OS in leiomyosarcoma and angiosarcoma patients receiving GEM [48]. However, in acute myeloid leukemia patients, the rs324148 CC genotype is associated with reduced postinduction OS and disease-free survival (DFS) [49]. Although there is 46% sequence identity between SLC29A1 and SLC29A2 [50], a big difference in the kinetics of GEM transport was observed [51]. The SLC29A2 gene also harbors some polymorphic variants with altered function, but they are unlikely to cause variations in drug response due to their low frequency [52]. A genome-wide association study on GEM + placebo identified a polymorphism in the SLC29A2 gene (rs2279861) that contributes to the GEM response variability [53]. Further pharmacogenomic studies are needed to validate the role of SLC29A2 polymorphisms in the kinetics of GEM transport.

GENES OF METABOLIZING ENZYMES Cytidine Deaminase The gene encoding cytidine deaminase (CDA) is located in locus 1p36.2-1p35 [54, 55]. CDA catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively, that results in the loss of their cytotoxic activity. Hence, decreased CDA activity is linked with improved efficacy and increased toxicity with GEM therapy [56]. Although >1000 polymorphic variants in the CDA gene have been identified, only few polymorphisms such as rs2072671, rs60369023, and rs1048977 were studied extensively [57–60]. The rs2072671 A > C is a nonsynonymous SNP that changes lysine to glutamine at amino acid position 27. Indeed, a series of in vitro studies reported the association between rs2072671 “A” allele and decreased CDA expression [59, 61–63]. In consensus to this, few studies reported an association between the AA genotype and greater risk for neutropenia in cancer patients treated with GEM [61, 64]. In contrast to the C allele was shown to be associated with higher risk of neutropenia [27, 65, 66]. Further, the association between rs2072671 and outcome measures of GEM therapy is also inconclusive [37, 44, 61]. Meta-analysis of 13 studies revealed that the CDA rs2072671 polymorphism was not significantly associated with OS; however, patients carrying the rs2072671C allele exhibited poor survival and experienced grade 3 leucopenia and severe neutropenia [67]. The rs60369023 G > A is a nonsynonymous SNP that changes alanine to threonine at amino acid position 70. Studies in Japanese cancer patients treated with GemCis demonstrated that the AA genotype of the rs60369023 is associated with the neutropenia [68, 69]. In both studies, the “A” allele was significantly associated with the decreased clearance of GEM. Further, the “A” allele was well correlated with the reduced CDA enzyme activity [69]. Few in vitro experiments also provide supporting evidence that the “A” allele is associated with deceased CDA enzyme activity [62, 70]. Although there is compelling evidence for an association between CDA rs60369023 and CDA enzyme activities, interethnic differences also exist in the distribution of this variant. The rs60369023 “A” allele is more common in Africans (13%) compared with Asians ( T is a synonymous polymorphism at amino acid position 145 of CDA. Studies to investigate the relationship between rs1048977 and CDA enzyme activity have

GENES OF METABOLIZING ENZYMES

183

yielded inconclusive results [59, 63]. The CC genotype was associated with response and mTTP without hematologic toxicity in NSCLC patients receiving GEM chemotherapy [37]. In biliary tract cancer patients receiving GEM-based chemotherapy, the rs1048977 variant allele in a dominant model was associated with tumor response [73]. Further studies failed to associate the C allele with patient outcomes [27, 44]. A pharmacokinetic study of GEM showed that the rs1048977 C > T is associated with lower clearance GEM and increased hematologic toxicities [40].

Deoxycytidine Kinase Deoxycytidine kinase (dCK) mediates phosphorylation of dFdC to dFdCMP. Overexpression of dCK enhanced the GEM sensitivity in various tumor cells [74]. High dCK protein expression is correlated with improved OS and DFS in PC patients [75]. siRNA-mediated downregulation of dCK in PC cells enhanced acquired resistance to GEM [76]. The gene coding for dCK (DCK) is mapped to chromosome 4q13.3–q21.1 [77]. Resequencing of the DCK gene revealed 28 polymorphic variants, of which two common variants rs66878317 and rs67437265 resulted in moderate decrease in DCK activity and dCK protein levels [78]. There are no well-established associations between DCK polymorphisms and response to GEM (Table 2). Two intronic polymorphisms (rs4694362 and rs12648166) showed association with treatment-related neutropenia toxicity and treatment response but not with OS in PC patients [79]. As a part of the haplotype with seven other SNPs, rs4694362 “T” allele is associated with rapid disease progression in PC patients treated with GEM [41]. Further in metastatic BC patients treated with GemTaxol, rs4694362 T allele is not associated with patient survival [44]. The in vitro evaluation of chemosensitivity of PC cell lines to GEM revealed that the cell lines with the rs12648166 AG genotype were more sensitive than the GG [33]. The rs12648166 AA genotype was significantly associated with increased OS, DFS, and reduced mortality in PDAC patients treated with GEM [75].

Deoxycytidylate Deaminase Deoxycytidylate deaminase (DCTD) catalyzes the conversion of dCMP to dUMP in the deamination process, thus providing the nucleotide substrate for thymidylate synthase (TS). The gene coding for DCTD is localized on human chromosome 4, at locus 4q35.1 [80]. Studies related to the association of these DCTD polymorphisms with toxicity/outcome measures of GEM therapy are represented in Table 2. Resequencing of DCTD gene revealed 29 polymorphic variants, of which 205A > G (rs35932500) Asn69Asp (also known as +172A > G; Asn58Asp) is the only nonsynonymous SNP [81]. Recombinant Asp69 showed reduced activity for GEM monophosphate compared with its wild-type allele Asn69 allele [81]. Another synonymous SNP rs4742 (V116V) is not associated with either risk of neutropenia or clinical outcome measures of GEM therapy in various cancers [27, 37]. However, a weak association between this variant and OS in PC patients treated with GEM was documented [79]. The DCTD rs1130902 and rs6834938 polymorphisms were associated with increased formation clearance of dFdCTP when treated with GEM in solid tumor patients [40]. Gene expression

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11. MOLECULAR MARKERS FOR TREATMENT AND TOXICITY OF GEMCITABINE

TABLE 2 Polymorphisms involved in genes coding for gemcitabine metabolizing enzymes Location on gene and rsID and characteristics

Affinity for GEM

Reference

CDA allozyme with “C” allele showed significantly higher Km than “A” allele

[81]

“C” allele is not associated with CDA activity. However, one patient with AC genotype had C (rs2072671) K27Q

+208G>A (rs60369023) A70T

+435C>T (rs1048977) T145T

185

GENES OF METABOLIZING ENZYMES

TABLE 2

Polymorphisms involved in genes coding for gemcitabine metabolizing enzymes—cont’d

Location on gene and rsID and characteristics

Affinity for GEM

Reference

The rs1048977 TT genotype is associated with increased expression of CDA

[59]

The rs1048977 C>T is associated with lower clearance of GEM and increased hematologic toxicities

[40]

The rs1048977 genotypes are not associated with expression of CDA

[63]

The rs1048977 is not associated with GEM clearance

[43]

The C allele was not associated with metastatic BC patients treated with GemTaxol

[44].

In a dominant model, the variant allele was associated with tumor response

[73]

Associated with neutropenia and tumor response to therapy but not with OS in PC patients

[79]

As a part of the haplotype, rs4694362 “T” allele is associated with rapid progression of PC

[41]

The “T” allele is not associated with metastatic BC patient survival

[44]

Associated with neutropenia and tumor response to therapy but not with OS in PC patients

[79]

Cell lines of PC patients with AG genotype were more sensitive to GEM than the GG genotype

[33]

Genotype “AA” significantly associated with longer OS, DFS, and reduced mortality

[75]

70A>G (rs66878317) Ile24Val

Decreased in DCK activity and dCK protein levels

[78]

364C>T (rs67437265) Pro122Ser

Decreased in DCK activity and dCK protein levels

[78]

+205A> G (rs35932500) Asn69Asp

The Asp69 showed reduced activity for GEM monophosphate in vitro (also known as +172A>G; Asn58Asp)

[81]

348T> C, T>A, and T> G (rs4742) (V116V)

Not associated with either risk of neutropenia or response in NSCLC patients receiving GEM therapy (also known as +315T>C; V105 V)

[37]

Not associated with neutropenia, PFS, tumor response to GEM therapy in patients with locally advanced PC

[27]

A weak association of the rs4742 SNP with OS in patients with PC was observed

[79]

DCK IVS6 1205C>T (rs4694362)

207 +9846A>G (rs12648166)

DCTD

Continued

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11. MOLECULAR MARKERS FOR TREATMENT AND TOXICITY OF GEMCITABINE

TABLE 2 Polymorphisms involved in genes coding for gemcitabine metabolizing enzymes—cont’d Location on gene and rsID and characteristics

Affinity for GEM

Reference

506G>A (rs1130902)

“A” allele carriers associated with increased formation clearance of dFdCTP

[40]

1739C>T (rs6834938)

“C” allele carriers associated with increased formation clearance of dFdCTP

[40]

The rs1044457 is not associated with response to GemCis in NSCLC patients

[84]

The rs1044457 is associated with decreased formation clearance of dFdCTP in patients with solid tumor treated with GEM

[40]

The rs1044457 was associated with OS, mTTP, and disease progression in PC patients treated with GEM

[41]

The rs4492666 wild-type genotypes associated with shorter OS in NSCLC patients treated with the GemCis

[84]

The rs4492666 is not associated with patient survival in metastatic BC patients treated with GemTaxol

[44]

The rs11211524 wild-type genotypes associated with shorter OS in NSCLC patients treated with the GemCis

[84]

The rs11211524 is not associated with patient survival in metastatic BC patients treated with GemTaxol

[44]

22G>C (rs7543016) Gly8Arg

Associated with improved OS and mTTP in PC patients

[41]

240G>T (rs35687416) Gln80His

Correlated with OS and mTTP in PC patients

[41]

1995G> A (rs3925058)

Not associated with mTPP in PC patients

[41]

28 bp VNTR (rs45445694) 50 UTR

Colorectal cancer patients with 3R/3R had higher TS mRNA levels and lesser side effects to 5-fluorouracil

[91]

+1494del6 (rs16430) 30 UTR

Patients with 1494ins6/ins6 genotype showed increased myelotoxicity from GemCarbo

[39]

CMPK1 360C>T (rs1044457)

171 +1057A> C (rs4492666) IVS1

928A>C (rs11211524) IVS1

TYMS

and methylation analyses in patients with malignant glioma demonstrated that high level of DCTD expression was correlated well with shorter survival [82].

Cytidine Monophosphate Kinase 1 Cytidine monophosphate kinase 1 (CMPK1) phosphorylates dFdCMP to dFdCDP and plays an important role in GEM activation. The gene coding for CMPK1 was localized to chromosome 1p34.1–1p33 [83]. Few studies have shown associations between cancer patient response to GEM and CMPK1 gene SNPs (Table 2). Analysis of four tagging SNPs in NSCLC

GENES OF DRUG TARGETS

187

patients treated with the GemCis revealed that the wild-type genotypes of CMPK1 rs4492666 and rs11211524 were associated with shorter OS [84]. The CMPK1 rs1044457 C > T variant was associated with decreased formation clearance of dFdCTP when treated with GEM in solid tumor patients [40]. In PC patients treated with GEM, the rs1044457 was associated with OS, mTTP, and disease progression, while rs35687416 was correlated with OS and mTTP [41]. Further, rs4492666 and rs11211524 SNPs are not associated with patient survival in metastatic BC patients treated with GemTaxol [44].

Thymidylate Synthase Thymidylate synthase (TS) catalyzes the conversion of dUMP to dTMP, a rate-limiting step in DNA synthesis. Inhibition of TS results in depletion of cellular nucleotide pools [85]. Further, the inhibition of TS activates hENT1 and increases antitumor response of GEM therapy [86]. siRNA-mediated knockdown of TS expression decreased resistance to GEM in PC cell lines [87]. The TS gene (TYMS) is located on chromosome 18p11.32 [88]. Two polymorphisms of TYMS gene were extensively studied (Table 2). The rs45445694 is a VNTR polymorphism in 50 -UTR, consisting of two or three repeats of 28 bp (2R or 3R). The rs16430 is a 6 bp insertion and deletion polymorphism (+1494del6) in the 30 -UTR of TYMSS gene [89, 90]. In patients with metastatic colorectal cancer treated with 5-fluorouracil, individuals homozygous for the 3R had higher TS mRNA levels and had less severe side effects compared with those homozygous for the 2R variant [91]. Asian breast cancer patients with the 1494ins6/ins6 genotype are associated with increased myelotoxicity from GemCarbo [39]. However, fewer studies investigated the association between TYMS variants and response to GEM are less.

GENES OF DRUG TARGETS Ribonucleotide Reductases 1 Ribonucleotide reductase (RNR) is an enzyme that regulates the cell cycle and represents an important target for cancer drugs [94]. When compared with the normal cells, the cancer cells need more amounts of dNTPs for proliferation, and hence, the RNR activity is much higher in cancer cells than normal cells [95]. Inhibition of RNR reduces the dNTP pool and favors the incorporation of dFdCTP into DNA. Mammalian RNRs consist of three homodimeric subunits (large subunit, RRM1; small subunit, RRM2; and RRM2B), which associate to form the holoenzyme [96]. As a nucleoside analogue, the phosphorylated metabolite of GEM (dFdCDP) reacts with the substrate-binding catalytic site of the RRM1 subunit, thereby inactivating the enzyme [97]. In NSCLC patients treated with GEM, there was an increased RRM1 expression associated with GEM resistance [98, 99]. Subsequent studies also confirmed the association between high levels of RRM1 and GEM resistance [100–102]. Further, low/negative expression of RRM1 correlated with an increased PFS, OS, and the efficacy of GEM therapy [103–107]. The gene coding for RRM1 was localized to the distal band 11p15 by in situ hybridization [108]. Polymorphic variants of the RRM1 gene were shown to be associated with susceptibility to GEM in cancer patients [100, 109–111]. The studies investigating the association between RRM1 gene polymorphisms and response to GEM have yielded inconclusive results (Table 3).

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11. MOLECULAR MARKERS FOR TREATMENT AND TOXICITY OF GEMCITABINE

TABLE 3 Polymorphisms involved in the genes coding for drug target RRM1 Location on gene and rsID and characteristics 524T>C (rs11030918) promoter

37A>C (rs12806698) promoter

1082C>A (33A>G) (rs183484) Arg284Arg

2927A>C (rs1042927)

2455A>G (rs9937) Thr741Thr

42G>A (2464G>A) (rs1042858) A744A

792 +287T>C (rs720106)

Response to GEM therapy

Reference

Associated with OS and PFS lung cancer patients

[98]

Not associated with response, mTTP, and toxicity

[37]

Not associated with OS

[84]

Decreased the occurrence of grade 2 nausea/vomiting

[65]

Not associated with OS, PFS, and neurotoxicity

[44]

CC genotype carriers showed significantly longer PFS

[29]

Associated with OS and PFS lung cancer patients

[98]

Allelotype rs11030918CT-rs12806698AC showed higher response rate

[34]

Not associated with response, mTTP, and toxicity

[37]

CC genotype is associated with higher median PFS in NSCLC patients

[112]

Not associated with OS

[84]

Decreased the occurrence of grade 2 nausea/vomiting

[65]

NSCLC patients with AA genotypes showed longer PFS, and CC genotype showed shorter OS

[29]

Genotypes are not different in PBMC samples of BC patients and healthy volunteers

[111]

Showed significant association with PFS and grade 3 and 4 neutropenia toxicity

[27]

Not associated with OS

[84]

Significantly decreased OS

[65]

Not associated with OS, PFS, and neurotoxicity

[44]

Genotypes are not different in PBMC samples, breast cancer patients, and healthy volunteers. The rs9937 and rs1042858 haplotype associated with low frequency of neutropenia

[111]

Showed significant association with PFS but not with neutropenia

[27]

Not associated with PFS in NSCLC patients

[112]

Significantly associated with neurotoxicity

[44]

Genotypes are not different in PBMC samples, breast cancer patients, and healthy volunteers

[111]

Not associated with PFS and neutropenia toxicity

[27]

Not associated with PFS NSCLC patients

[112]

Associated with OS and PFS but not with neurotoxicity

[44]

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189

Two promoter polymorphisms (rs11030918 and rs12806698) that impact the promoter activity in vitro were associated with overall and disease-free survivals in lung cancer patients [98]. However, these polymorphisms were not associated with RRM1 expression in the tumors [98]. In contrast to this, these promoter polymorphisms are not associated with response, mTTP, and toxicity in Asian NSCLC patients [37]. Among the RRM1 promoter allelotypes, the rs11030918CT-rs12806698 AC showed higher response rate, but the OS and PFS did not differ significantly by allelotype in NSCLC patients treated with GEM [34]. In NSCLC patients treated with platinum compounds and GEM, the carriers of rs11030918 CC and rs12806698 AA showed significantly longer PFS [29]. Furthermore, the rs12806698 CC genotype carriers were associated with shorter OS in [29]. The distribution of RRM1 polymorphisms in peripheral blood mononuclear cells (PBMC) from advanced BC patients treated with GEM is not different from PBMC from healthy volunteers [111]. The haplotype of rs9937 A > G and rs1042858 G> A SNPs were associated with susceptibility of GEM and showed lower frequency of neutropenia [111]. In NSCLC patients, sequencing of three RRM1 SNPs (rs12806698, rs9937, and rs1042858) revealed that the patients with only the rs12806698AC genotype had higher median PFS [112]. In PBMCs, RRM1 mRNA expression is not associated with the efficacy of GEM [112]. Tag SNPs from the RRM1 gene are not associated with the OS of NSCLC patients receiving GemCis therapy [84]. The ATAA and ATGA haplotypes formed out of rs11030918, rs1042927, rs9937, and rs720106 SNPs showed association with the neurotoxicity [44]. Given the known prognostic or predictive value of RRM1 expression in patients receiving GEM therapy, the inconsistent associations observed indicate the lack of predictive value for RRM1 polymorphisms.

CONCLUSIONS Effectiveness of GEM chemotherapy is often limited by treatment-related toxicity. Predicting response and identification of nonresponders before chemotherapy may help to reduce the adverse effects. A number of studies have analyzed the association between chemoresistance and genes that alter uptake, metabolization, and catabolization of GEM. On the basis of the data available, SLC29A1, CDA, and RRM1 gene expressions and protein levels are connected with cancer patient responses to GEM. Determination of polymorphisms in genes of these metabolizing enzymes offered very little information to individualize and optimize treatment. Due to inadequate evidence of clinical utility for pharmacogenetic testing, more research is warranted to uncover clinically relevant SNPs associated with efficacy of GEM therapy. Further computational data analysis approaches linking pharmacogenomics with informatics are required for translating pharmacogenomics into clinical testing panels that allow clinicians to better predict the potential efficacy of chemotherapy.

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Index Note: Page numbers followed by f indicate figures, and t indicate tables.

A Abraxane, 76, 167–170t ACS. See American Cancer Society (ACS) Activated macrophages, 120 Active targeting, 163 Adjuvant chemoradiation, 63–64 Adjuvant therapy in borderline removable tumor, 64–65 gemcitabine and capecitabine, 63 gemcitabine and 5-fluorouracil, 62 gemcitabine and S-1, 63 ADP-ribosyl transferases (ADPRT), 134–135 Alcohol consumption, 14–15, 119 AMD3100, 121 American Cancer Society (ACS), 59, 118, 160 Angiogenesis, 74, 96–98 APACT trial, 64 Apoptosis, 7, 18, 46–48, 92–93, 98, 122 Apoptotic proteins, 114 ATP hydrolysis, 125 Aucubin, 113 Axitinib, 74 AXP107–11, 102

B Bax, 93–96, 98 β-catenin, 32 B-cell lymphoma 2 (Bcl-2), 47, 92–98, 122 B-cell lymphoma-extra-large (bcl-xL), 47 Bcl-2. See B-cell lymphoma 2 (Bcl-2) β-ionone, 114 Bevacizumab, 74 Bisdemethoxycurcumin (BDMC), 99, 101–102 Borderline removable tumor, 64 BRACA1, 9–10 BRACA2, 9–10 β-Trcp, 32 BxPC-3 cells, 36, 50

C Cadherins, 122–123 Cancer, 2–3, 112, 160 causes of deaths, 2–3, 2f

Cancer stem cells (CSCs), 30, 91, 99–100 Candidate genes, 179 CAPAN-1 cells, 52 Capecitabine, 63, 69, 72, 90 Carcinoembryonic antigen (CEA), 18 Carcinoembryonic antigen 19-9 (CA19-9), 14, 18, 112 Caspase activity regulation, terpenoids in, 113–114 Caspases, 47, 92t, 96 CD44, 91 CDA. See Cytidine deaminase (CDA) CDH1 gene, 29 CDKN2A, 6–7 Cetuximab, 73 Chemoresistance age-related effects, 20f association with age and gender, 16–17 epithelial-mesenchymal transition in, 33–35 factors modulated in age groups, 18–19 5-flurouracil, 34 gemcitabine and, 34–35, 48–50 mechanisms associated with, 17–18 molecular markers, 19t tumor microenvironment, 90–91 Chemosensitizers, 91 curcumin as, 92–93, 92t genistein as, 93–94 Chemotherapy, 59–60, 90, 120–121, 161 Chemotherapy resistance pathways, 123–124 Chromatin, 139 Chromatin immunoprecipitation (ChIP) analysis, 134–135 Chronic pancreatitis (CP), 14, 119–120, 138 C-ion RT, for locally advanced pancreatic cancer (LAPC), 121 Circulating tumor cells (CTCs), 26–27 Claudin 5 (CLDN5), 137 Clinical classification borderline resectable tumors (stages IIA–IIB), 61 localized surgically resectable (stages I–IIB), 61 locally advanced unresectable tumors (stage III), 61 metastatic tumor (stage IV), 61 Clonal expansion, 4–5 CMPK1. See Cytidine monophosphate kinase 1 (CMPK1)

197

198

INDEX

C-myc, 46–47 Computed tomography (CT), 59, 61 Concentrative nucleoside transporters (CNTs), 179 Conjugated drugs EndoTag-1, 76 nab-paclitaxel, 76 PEP02 (MM-398), 76 trastuzumab emtansine, 76–77 CONKO-003 trial, 70 COX-2. See Cyclooxygenase-2 (COX-2) CpG islands (CGIs), 135–136, 138 C-reactive protein (CRP), 73 Cross talk, 94–95, 135 CSCs. See Cancer stem cells (CSCs) Cullin3, 99 Curcumin, 92–93, 92t, 99, 101–102 Curcuminoids, 115 Cyclin D1, 46–47 Cyclin-dependent kinases (CDKs), 7, 137 Cyclooxygenase-2 (COX-2), 47, 92–97 Cytidine deaminase (CDA), 91 genes coding for gemcitabine, 182–183, 184–186t Cytidine monophosphate kinase 1 (CMPK1), 177–178 genes coding for gemcitabine, 184–186t, 186–187 Cytokeratin 81 (KRT81), 112

D Deoxycytidine kinase (dCK), 18, 20–21, 34–35 deficiency, 49 genes coding for gemcitabine, 183, 184–186t phosphorylation of gemcitabine, 177–178 Deoxycytidylate deaminase (DCTD), genes coding for gemcitabine, 183–186, 184–186t Desmethoxycurcumin (DMC), 101–102 Desmoplasia, 74–75 Desmoplastic stroma, 3 20 ,20 -Difluoro-20 -deoxycytidine (dFdC). See Gemcitabine 20 ,20 -Difluorodeoxyuridine (dFdU), 177–178 Dihydrofluorouracil (DHFU), 34 Dihydropyrimidine dehydrogenase (DPD), 34 Diterpenoids, 113–114 DNA damage, 114, 122 DNA damage response (DDR), 122 DNA demethylation, 135–139 DNA methylation, 123, 134–139 DNA methyltransferases (DNMTs), 101, 135–137 DNA replication, 46 DNMT1, 137 DNMT3b, 137 DNMTs. See DNA methyltransferases (DNMTs) Dose, of gemcitabine (GEM), 178 Doxycycline, 20 Drug delivery, 163–167

E Eastern Cooperative Oncology Group Performance Status (ECOG) score, 66 E-cadherin, 26–27, 29, 122–123 Efflux, 122 EGFR. See Epidermal growth factor receptor (EGFR) EMT. See Epithelial-mesenchymal transition (EMT) Endocrine, 118 Endocrine pancreatic cancer, 119 EndoTag-1, 76 Enhanced permeability and retention phenomenon (EPR), 163 Enhancer of zeste homologue 2 (EZH2), 101, 142–143 Epidermal growth factor receptor (EGFR), 51–52, 94–97 inhibitors, 73 Epithelial-mesenchymal transition (EMT), 9, 124 cancer metastasis, 26 in chemoresistance, 33–35 5-flurouracil, 34 gemcitabine, 34–36 developmental, 26 methyl-CpG-binding domain proteins, 137 paired related homeobox 1, 31 signaling pathways, 28f Notch, 32 TGF-β, 32 TNF-α, 33 Wnt/β-catenin, 32 SNAIL, 30 solid tumors, 122–123 Twist, 30–31 wound healing/tissue regeneration, 26 ZEB1, 29 Epoxyeicosatrienoic acids (EETs), 92–93 Equilibrative nucleoside transporters (ENTs), 179 Erlotinib, 60, 73 ESPAC-3 trial, 62–63, 181–182 Etoposide, 113 E3 ubiquitin ligases, 134–135 Eukaryotic DNA, 134–135, 139 European Organization for Research and Treatment of Cancer (EORTC) trial, 63, 161–162 Evofosfamide, 77 Exocrine, 118 Exocrine pancreatic cancer, 119 Exome sequencing, 138, 145 External beam radiation therapy (EBRT), 161 Extracellular signal-regulated kinase (ERK), 5, 50

F Familial adenomatous polyposis (FAP), 112–113 Familial atypical multiple mole-melanoma (FAMMM), 9–10

INDEX

Familial malignant melanoma, 112–113 Familial pancreatic cancers, 9–10. See also Pancreatic cancer (PC) Fanconi’s anemia, 9–10 Farnesylation, 72 5-flurouracil (5-FU), 34, 121 5-hydroxymethylcytosines, 136–137 5-methylcytosine (5mC), 135–136 Fixed-dose rate (FDR) approach, 66 Fluorodeoxyuridine monophosphate (FdUMP), 34 5-Fluorouridine-50 -triphosphate (FUTP), 34 FOLFIRINOX, 16–18, 60, 67, 120–121 vs. gemcitabine/nab-paclitaxel, 68 metastatic pancreatic cancer, 66 as neoadjuvant therapy, 65 Forkhead box protein M1 (FoxM1), 93–94 5-FU. See 5-flurouracil (5-FU)

G Galunisertib, 74 Ganetespib, 126–128 Gastrointestinal Tumor Study Group (GITSG), 161–162 Geldanamycin, 125–126t, 126–128, 127f GEM. See Gemcitabine (GEM) GemCarbo, 178–179, 187 Gemcitabine (GEM), 16–18, 46, 90 candidate genes affecting, 179 and chemoresistance, 34–35, 48–50 dose, 178 drug targets, genes of ribonucleotide reductases 1, 187–189, 188t efficacy, 36 mechanism, 46 metabolism of action, 177–178, 177f metabolizing enzymes, genes of cytidine deaminase, 182–183, 184–186t cytidine monophosphate kinase 1, 184–186t, 186–187 deoxycytidine kinase, 183, 184–186t deoxycytidylate deaminase, 183–186, 184–186t thymidylate synthase, 184–186t, 187 neucleoside transporter genes SLC29 family, 180–181t, 181–182 SLC28 family gene polymorphisms, 179–181, 180–181t nucleotide transporters, 36 phosphorylation of, 177–178 resistance, EMT in, 36 sensitivity, 46–48 structure, 46 in targeting signaling pathways, 50–52 toxicitiy, 178 transport, 177–178

199

Gemcitabine and S-1 trial (GEST), 69 Gemcitabine-cisplatin, 120–121 Gemcitabine-oxaliplatin, 120–121 Gemcitabine plus nab-paclitaxel, 64, 67 GemTaxol, 178, 181–182 Genistein, as chemosensitizers, 93–94 Genome-wide sequencing, 144–145 Glucose-regulated protein 78 (GRP78), 99 Guanine nucleotide exchange factor (GEF), 138–139

H HATs. See Histone acetyltransferases (HATs) H2BK5, 134–135 HDACs. See Histone deacetylases (HDACs) Heat shock proteins, 124–125 Hedgehog inhibitors, 75 hENT1. See Human equilibrative nucleoside transporter 1 (hENT1) Hepatocyte nuclear factor 1A (HNF1A), 112 Hereditary breast and ovarian cancer syndrome (HBOC), 112–113 Hereditary pancreatitis (HP), 112–113, 119–120 HER-2 inhibitors, 72 Heterochromatin protein 1 (HP1), 141–142 Histone acetylation, 139–141 Histone acetyltransferases (HATs), 101, 134–135, 139, 141 Histone deacetylases (HDACs), 101, 134–135, 139 class I, 139 class II, 139 Histone deacetylation, 139–141 Histone demethylases (HDMs), 134–135 Histone H3 lysine 9 acetylation (H3K9Ac), 141 Histone H3 lysine 27 acetylation (H3K27Ac), 141 Histone methylations activation, 143–145 inactivation, 141–143 Histone methyltransferases (HMTs), 134–135 H3K9, 134–135 H3K14, 134–135 H3K27, 134–135 H4K20, 134–135 H3K4me3, 143–144 H3K9me3, 141–142 H3K27me3, 142–143 HMGA1, 34–35 HSP40, 125 HSP70, 125 HSP90, 124–125, 125–126t drawbacks in clinical trials, 125–126t inhibitors in clinical trials, 125–126t, 126–128 Human concentrative nucleotide transporter (hCNT), 177–178

200

INDEX

Human equilibrative nucleoside transporter 1 (hENT1), 18, 48, 177–178 Hyaluronic acid (HA), 75 Hydralazine, 20–21 Hypoxia, 91 Hypoxia-inducible factor-1α (HIF-1α), 91

I IAP repeat-containing protein 2 (c-IAP1), 47 IL6, 52 Immunotherapy, 121 IMPACT trial, 68 Inducible nitric oxide synthase (iNOS), 96–97 Inflammation, 30 Inorganic carbon nanotubes (CNTs), 166 Insulin-like growth factor 1 (IGF-1) receptors, 73–74 Integrins, 122–123 Interferon-stimulated gene 15 (ISG15), 91 Irinotecan, 113

J JAK/STAT inhibitors, 73 JAPAC 01 trial, 63 c-Jun N-terminal kinases (JNK), 91

K KDM2B, 145 Ki67 antigen, 18 Kinases, 134–135 Kirsten rat sarcoma virus oncogene (KRAS) inhibitors, 72 mutation, 5–6, 6f nuclear factor kappa B signaling cascade, 6 phosphoinositide 3-kinase/ATK cascade, 5 Raf/mitogen-activated protein kinase cascade, 5 KLM1, 49

L Li-Fraumeni syndrome (LFS), 7, 112–113 Liposome, 165 Lipoxygenase (LOX), 96–97 Luminespib, 125–126t, 126–128, 128f Lymphoid enhancer factor (LEF), 32 Lynch syndrome, 112–113 Lysine acetylation, 141 Lysine methyltransferase, 141–142

M Magnetic nanoparticles, 166–167 Magnetic resonance imaging (MRI), 59, 61 Matrix metallopeptidase 9 (MMP-9), 47–48 Matrix metalloproteinases (MMPs), 8–9, 75, 93–94 MDR. See Multidrug resistance (MDR)

Mesenchymal-epithelial transition (MET), 26–27 Mesoporous inorganic nanoparticle, 165–166 Metabolizing enzymes, genes of cytidine deaminase, 182–183, 184–186t cytidine monophosphate kinase 1, 184–186t, 186–187 deoxycytidine kinase, 183, 184–186t deoxycytidylate deaminase, 183–186, 184–186t thymidylate synthase, 184–186t, 187 Metal oxide nanoparticle, 166 Metastasis, 8, 27, 31, 122–123 Metastatic Pancreatic Adenocarcinoma Clinical Trial (MPACT), 60, 67–69 Metastatic pancreatic cancer early period, 65–66 first-line therapies clinical parameters, 68–69 FOLFIRINOX, 66 gemcitabine plus nab-paclitaxel, 67 MPACT trial, 67–68 precautionary and toxicity reducing strategies, 67 PRODIGE-4/ACCORD-11 trial, 66–67 regimen selection, 69 second-line therapies 5-FU/leucovorin, 70–71 5-FU plus oxaliplatin-based combination, 70 nanoliposomal irinotecan, 70–71 NAPOLI-1 trial, 71 Methyl-CpG-binding domain proteins (MBDs), 135–137 Microenvironments, 8–9 MicroRNAs (miRNAs), 9, 99–100, 115 Mi2/nucleosome remodeling and deacetylase (Mi2/ NuRD) complex, 30–31 miRNAs. See MicroRNAs (miRNAs) Mitochondrial pathway, 98–99 Mitogen/extracellular signal-regulated kinase (MEK), 72 MLL1, 143–144 MMPs. See Matrix metalloproteinases (MMPs) Molecular markers, 19–20, 19t Molecular targets of curcumin, 92t of genistein, 92t Monoclonal antibodies, 33 Monoterpenoids, 113 MPACT trial (Metastatic Pancreatic Adenocarcinoma Clinical Trial), 67–69 Mucin1, 142 Mucin 4 (MUC4) nanovaccine, 121 Multidrug resistance (MDR), 90–91, 122–123 Multiwalled carbon nanotubes (MWNT), 166 Myeloid-derived suppressor cells (MDSCs), 120

INDEX

N Nab-paclitaxel, 16–17, 76 Nanocapsules, 163–164 Nanocarriers, 162–163 physicochemical properties, 163, 164f with selected agent on pancreatic cancer therapy, 167, 167–170t Nanogels, 165 Nanoliposomal irinotecan (Nal-IRI), 70–71 Nanoparticles, 166–170 magnetic, 166–167 mesoporous inorganic, 165–166 metal oxide, 166 Nanoscience, 162–166 Nanotechnology, 162–166 Nanovehicles, 166–167 NAPOLI-1 trial, 71 N-cadherin, 26–27, 30–31 Neoadjuvant chemotherapy-gemcitabine, S-1, LV (NAC-GSL), 65 Neoadjuvant therapy, 64–65 Neucleoside transporter (NT) genes SLC28 family gene polymorphisms, 179–181, 180–181t SLC29 family gene polymorphisms, 180–181t, 181–182 Neuroendocrine tumors (NETs), 119 Neuronal pentraxin II (NPTX2), 137 NF-κB. See Nuclear factor-kappa B (NF-κB) Nonsteroidal anti-inflammatory drugs (NSAIDs), 119 Notch intracellular domain (NICD), 32, 100 Notch signaling pathway, 100, 123 Nuclear factor-kappa B (NF-κB), 6, 19–20, 20f, 96, 123 Nuclear protein 1 (Nupr1), 137 Nucleoside diphosphate kinase (NDPK), 177–178 Nutraceuticals, 91

O Oncogene-induced senescence (OIS), 143 Oncogenesis, 5–6 KRAS mutation, 5–6, 6f nuclear factor kappa B signaling cascade, 6 phosphoinositide 3-kinase/ATK cascade, 5 Raf/mitogen-activated protein kinase cascade, 5 Oridonin, 113–114 Oxidative stress, 99

P P53, 7, 18, 31 Paclitaxel, 47, 113–114 Paired related homeobox 1 (PRRX1), 31 Pancreas, 118 Pancreatic adenocarcinoma, treatment for adjuvant chemoradiation

201

gemcitabine plus nab-paclitaxel, 64 adjuvant therapy in borderline removable tumor, 64–65 gemcitabine and capecitabine, 63 gemcitabine and 5-fluorouracil, 62 gemcitabine and S-1, 63 localized surgically resectable stage I and II disease, 62 neoadjuvant therapy, 64–65 FOLFIRINOX as, 65 with GSL, 65 Pancreatic cancer (PC), 89–90 association of age and gender, 15, 16f biological stages of development, 3–4, 3f clonal expansion, 4–5 initiation, 4 causes and prevention, 118–121 chemotherapy resistance pathways, 123–124 familial, 9–10 incidence, 15, 16f microenvironments, 8–9 molecular biology, 3–5 mortality, 2–3 oncogenesis, 5–6 pathogenesis, 3–5 prevalence in U.S, 15, 15t prognosis, 15 risk factor of, 112–113 and small molecules, 124–128 therapeutic treatments, 118–121 treatment resistance, 121–123 tumor suppressor genes, 6–8 Pancreatic ductal adenocarcinoma (PDAC), 15, 34–35, 135 CLDN5, 137 genes under expressed in, 136f HDACs, 139–140 histone modifications, 139, 140f Mucin1, 142 p16 tumor suppressor gene promoter inactivation via methylation, 137 Pancreatic intraepithelial neoplasias (PanINs), 4–5 Pancreatic neoplasms, 4 Pancreatic neuroendocrine tumors (PanNETs), 138 Pancreaticoduodenectomy. See Whipple procedure Pancreatic stellate cells (PSCs), 91 PANCREOX trial, 70 Panitumumab, 73 p19ARF, 7 Passive targeting, 163 PDAC. See Pancreatic ductal adenocarcinoma (PDAC) PEGPH20, 75 PEP02 (MM-398), 76 Pericytes, 47–48

202

INDEX

Peripheral blood mononuclear cells (PBMC), 189 Peutz-Jeghers syndrome (PJS), 112–113 Phosphatases, 134–135 Phosphatidylinositol-3-kinase (PI3K), 95–96, 123 Phosphoinositide 3-kinase/ATK cascade, 5 Pimasertib, 72 P16INK4A, 7 Platelet-derived growth factors (PDGF), 74–75 PODXL, 52 Poly(curcumin-dithiodipropionic acid) (PCDA), 101–102 Polycomb repressive complex 2 (PRC2), 142–143 Polyethylene glycol (PEG), 165 Polymeric micelles, 164–165 Polymeric nanoparticles (PNPs), 163–164 Positron emission tomography (PET), 61 PRODIGE-4/ACCORD-11 trial, 66 outcomes, 67 Prostaglandins (PGE), 47, 96–97 Protease, serine 1 (PRSS1), 9 Protein-protein interactions, 124–125 Proto-oncogenes, 4

R Radiation therapy, 121, 161–162 Raf/mitogen-activated protein kinase (MAPK) cascade, 5 Ras association domain family 1 (RASSF1), 138 RAS proto-oncogenes, 72 Refametinib, 72 Regulatory T cells, 120 Resectable surgery, 14 Reversin 121 (R121), 123 Ribonucleotide reductase (RNR), 46, 49, 122, 177–178 genes coding for, 187–189, 188t ROBO, 138 Ruxolitinib, 73

S S-1, 63 Secreted apoptosis-related protein 2 (SARP2), 137 Selumetinib, 72 Serine peptidase inhibitor, kazal type 1 (SPINK1), 9 SET domain-containing 1A (SET1a), 143–144 SET domain-containing 1B (SET1b), 143–144 Signaling pathways in EMT regulation, 28f Notch, 32 TGF-β, 32 TNF-α, 33 Wnt/β-catenin, 32 epidermal growth factor receptor, 94–97 gemcitabine in targeting, 50–52

Signal transducer and activator of transcription proteins (STAT) pathway, 98 Single-walled carbon nanotubes (SWNT), 166 Sirtuins (SIRTs), 134–135, 139–140 SLC28 family gene polymorphisms, 179–181, 180–181t SLC29 family gene polymorphisms, 180–181t, 181–182 SMAD4, 8 Small interference RNA (siRNA), 93–94 Small molecules, 124 Small ubiquitin-related modifier (SUMO)-conjugating enzymes, 134–135 Smoking, 4, 14, 119 Smoothened homologue (SH), 75 SMYD2, 144–145 SNAIL, 30 Solid tumors, 122–123 S727 phosphorylation, 51–52 Stellate cells, 8–9 Stem cell compartment, 3 Stereotactic ablative radiotherapy (SART). See Radiation therapy Sulforaphane (SFN), 123 Sunitinib, 74–75 Survivin, 18

T Tanespimycin, 125–126t, 126–128, 127f Telomere shortening, 4, 7 Ten-eleven translocation (TET) protein, 136–137 Terpenoids in caspase activity regulation, 113–114 with differential targets, 115 isoprene subunits, 113 molecular targets, 113–115, 114f as NF-κB signaling inhibitors, 113 targeting apoptotic proteins, 114 with targeting DNA damage, 114 TGF-β. See Transforming growth factor-β (TGF-β) TGF-βRII, silencing of, 51 Therapeutic treatments, 162f chemotherapy, 120–121, 161 C-ion RT, for locally advanced pancreatic cancer (LAPC), 121 5-flurouracil, 121 immunotherapy, 121 radiation therapy, 121, 161–162 total pancreatectomy, 120 Whipple procedure, 120 Thioredoxin, 49 Thymidylate synthase (TYMS), genes coding for gemcitabine, 184–186t, 187 Tipifarnib, 72 TNF. See Tumor necrosis factor (TNF)

INDEX

Total pancreatectomy, 120 Toxicitiy, with gemitabine, 178 Transcription start site (TSS), 135–136 Transforming growth factor-β (TGF-β), 8, 32, 74 Trastuzumab, 72 Trastuzumab emtansine (T-DM1), 76–77 Treatment resistance, causes of, 121–123 Trichostatin A (TSA), 139 Triphendiol, 102 Triterpenoids, 113–114 Tumor-associated macrophages (TAMs), 91 Tumor hypoxia, 77 Tumor microenvironment, 90–91 Tumor necrosis factor (TNF), 18–20, 20f Tumor necrosis factor-alpha (TNF-α), 33 Tumor-node-metastasis (TNM) staging, 60–61 Tumor stroma, 74–75 Tumor suppressor genes (TSG), 4 CDKN2A, 6–7 SMAD4, 8 TP53, 7 Twist, 30–31 Type II diabetes, 4

203

U Ultrasound (US), 61

V Vascular endothelial growth factor (VEGF), 74, 97–98 Vascular endothelial growth factor receptor (VEGFR) inhibitors, 74 Vav1, 138–139 Vincristine, 113 Vismodegib, 75

W Whipple procedure, 62, 120 Wnt-1 gene, 32 World Health Organization (WHO), 178

X X-linked inhibitor of apoptosis protein (XIAP), 18, 95–96

Z ZEB1, 29